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AUTOSOMAL RECESSIVE POLYCYSTIC KIDNEY DISEASE EPITHELIAL CELL MODEL REVEALS MULTIPLE BASOLATERAL EGF RECEPTOR SORTING PATHWAYS

By

SEAN P. RYAN

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Thesis Advisor: Cathleen R. Carlin, Ph.D.

Molecular Virology Program Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY

August, 2010

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Sean P. Ryan _____________________________________________________

Ph.D. candidate for the ______________________degree *.

Jonathan Karn

(signed)_______________________________________________ (chair of the committee)

Cathleen Carlin

________________________________________________

Calvin Cotton

________________________________________________

Phil Howe

________________________________________________

________________________________________________

________________________________________________

3/4/2010

(date) _______________________

*We also certify that written approval has been obtained for any proprietary material contained therein.

Dedication For my wife and family.

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Table of Contents Page

Dedication

iii

Table of Contents

iv

List of Figures

vi

Acknowledgements

viii

List of Abbreviations

ix

Abstract

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Chapter 1. Introduction

1

1.1 Overview

1

1.2 Development of polarity

2

1.3 Polarized epithelial structure

4

1.4 Epithelial sorting and trafficking

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1.5 The kidney and polycystic kidney disease

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1.6 Epidermal Growth Factor Receptor

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1.7 Figures

23

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Chapter 2. Methods and Materials

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2.1 Experimental methods and materials

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Chapter 3. Autosomal recessive polycystic kidney disease epithelial cell model reveals multiple basolateral EGF receptor sorting pathways

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3.1 Introduction

52

3.2 Results

55

3.3 Figures

65

Chapter4. Discussion and Future Directions

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4.1 Discussion

81

4.2 Future Directions

89

4.3 Conclusions

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4.4 Figures

97

References

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v

List of Figures Page Figure 1.1

Epithelial Cell Morphology.

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Figure 1.2

Protein complexes involved in polarity.

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Figure 1.3

Apico-basolateral Junctions in Polarized Epithelia.

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Figure 1.4

Desmosomes and Hemidesmosomes in Polarized Epithelia.

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Figure 1.5

Sorting in Polarized Epithelia.

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Figure 1.6

Exocyst Complex.

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Figure 1.7

GTPase Cycle.

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Figure 1.8

PKD proteins.

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Figure 1.9

Bicadual-C and BPK mutation.

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Figure 1.10

Epidermal Growth Factor Receptor Sorting Signals.

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Figure 3.1

Conditionally immortalized cell lines from ARPKD mouse model.

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Epithelial cell markers distributed normally in conditionally immortalized cell lines.

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Figure 3.3

EGFR expression in conditionally immortalized cell lines.

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Figure 3.4

EGFR 658-LL basolateral sorting signal interacts with AP-1B.

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Figure 3.5

EGFR follows multiple basolateral sorting pathways in MDCK cells.

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T654D mutation reconstitutes basolateral EGFR expression in cystic cells.

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EGFR co-localizes with Rab11-positive sub-apical compartments in cystic cells.

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The T654D substitution does not enhance in vitro AP-4 binding and PMA does not alter EGFR localization in cystic cells.

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Figure 3.2

Figure 3.6

Figure 3.7

Figure 3.8

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Figure 4.1

EGFR sorting pathways in polarized epithelial cells

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Acknowledgements First I would like to thank my mentor Dr. Cathy Carlin for her guidance and support in finishing this project. I would like to thank my committee member past (Dr. Josephine Adams and Dr. Catherine Patterson) and present (Dr. Calvin Cotton, Dr. Jonathan Karn and Dr. Phil Howe) for their questions, comments, and encouragement during this project.

I would like to thank my lab members Dr. Nicholas Cianciola, Dr. Song Jae Kil, Dr. Ankur Shah, Dr. Xuehuo Zheng, Mr. Nikolas Balanis, and Mr. Akash Kataruka for their help with both technical and experimental issues and well as lively scientific discussions.

I would like to thank the Department of Molecular Biology and

Microbiology staff and faculty for their administrative help and support. I would also like to thank the Neurosciences Imaging Center and Maryanne Pendergast for the patient technical support.

Lastly, I would like to thank my friends and family that have

supported and encouraged me throughout this project.

This work was supported by Public Health Service grants P50 DK54178 and RO1GM081498 awarded to Dr. Cathy Carlin and in part by NIH grant T32 HL-007415.

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List of Abbreviations A

alanine

AA

dialanine

ADPKD

autosomal dominant polycystic kidney disease

AEE

apical early endosome

AJ

adherens junctions

Ap

apical

aPKC

atypical protein kinase C

ARE

apical recycling endosome

ARPKD

autosomal recessive polycystic kidney disease

ASE

apical sorting endosome

BEE

basolateral early endosome

BicC

bicaudal C

BL

basolateral

BPK

Balb/C polycystic kidney

BSE

basolateral sorting endosome

Crb

crumbs

CRE

common recycling endosome

D

aspartic acid

Des

desmosomes

Dgl

discs large

DMSO

dimethyl sulfoxide

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E-cad

E-cadherin

ECM

extracellular matrix

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

EMT

epithelial-to-mesenchymal transition

ER

endoplasmic reticulum

ERK

extracellular-signal regulated kinase

FBS

fetal bovine serum

FCY

fibrocystin

FGF

fibroblast growth factor

GAP

GTPase activating protein

GDI

guanine dissociation inhibitor

GDP

guanosine-5'-diphosphate

GEF

guanine nucleotide exchange factor

GJ

gap junction

GPI

glycosyl phosphatidylinositol

GST

glutathione S-transferase

GTP

guanosine-5'-triphosphate

HD

hemidesmosomes

HGF

hepatocyte growth factor

HPV

human papillomavirus

IFT

intraflagellar transport

IGF

insulin-like growth factor

x

IP

immunoprecipitation

JAM

junctional adhesion molecule

Jx

juxtamembrane

KH

K-homology

L

leucine

Lgl

lethal giant larvae

LL

dileucine

MAPK

mitogen-activated protein kinase

MET

mesenchymal-to-epithelial transition

N

nucleus

P

proline

PALS1

protein associated with lin seven 1

Par

partition-defective

PATJ

PALS1-associated TJ protein

PCR

polymerase chain reaction

PKC

protein kinase C

PKD

polycystic kidney disease

PM

plasma membrane

PMA

phorbol 12-myristate 13-acetate

PTEN

phosphatase and tensin homolog on chromosome 10

PtdIns

phosphatidylinositol

SAM

sterile alpha motif

Scr

scribble

xi

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SV40

simian virus 40

T

threonine

TfR

transferrin receptor

TGF

transforming growth factor

TGN

trans-Golgi network

TJ

tight junctions

Thr

threonine

TM

transmembrane

TX-100

Triton X-100

WT

wild-type

ZO

zona occludens

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Autosomal Recessive Polycystic Kidney Disease Epithelial Cell Model Reveals Multiple Basolateral EGF Receptor Sorting Pathways

Abstract by SEAN P. RYAN

Polarized epithelial cells play a vital role as a selective barrier in many tissues and organs in the body. Maintenance of polarity requires a wide array of proteins working in balance and any disruption in this tight regulation can lead to an aberrant phenotype. The goal of this project was to better understand how the epidermal growth factor receptor (EGFR) becomes mislocalized to the apical surface in a mouse cell culture model for autosomal recessive polycystic kidney disease (ARPKD). While the EGFR exhibited this abnormal phenotype, other polarized membrane proteins sorted correctly in cystic BPK cells. We show that the EGFR mislocalization seen in the cystic cells is the result of a defect in the EGFR-specific sorting machinery and not the receptor itself. Even when a wild-type human EGFR was expressed in the cystic cells, the receptor mislocalization phenotype persisted. In vitro, we found an interaction between the EGFR juxtamembrane domain and the epithelial specific clathrin adaptor AP-1B. We report that there is an AP1B-dependent basolateral sorting pathway for the receptor and expression of the wild-

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type receptor in AP-1B deficient LLC-PK1 cells leads to mislocalization of the EGFR. Mutation of the AP-1B binding site of the EGFR also leads to mislocalization of the receptor in both normal mouse kidney and MDCK cells. When this mutant receptor was expressed in cystic cells there was no exacerbation of the abnormal EGFR phenotype, which indicates that the BPK defect interferes with the AP-1B pathway. We discovered that by introducing an aspartic acid mutation at threonine 654 (Thr654) of the EGFR resulted in restoration of basolateral sorting of the EGFR in cystic cells and LLC-PK1 cells. We found that though this receptor could not bind AP-1B, it was instead routed through a Rab11-positive endocytic compartment, possibly the apical recycling endosome (ARE), before reaching the basolateral surface. We conclude that the EGFR is mislocalized to the apical surface in cystic cells through a defect in some co-factor needed for AP-1B-dependent EGFR sorting, but also that there appears to be a Rab11dependent recovery pathway for basolateral sorting of the receptor.

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Chapter 1 Introduction

1.1 OVERVIEW

In this introduction I will describe both how polarity is developed and maintained in polarized epithelia as well as the major structural components that make polarization possible. Polarized epithelial cells play a vital role in the form and function of many organs and tissues in the body. The development and continued maintenance of polarity is crucial in creating the homoeostatic balance required by multiple tissues and organs in the human body. Various environmental cues encountered by polarized epithelial cells can induce the signaling complexes that lead to the organization of a variety of structures which make the tight epithelial monolayer possible. Once established, preservation of this state is aided by various proteins and GTPases which maintain distinct apical and basolateral plasma membrane environments. When the mechanisms that sustain polarity in epithelial cell fail it can cause a variety of diseases such as cancer, polycystic kidney disease, or blindness. Polycystic kidney disease in a prime example of what happens when defects in cell polarity occur in epithelial cells. Proper basolateral localization of the epidermal growth factor receptor also plays an important role in polarity in epithelial cells, but there is a loss of epidermal growth factor receptor polarity in polycystic kidney. This loss of polarity leads to abnormal signaling which reinforces the cystic phenotype.

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1.2 DEVELOPMENT OF POLARITY

Morphology & Function

Epithelial cells are characterized by their ability to form tight cell-cell contacts and act as a barrier which allows distinct biological compartments to exist. The first step in the initiation of epithelial polarity is the interaction of cells with the extracellular matrix (ECM) which is facilitated by Rho GTPases and cell surface proteins such as integrins [1].

Cells are then able to modify the surrounding ECM via matrix

metalloproteinases [2]. The next step is the development of cell contacts between other epithelial cells and the creation of two distinct microenvironments, apical and basolateral (Fig. 1.1). These contacts are initiated by cell surface proteins such as cadherins, which lead to a variety of intracellular protein interactions and signal transduction. The apical surface is important in absorption and secretion in the lumen of different organs such as the stomach, kidney, and vascular system. The apical surface acts as a site for regulation of extracellular cues via cell signaling molecules found in the lumen. The basolateral surface is responsible for anchoring the cells via both cell-cell and cell-matrix attachments via a variety of surface proteins as site for regulation of cell signaling from underlying tissue and adjacent cells.

These two microenvironments allow the

development of the complex organ systems in the body. Even plasma membrane lipid composition differs between the apical and basolateral surfaces (Fig. 1.5).

The apical

surface is highly enriched in PtdIns(4,5)P2, while the basolateral surface is enriched in 2

PtdIns(3.4.5)P3. The apical surface is enriched in phosphatase and tensin homolog on chromosome 10 (PTEN), which converts PtdIns(3.4.5)P3 to PtdIns(4,5)P2 and its activity is important for maintaining plasma membrane polarity. Studies have shown that adding the opposite phosphoinositide to either surface reroutes domain-specific cargoes to the incorrect surface [3, 4]. Studies have also recently shown that PtdIns(3.4.5)P3 is required in recycling endosomes for basolateral sorting of some proteins in polarized epithelia [5].

Polarity Complexes

Three main complexes work together and play a major role in the development of apico-basolateral polarity in epithelial cells (Fig. 1.2).

Par/aPKC complex, which

includes Par3, Par6, and aPKC, come together near the newly formed adherens junctions and link to the Rac1/Cdc42 pathways [6-9].

Crumbs complex containing Crumbs,

PALS1, and PATJ has been shown to be an important apical membrane determinant in Drosophila [10]. Scribble complex which includes Scribble (Scr), lethal giant larva (Lgl), and discs large (Dlg) has been shown to be a basolateral determinant [11, 12]. Some studies have suggested that Cdc42 conveys polarity signals to the aPKC complex through Par6, playing a key role in establishing polarity [7, 8, 13-15]. Par6 also is linked to the Crumbs and Scribble polarity complexes [16-18]. Par6 acts to stabilize the Crumbs protein Crb, while aPKC can phosphorylate and inactivate Lgl of the Scribble complex

3

[19].

Lgl and PALS1/PATJ can both inactivate the Par/aPKC complex, creating a

feedback loop that helps to regulate polarity [20].

1.3 POLARIZED EPITHELIAL STRUCTURE

Tight Junctions

Tight junctions form one of the integral barriers between the apical and lateral membranes and are comprised a number of proteins including occludins, claudins, and JAM-1 (Fig. 1.3). Also part of the tight junctions are members of the zona occluden (ZO) family (ZO-1, ZO-2, and ZO-3) that link these proteins to the actin cytoskeleton. [21-25]. Tight junctions act as a barrier to both molecules outside the cell as well as to block the movement of integral membrane proteins between the apical and basolateral subdomains.

Occuldins and claudins are tetraspan proteins which contain two

extracellular loops and intracellular domains responsible for binding ZO-1, ZO-2, and ZO-3 [26].

Another tight junction component, JAM-1, is a member of the IgG

superfamily and creates homophilic cell-cell contacts with JAM-1 molecules on adjacent cells [27, 28]. ZO-1, ZO-2, and ZO-3 all contain PDZ domains which are responsible for binding various amino acid motifs on proteins such as claudins and occludins, as well as the JAM-1 [26, 29]. 4

Adherens Junctions

Below the tight junctions is another cell polarity landmark in the adherens junctions. Adherens junctions form cell-cell contacts through interactions with a subset of cadherins, E-cadherin, on the surface of one cell to another (Fig. 1.3) E-cadherin is a member of the large cadherin superfamily of cell adhesion proteins and is primarily expressed in epithelial cells where it forms homophilic interactions with E-cadherin molecules on neighboring cells [30, 31]. E-cadherin interacts directly with β-catenin and p120-catenin and α-catenin acts a cytoskeletal linker through direct interactions with βcatenin and the actin filament network [32-34]. Nectins also play an important role in cell-cell adhesion in adherens junctions. Nectins can form homophilic or heterophilic interactions with nectins on adjacent cells and are linked intracellularly to the actin cytoskeleton by the linker protein afadin [35]. Nectins have also been shown to be necessary for both adherens junction and tight junction formation, suggesting that they may act as a landmark for junction formation [36].

Gap Junctions

The gap junction is another structure involved in cell-cell contact and it facilitates intercellular communication via transport of various ions and other small signaling proteins through the gap junction channels (Fig. 1.3). Each gap junction hemichannel is

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comprised of six connexin subunits and each subunit consists of a multi-pass transmembrane protein [37]. Each hemichannel can be either homomeric or heteromeric in connexin composition and when hemichannels from neighboring cells interact, the completed channels can also be homotypic or heterotypic in nature. Over 20 different connexins have been discovered in mouse and humans and it’s believed that different connexins confer varied selectivity to gap junctions [38, 39]. Studies have also shown that connexins can interact either directly or indirectly to cytoskeletal proteins [40, 41].

Desmosomes

Much like adherens junctions, desmosomes form strong intercellular contacts and are linked to the cytoskeletal network (Fig. 1.4). Desmosomes are composed of multiple subunits which together link to the desmosome–intermediate filament complex that provides a scaffolding that affords structural integrity to withstand various mechanical stresses [42-44].

Desmosomes are comprised of a few basic structures including

desmosomal cadherins, desmosomal plaque, and intermediate filament linker proteins. Desmocollin and desmoglein are the two cadherin family members that form the cell-cell contacts in desmosomes while the desmosomal plaque contains plakoglobin and plakophilins which interact with desmoplakin to link desmosomes to the cell cytoskeleton via intermediate filaments [45, 46].

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Hemidesmosomes

While desmosomes form tight cell-cell contacts between cells, hemisdesmosomes are responsible for forming cell-ECM contacts (Fig. 1.4). Type II hemidesmosomes which are found in epithelia, such as those in the intestinal lining and kidney nephron tubules and collecting ducts, and consist of α6β4 integrin and the plakin plectin [47, 48]. α6β4 integrin binds to laminin 10 on the extracellular matrix and, much like desmosomes, connects with the intermediate filament cytoskeletal network through interactions with plectin.

Plectin connects the hemidesmosome to the intermediate filament network

through interactions with the β4 integrin subunit [49].

1.4 EPITHELIAL SORTING AND TRAFFICKING

Trafficking in Polarized Epithelia

Newly synthesized polarized cargo can leave the trans-Golgi network (TGN) for the plasma membrane either directly or through intermediate sorting endosomes (Fig. 1.5). Apically-targeted cargo can be effectively divided into either lipid raft-dependent or

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lipid raft-independent apical cargo [50]. GPI-anchored proteins are known to associate with lipid rafts and are believed to follow this lipid raft-dependent apical delivery pathway and may be sorted from the TGN to the apical early endosome (AEE) before reaching the apical surface [51, 52]. N- and O-glycosylated proteins have been shown to follow a lipid raft-independent route to the apical surface and are believed to go through the apical recycling endosome (ARE) before reaching the apical plasma membrane via a myosin-Vb-dependent mechanism [53-56]. Other apical sorting pathways include a route through the common recycling endosome (CRE) either directly from the TGN or via transcytosis from the basolateral surface [57-59]. Basolateral cargo is typically sorted via short cytoplasmic tyrosine or dileucine motifs which interact with a variety of clathrin adaptor proteins (APs) to facilitate sorting [60-62]. These tyrosine or dileucine motifs are also generally flanked by acidic clusters of amino acids. Tyrosine-based motifs can be divided into either NPxY or YxxΦ motifs. Dileucine motifs also exhibit some variability as acidic amino acid residues upstream from the dileucine can confer differential binding to adaptor proteins associated with intracellular trafficking [63]. There have also been reports of a hybrid tyrosine/dileucine motif YRLL in AQ3, a member of the aquaporin family, that appears to be involved in basolateral sorting of the protein [64].

Another

basolateral sorting signal that has been discovered is the proline-rich PLTP motif of the epidermal growth factor receptor (EGFR), although the exact mechanism for basolateral sorting remains unclear [65]. Polarized epithelia express four common APs, AP-1A, AP2, AP-3, and AP4, as well as an epithelial specific adaptor AP-1B [66]. AP-1B has been shown to localize to the CRE to facilitate sorting of cargo to the basolateral surface from this compartment [67, 68]. Transferrin receptor is the most commonly cited AP-1B-

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dependent protein which associates with the CRE en route to the basolateral surface [69]. Another indirect route to the basolateral surface has been discovered which leads from the ARE to the basolateral surface. E-cadherin has been shown to sort from the ARE to the basolateral surface in a Rab-11a-dependent manner [70, 71]. Sorting pathways from TGN to the basolateral surface have also been described which sort directly to the basolateral surface or first briefly through the basolateral early endosome (BEE) and may be dependent on another adaptor protein than AP-1B [72, 73].

Exocyst

The exocyst is a conserved multi-protein complex that has been shown to be involved in trafficking, tethering, and fusion of endosomes to the plasma membrane in polarized cells (Fig. 1.6). Although initially shown to be involved in basolateral sorting, studies have confirmed that some apically sorted cargo also utilize the exocyst [74]. The multi-protein complex that makes up the exocyst contains 8 subunits: Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p. This complex works in concert with SNARE proteins to regulate the tethering and subsequent fusion of vesicles destined for the plasma membrane. The exocyst complex was first described in budding yeast and plays an important role in directing cargo to the newly formed bud[75]. Sec3p has been shown to serve as landmark for tethering of specific cargo to specialized subdomains on the plasma membrane in S. cerevisiae [76, 77]. It has also been shown that while secretion is not disrupted in Sec3p mutants, polarized secretion is lost resulting in a more 9

ubiquitous distribution of exocytic markers [77, 78]. While the exact function of the yeast protein Sec5p is unclear, it has been shown to interact with Exo70p in vitro [79]. Sec6p also has been shown to interact with Exo70p as well as being involved anchoring the assembled exocyst complex to sites of secretion through interaction with t-SNARE Sec9p [80, 81]. Along with interacting with Sec3p and providing a landmark for the exocyst on the plasma membrane, Exo70p has been shown to be a Rho GTPase effector and binds to PtdIns(4,5)P2 playing a vital role in secretion [82-84].

Mammalian

homologues of the yeast exocyst proteins have also been discovered. Blocking Sec8 using function-blocking antibodies inhibited delivery of newly synthesized proteins from the TGN to basolateral surfaces in MDCK cells [85]. Sec10 has been shown to be involved in primary ciliogenesis and cystogenesis in MDCK cells, and when Sec10 was knocked down the levels of other exocyst subunits were also reduced [86]. Sec15 has been shown to interact with Rab11a in polarized epithelial cells and is a crucial component in basolateral-to-apical transcytosis [74, 87, 88]. Exo84 has proven to be a key regulator of apico-basolateral polarity as mutant Exo84 result is missorted apical proteins and a loss of polarity similar to that seen in Crumbs mutants [89].

Rab GTPases

Rab GTPases represent the largest family of small GTPases. Like other members of the GTPase family, Rab GTPases function by switching between both an active GTP bound state and an inactive GDP bound state. GTPase activation is initiated when the 10

GDP-bound form of the protein encounters a guanine nucleotide exchange factor (GEF) that displaces the GDP for GTP allowing the protein to interact with effector proteins. Once the Rab interacts with multiple effectors, the GTP is then hydrolyzed by a GTPaseactivating protein (GAP) converting it back to the GDP-bound form (Fig. 1.7). The GDP-bound Rab GTPase can also be geranylgeranylated when presented to a geryanylgeranyl transferase by a rab escort protein. This geranylgeranylation allows the Rab GTPase to be recognized by a GDP dissociation inhibitor (GDI) and different Rab GTPases are then targeted to specific membranes [90-92]. This allows Rab proteins to function on a variety of membrane domains in the cell. Rab5 is associated with early endosomes, Rab4 and Rab11 with recycling endosomes, and Rab7 and Rab9 with late endosomes [92-94].

Rab proteins have been implicated in both development and

maintenance of polarity in polarized epithelia. Rab3B, Rab8, and Rab13 have been shown to localize to the apical junctions in polarized cells [95-97]. Rab proteins also play a key role in the sorting of polarized cargo to the apical and basolateral surfaces. Rab3, Rab11, Rab 14, Rab17 and Rab25 have been shown to be involved in sorting of apical cargo, while Rab8, Rab10, Rab11, and Rab13 have been shown to be involved in basolateral sorting of proteins [58, 70, 95, 98-103].

Epithelial-to-Mesenchymal Transition

During both cell development and repair, polarized epithelial cells undergo an epithelial-to-mesenchymal transition (EMT), as well as the reverse process of 11

mesenchymal-to-epithelial transition (MET). In development, multiple rounds of EMT are required in the transformation of cells to various systems and organs in the body. These ordered rounds of differentiation can be classified into primary, secondary and tertiary EMT.

Examples of primary EMT include gastrulation in invertebrates and

vertebrates and the formation of the neural crest in vertebrates. An example of secondary EMT includes the differentiation of cells into specific subsets cells such as neurons, bone, and cartilage cells in the case of the neural crest or axial, paraxial, intermediate and lateral in the case of mesodermal cells in gastrulation [104]. Finally, tertiary EMT examples include further differentiated cells such as those of the atrioventricular canal endothelial cells which infiltrate the cardiac jelly and eventually form the valvulo-septal complex [105]. A variety of extracellular ligands have been shown to induce EMT, such as transforming growth factor-β, epidermal growth factor (EGF) family members, fibroblast growth factors (FGF), hepatocye growth factor (HGF), insulin-like growth factor (IGF), Wnts, and Notch [104, 106].

Binding of these ligands to membrane

receptors leads to protein-protein interactions of receptors to integral membrane proteins involved in maintenance of polarity or signal transduction leading to modification of polarity complexes [107]. Induction of EMT by these extracellular signals also can affect gene expression in polarized cells which contribute to the EMT. Genes of members of the Snail superfamily of transcriptional repressors are induced via signal transduction and lead to loss of epithelial marks as well as increase in mesenchymal markers, cytoskeletal remodeling, and ECM degradation[108-115]. One defining characteristic of EMT is a cadherin switch that occurs where there is decreased expression of E-cadherin and an

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increase in N-cadherin expression, which is more commonly found in mesenchymal cells [116-120].

Diseases involving dysfunctional polarity

Maintenance of epithelial cells in their respective locations is crucial to creating a healthy environment. There is a network of different proteins and complexes involved in the formation and maintenance of polarity in epithelial cells and dysfunction at any point in this network can create potential problems. Various epithelial polarity proteins have been implicated in tumorigenesis in both animal models and cell culture. Overexpression or loss of certain polarity proteins can cause faulty morphogenesis by disturbing polarity. Loss of E-cadherin expression has been shown to be a characteristic of metastasis in both humans and mice [121, 122]. Downregulation of Scribble via RNAi in immortalized pluripotent mouse mammary epithelial cells lacking the tumor suppressor p53 leads to the formation of mammary tumors [123]. Loss of another Scribble complex protein, Lgl1, via knockout caused severe dysplasia in the brains of mice [124]. Loss of the Crumbs protein Crb3 in mouse kidney epithelial cells causes disruption of tight junctions and apico-basal polarity as well as loss of contact-inhibited growth [125].

Human

papillomaviruses (HPVs), the primary cause of cervical cancer in humans, affects a range of polarity proteins. HPV E6 oncoproteins contain a PDZ recognition motif which has been shown to cause ubiquitin-mediated degradation of PDZ-containing polarity proteins such as Par3, Scribble, Dlg, and PATJ [126-130]. Defects in CRB1 which encodes Crb1 13

of the Crumbs polarity complex have been shown to result in various human retinal dystrophies [131].

Further studies in Drosphilia, zebrafish, and mice have revealed that

mutations or complete loss of certain Crumbs complex proteins leads to dysfunction of photoreceptor cells and blindness [132-134].

1.5 THE KIDNEY AND POLYCYSTIC KIDNEY DISEASE

Kidney

The kidney is a major regulator of homeostasis in the circulatory system, including regulation of pH and salts in blood as well as blood volume and pressure. The functional unit of the kidney is the nephron. The nephron consists of the Bowman’s capsule which encases the glomerulus, the proximal convoluted tubule, the loop of Henle, the distal convoluted tubule, and finally the collecting duct before emptying into the bladder. Filtrate from the blood containing small molecules enters the glomerulus and then goes to the proximal convoluted tubule where amino acids, sugar, and vitamins can be reabsorbed. The filtrate then continues through the loop on Henle which acts to increase salt concentration.

Salt can then be reabsorbed as necessary in the distal

convoluted tubule to maintain both salt and pH balance. Finally, water can be reabsorbed from the filtrate in the collecting duct via stimulation with anti-diuretic hormone, which 14

acts to increase permeability of the cells of the collecting duct to water. Together, thousands of nephrons act in the kidney to maintain the osmotic homeostasis required in the circulatory system.

Polycystic Kidney Disease

Polycystic Kidney Disease (PKD) is a common disease that affects over 600,000 people in the United States alone. PKD can be divided into two distinct inherited forms, autosomal dominant PKD (ADPKD) and autosomal recessive PKD (ARPKD). Both forms of PKD contain defects which result in the formation of cysts in the kidney. Over time cysts grow and block nephron function resulting in end stage renal disease. The majority of ADPKD, which affects ~1:1,000 live births, is caused by mutation in one of two known genes PKD1 or PKD2. These genes encode two membrane proteins known as polycystin-1 and polycystin-2, respectively (Fig. 1.8). Polycystin-1 is a multi-pass transmembrane protein with a

large extracellular

domain that

includes 12

immunoglobulin-like repeats called PKD domains which are possibly involved in protein-protein interactions [135-137]. Polycystin-2 is a calcium permeable nonselective cation channel and shares some homology within its final six multi-pass transmembrane domains with polycystin-1 [138, 139]. ADPKD is distinguished by renal cyst formation in all tubular segments of the kidney, as well as cyst formation in the liver and pancreas [140]. ARPKD, which is less common and affects ~1:20,000 live births, is caused by a mutation in a single known gene PKHD1. PKHD1 encodes a protein called fibrocystin, 15

which has been implicated in both calcium signaling and is thought to associate with polycystin-2 [141, 142] (Fig. 1.8). ARPKD is characterized by renal collecting tubule cyst formation, in addition to biliary ductal plate abnormalities and fibrosis of the liver [143]. Another characteristic phenotype of cystic cells involves EGFR, which is present on both apical and basolateral membranes in contrast to normal renal collecting duct cells where it is predominantly basolateral. The EGFR phenotype is observed in all forms of ADPKD and ARPKD as well as various mouse models including the BPK model for ARPKD [144-146]. While the triggers for cystogenesis are poorly understood in all forms of PKD, once initiated epithelial cells break off from the kidney tubules and form cysts in the kidney cortex and medulla. Cells that were once absorptive begin secreting various growth factors into the newly formed cysts. Studies have shown that all three PKD susceptibility proteins polycystin-1, polycystin-2, and fibrocystin localize to the primary cilia and are functionally related, suggesting that this organelle plays a key role in cyst formation [147, 148]. The primary cilium consists of a core of microtubules called an axoneme which extend from a mother centriole that forms the basal body [149, 150]. The basal body acts as a gate to proteins entering the cilium via the intraflagellar transport (IFT) system en route to the tip of the cilium [151]. In renal epithelial cells the primary cilia act as a mechanosensory organelle which responds to flow on the apical surface leading to changes in calcium flux and also cleavage of both C-terminal and Nterminal domains of fibrocystin and the C-terminal domain of polycystin-1[149]. Deciliation via chemical means has been shown to affect both epithelial junctions and domain-specific surface expression of proteins in MDCK cells [152]. Rodent models have also been identified which exhibit PKD phenotypes, but do not contain mutations in

16

PKD1, PKD2, or PKHD1. Two of the mouse models which affect the Bicc1 gene which encodes the Bicaudal C (BicC) protein are the bpk and jcpk mouse models, which are the result of mutation in different regions of the Bicc1 gene [153-155]. The Oak Ridge Polycystic Kidney mouse, a mouse model for ADPKD, contains a defect in the protein IFT88 (also known as polaris), which is required for cilia formation and IFT [147]. A rat model for ADPKD, Han:SPRD, results from mutations in the poorly defined Cy allele of chromosome 5[156].

BPK Mouse Model

The BPK mouse was identified as a random mutation in a BALB/C mouse which gave rise to a phenotype in mice similar to that of ARPKD in humans [157]. While human ARPKD involves a mutation in fibrocystin, the BPK mouse contains a mutation which has been mapped to the bicc1 gene, which encodes an RNA-binding protein Bicaudal C (BicC).

BicC is important in cell polarity in embryonic development,

although its role in the polarized cell has yet to be clearly understood. BPK mice were mated with an Immorto mouse (H-2Kb-ts-A58), a mouse which has a temperaturesensitive mutant of SV40 T-antigen integrated into all of its cells. Primary collecting tubule cells from the progeny were harvested and developed into either the cystic (homozygous

cystic

BPK/Immorto

mouse)

or

normal

(heterozygous

normal

BPK/Immorto mouse) cell lines. These cells then have a specific growth regime which includes a permissive condition (33˚C) which uses the SV40 T-antigen to actively 17

proliferate the cells, and then a switch to a non-permissive condition (37˚C) which allows the cells to develop into a tight epithelial sheet[146].

Bicaudal C

Bicaudal C (BicC) consists of three conserved N-terminal K-Homology (KH) domains and one C-terminal sterile alpha motif (SAM) (Fig. 1.9). The KH domains of BicC are involved in binding of RNAs while the SAM domain is a protein-binding motif [158-160]. Mouse and human homologues of BicC are approximately 90% identical and it was found the expression of BicC was strong in both heart and kidney tissues in mice [161]. BicC has been shown to regulate its own expression through cis-interactions and is involved in regulation of deadenylation and polyadenylation [162]. BicC was also shown to inhibit Dvl2, a member of the canonical Wnt pathway, and Dvl-induced inhibition of the Gsk3β β-catenin destruction complex [163]. Mutations in β-catenin have also been shown to induce cystic growth in transgenic mice [164, 165]. The endoplasmic reticulum (ER) has recently been found to require BicC to regulate exit site homeostasis needed for normal protein sorting. Bicc1-null embryos result in defective exocytosis. Both ADPKD susceptibility gene products PKD1 and PKD2 have also been shown to be involved in maintenance of structure and function of the ER as well as PKD2 localizing to the ER[139, 166, 167]. This may suggest that the ER may play a role in PKD via the PKD susceptibility genes.

18

1.6 EPIDERMAL GROWTH FACTOR RECEPTOR

EGFR

The EGFR is a member of the ErbB family of receptor tyrosine kinases which also includes ErbB2, ErbB3, and ErbB4 (Fig. 1.10). Newly synthesized ErbBs are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where the 5-7 Nlinked glycans acquired in the ER are co-translationally modified before being delivered to the plasma membrane [168, 169]. When members of the ErbB family bind ligands such as EGF, they induce receptors to form homo- and heterodimers [170, 171]. Once dimerized, autophosphorylation of tyrosine residues occurs via the kinase domains on each receptor. These phosphorylated residues create various sites which bind proteins related to specific signaling pathways. ErbBs also exhibit specificity in ligand binding. EGF and TGF-α have a high affinity for the EGFR, while various neuregulins bind to ErbB3 and ErbB4.

ErbB2 has no known ligand, but can be activated via

heterodimerization with other ErbB family members [172, 173].

These ligands are

synthesized as precursor membrane proteins which are sorted to the plasma membrane and released via proteolytic cleavage at the cell surface [174, 175]. The general domains of the EGFR include an extracellular domain containing two ligand-binding domains (L1 and L2) and two cysteine-rich domains (C1 and C2), a single-pass transmembrane domain (TM), an intracellular juxtamembrane region (Jx), a tyrosine kinase domain, and a C-terminal region (Fig. 1.10) [176]. The L1 and L2 domains act in concert to bind a 19

single molecular of ligand, while the C1 and C2 domains are believed to play roles in ligand affinity, autoinhibition of the receptor, dimerization and conformation [177, 178]. As well as anchoring the EGFR in the membrane, the TM domain is has been shown to be involved in dimerization in erbB2 and based on sequence homology it is believed that other EGFR family members also contain dimerization motifs in this region [179]. The Jx domain of the EGFR contains motifs shown to be important in endosomal and lysosomal trafficking of the receptor in cells, and has recently been shown to be involved in the formation of an asymmetric dimer of the kinase domains of dimerized EGFRs [65, 180-183].

The kinase domain of the EGFR, once induced via ligand binding,

phosphorylates tyrosine residues in the C-terminal region.

Once these C-terminal

tyrosines are phosphorylated, they bind to specific binding motifs of various proteins involved in downstream signal transduction [184].

Polarized EGFR Trafficking

The EGFR is involved in various cellular processes including development, proliferation and differentiation. Tight regulation of EGFR function is critical to ensure that none of these cellular functions is disrupted. While a small fraction of receptor is detectable on the apical surface, the EGFR is predominantly found on the basolateral surface in polarized epithelial cells [185-187]. Basolateral sorting determinants for the EGFR in polarized cells were found on the juxtamembrane domain and a series of truncations of the C-terminus of the receptor found that removing residues between 65120

674 resulted in apical delivery and localization of all EGFR [180]. This indicated that not only was there basolateral sorting information in the 651-674 region, but that there was also apical sorting information that directs the receptor to that surface in the absence of the basolateral signals. The basolateral sorting information of the EGFR juxtamembrane can also route a normally apical GPI anchored protein to the basolateral surface when fused with the EGFR intracellular domain via a chimeric protein [188]. Further studies indicated that a dileucine-motif at residues 658 and 659 and a PLTP-motif at 667-670 were necessary for proper basolateral sorting of the receptor (Fig. 1.10) [65, 188]. Alanine mutations in either proline of the PLTP-motif or truncation of the receptor to residue 663 resulted in increased apical receptor localization and delivery. When the dileucine-motif at 658-659 was mutated to dialanine in the absence of the PLTP motif there was an increase in the apical delivery of the 663 truncated receptor, but this mutation had no effect in a 674 truncated receptor which contained the PLTP [65]. This suggests that the PLTP motif was dominant over the dileucine motif, but that the dileucine motif was also involved in basolateral sorting of the EGFR.

EGFR, the Kidney, and PKD

While the EGFR is localized to the basolateral surface in the epithelial cells of the nephron, EGF has been found to be highly enriched in urine due to release via proteolytic cleavage of proEGF from the apical surface [189]. This sequestration of ligand and receptor plays a key role in maintaining homeostasis in the collecting duct. Upon injury 21

to the epithelial cells surrounding the collecting duct, EGF leaches into the basolateral interstitium and can then interact with the EGFR leading to cell repair[190, 191]. Extended EGFR activation, however, is thought to play a key role in the development of various renal diseases, including PKD [192, 193]. In PKD, the EGFR mislocalizes to the apical surface where it can become activated by the EGF. Upon cyst formation, EGF is found to be highly enriched in cyst fluid and cells that were once absorptive begin secreting various growth factors into this cyst fluid promoting further EGFR activation and cellular proliferation that accompanies PKD.

Summary My goal in this thesis was to obtain a better understanding of how the EGFR becomes mislocalized to the apical surface in the cystic BPK mouse cells. I found that the EGFR-specific sorting machinery was at fault in these cystic cells, and lack of AP-1B can create a similar phenotype in LLC-PK1 cells. In vitro binding assays also show that the EGFR can bind specifically to the AP-1B mu subunit, further suggesting a role for AP-1B in EGFR sorting.

Finally, introduction of an aspartic acid point mutation at

threonine 654 of the EGFR resulted in basolateral recovery of receptor in both cystic and LLC-PK1 cells through a Rab11-positive endocytic compartment.

22

1.7 FIGURES

Figure 1.1 Epithelial Cell Morphology General morphology of a polarized epithelial cell. From Top to Bottom: Primary Cilia (red), Apical Surface (Green), Basolateral Surface (Orange). Proteins or sorting signals involving specific membranes are colored accordingly.

23

Figure 1.1 Epithelial Cell Morphology

24

Figure 1.2 Protein complexes involved in polarity There are three main polarity complexes in polarized epithelial cells. Par3/Par6/aPKC polarity complex associates with the tight junctions. The Crumbs complex associates with the apical membrane.

The Scribble complex associates with the basolateral

membrane. (A) Cdc42 regulates the Par3/Par6/aPKC complex through Par6 which leads to activation of aPKC. (B) Par6 either directly or indirectly interacts with Crb3 of the Crumbs polarity complex. (C) aPKC phosphorylates and inactivates Lgl of the Scribble polarity complex. (D) Activated aPKC and Par3/Par6 regulate tight junction formation. (E) Dominant negative PATJ negatively regulates tight junction formation. (F) Lgl and the Scribble complex are required for Par3/Par6/aPKC complex dissociation.

25

Figure 1.2 Protein complexes involved in polarity

26

Figure 1.3 Apico-basolateral Junctions in Polarized Epithelia (A) Tight Junction complex proteins. Occludins and Claudins create homophilic cell-cell adhesions and link to the actin cytoskeleton via ZO-1, ZO-2, and ZO-3. JAM-1 also creates homophilic cell-cell contacts in the tight junction.

(B)

Adherens Junction

complex proteins. E-cadherin forms homophilic cell-cell adhesions which link to the actin cytoskeleton via the catenin complex of α-catenin, β-cateinin, and p120 catenin. Nectins also create cell-cell contacts and link to the actin cytoskeleton via afadin. (C) Gap Junction complex proteins. Six connexin molecules form to create one hemichannel which interacts with a hemichannel of an adjacent cell to create a complete gap junction.

27

Figure 1.3 Apico-basolateral Junctions in Polarized Epithelia

28

Figure 1.4 Desmosomes and Hemidesmosomes in Polarized Epithelia (A) Desmosome complex proteins. Desmocollin binds desmoglein from an adjacent cell creating a cell-cell adhesion and linking to the intermediate filament network via plakophilin, plakoglobin, and desmoplakin. (B) Hemidesmosome complex proteins. Integrin α6β4 binds extracellularly to Laminin on the basement membrane and is intracellularly linked to the intermediate filament network via plectin.

29

Figure 1.4 Desmosomes and Hemidesmosomes in Polarized Epithelia

30

Figure 1.5 Sorting in Polarized Epithelia Proteins are sorted from the TGN either directly to the apical (C) or basolateral surface (D) or first through intermediate sorting endosomes to the apical (A, E, O) or basolateral (A, K). From the CRE proteins are sorted to the apical surface via the ARE (F to G) or to the basolateral surface either directly (B) or indirectly through the BEE (H to I). Recycling occurs from both the apical and basolateral surfaces into early endosomes (I’, J’)

and proteins can be sorted back to the home surface or sorted either directly to the

CRE (H’, M) or apical cargo can be first directed through the ARE en route to the CRE or back to the apical surface (N to F’ or G). Some proteins have also been shown to sort from the ARE to the basolateral surface (L).

31

Figure 1.5 Sorting in Polarized Epithelia

32

Figure 1.6 Exocyst Complex Activated Rab and Ral GTPases promote initial exocyst docking. Rho or Cdc42 GTPases then activates the completed exocyst complex, promoting t-SNARE assembly and SNARE-mediated fusion at the exocyst site. (Adapted from [194])

33

Figure 1.6 Exocyst Complex

34

Figure 1.7 GTPase Cycle (A) Inactive GDP-bound GTPase interacts with GEFs which replace the GDP with GTP creating an active GTPase. (B) Active GTPase binds effector proteins. (C) After effector binding, GAPs hydrolyzes the GTP on the GTPase and inactivates it. (D) GDIs bind inactive GTPases and prevent further activation until shuttled to the proper place for additional rounds of activation. GEF – guanine nucleotide exchange factor, GAP – GTPase-activating protein, GDI – GTP dissociation inhibitor

35

Figure 1.7 GTPase Cycle

36

Figure 1.8 PKD proteins Schematics of proteins related to PKD in human. Polycystin-1 (Left) and Polycystin-2 (Middle) are the proteins related to ADPKD in humans. Fibrocystin (Right) is the protein related to ARPKD in humans. (Adapted from [195])

37

Figure 1.8 PKD proteins

38

Figure 1.9 Bicaudal-C and BPK (A) Schematic of the Bicc1 gene. Exons responsible for the SAM domain (blue), an alternatively spliced exon (green), and mutated exon in BPK mouse (red).

Arrow

indicates the region where a GC insertion creates a frame-shift mutation in the BPK mouse. (B) Transcripts associated with Bicc1 gene. In the BPK mouse the GC insertion creates an elongated transcript A versus the wild-type product, while the alternatively spliced transcript B is unaffected by the insertion. (C) Schematic of the BicC protein. BicC contains RNA-binding N-terminal KH-homology domains and a protein-binding Cterminal SAM domain. KH – K homology, SAM – sterile alpha motif

39

Figure 1.9 Bicaudal-C and BPK

40

Figure 1.10 Epidermal Growth Factor Receptor Sorting Signals (A) Schematic of the domains of the EGFR. (B) EGFR juxtamembrane region between residues 652-674. Residues in the 658-LL and 667-PLTP (blue) have been shown to be involved in basolateral trafficking of the EGFR and just upstream of these two signals is a threonine which is a putative PKC phosphorylation site (green).

41

Figure 1.10 Epidermal Growth Factor Receptor Sorting Signals

42

Chapter 2 Methods and Materials

2.1 EXPERIMENTAL METHODS AND MATERIALS

Antibodies and Reagents

The following antibodies were purchased: actin, AP-1 γ-subunit, AP-2 α-subunit, and ezrin mouse monoclonal antibodies from Sigma (St. Louis, MO); AP-1A, AP-1B, and AP-4 µ-subunit specific rabbit polyclonal antibodies from Proteintech (Chicago, IL); βcatenin, CD73, E-cadherin, and pan-Erk mouse monoclonal antibodies from BD Biosciences (San Diego, CA); CD73 and c-MET rabbit polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); EGFR rabbit polyclonal antibody from Research Diagnostic (Flanders, N.J); mouse-specific EGFR goat polyclonal antibody from R & D Systems (Minneapolis, MN); phospho-specific EGFR (Thr654) mouse monoclonal antibody from Abcam (Cambridge, MA); phospho-specific MAPK rabbit and biotin HRP-conjugated goat polyclonal antibodies from Cell Signaling (Beverly MA); Rab11 rabbit polyclonal antibody and transferrin mouse monoclonal antibody from Zymed (San Francisco, CA); and transferrin receptor mouse monoclonal antibody from Leinco (Ballwin, MO).

ZO-1 rat monoclonal antibody was obtained from Developmental

Studies Hybridoma Bank developed under the auspices of NICHD and maintained by The University of Iowa Department of Biological Sciences (Iowa City, IA). Human43

specific EGFR1 mouse monoclonal antibody was produced using the ascites method [196].

Detyrosinated tubulin ID5 mouse monoclonal antibody was a gift from J.

Wehland (Gesellschaft für Biotechnologische Forschung, Braunschweig, GER). Rabbits were immunized with synthetic peptides corresponding to sequences from mouse Bicc-1 (NCBI Reference Sequence NM_031397) and human fibrocystin (NCBI Reference Sequence NP_619639 ). Conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (Fort Washington, PA), biotin and streptavidin reagents from Pierce (Rockland, IL), and receptor-grade mouse EGF and phorbol 12-myristate 13acetate (PMA) from Sigma.

Cloning and Mutagenesis

Nucleic acids coding for human EGFR juxtamembrane residues 652 to 697 were PCRamplified from a full-length EGFR/pCB6+ template described in [180]. The following primers replaced the alanine residue at position 698 with a stop codon (bold) and incorporated novel Bam H1 (underlined) and Eco RV sites (italics): Forward

5’-GTAATCGGATCCAAAAGAGAACACTGCGGAGGCTG

Reverse

5’-ATCGATATCTTAACCGGAGCCCAGCACTTTG-3’

Gel-purified PCR product was digested with Bam H1 and Eco RV, and ligated to Bam H1 and Sma I sites in pGEX-3X to produce a glutathione S-transferase (GST) fusion protein in E. coli (Amersham-Pharmacia; Piscataway, NJ). Nucleic acids coding for the dileucine motif at positions 658 and 659 were mutagenized to a di-alanine, and threonine residue 654 to alanine or aspartic acid, using the EGFR-Jx/pGEX-3X template, a Quick Change Mutagenesis kit (Invitrogen), and the following primers (mutations in bold): 44

(658-AA) forward

5′-AGAACACTGCGGAGG GCGGCGCAGGAGAGGGAGCTT-3′

(658-AA) reverse

5′-AAGCTCCCTCTCCTGCGCCGCCCTCCGCAGTGTTCT-3′

(T654A) forward

5′-CGTGGGATCCAAAAGAGCACTGCGGAGGCTGCTGCAG-3′

(T654A) reverse

5′-CTGCAGCAGCCTCCGCAGTGCTCTCTTTTGGATCCCACG-3′

(T654D) forward

5′-CGTGGGATCCAAAAGAGATCTGCGGAGGCTGCTGCAG-3′

(T654D) reverse

5′-CTGCAGCAGCCTCCGCAGATCTCTCTTTTGGATCCCACG-3′

The 658-AA and T654D substitutions were also introduced to full-length human EGFR cloned in the eukaryotic expression plasmid pBK∆lacP-CMV [182] and these mutagenic primers (mutations in bold). (658-AA)

forward

5'-

AGCGCACGCTGCGGAGGGCTGCTCAGGAGAGGGAGCTTGTGGAG-3' (658-AA)

reverse

5'-

CTCCACAAGCTCCCTCTCCTGAGCAGCCCTCCGCAGCGTGCGCT-3' (T654D) forward

5'-ACATCGTTCGGAAGCGCGATCTGCGGAGGCTGCTGCA-3'

(T654D) reverse

5'-TGCAGCAGCCTCCGCAGATCGCGCTTCCGAACGATGT-3'

Wild-type and mutant EGFR constructs were then sub-cloned into the pQCXIN bicistronic retroviral packaging vector (BD Biosciences) using the Not I restriction sites upstream of the IRES element and Neomycin resistance gene. PCR primers were designed using the DNASTAR software package (DNASTAR, Inc., Madison, WI), and all PCR products were sequenced in their entirety by automated DNA sequencing.

45

Renal Epithelial Cell Culture Models

The conditionally immortalized cell lines used in this study were isolated from normal and cystic animals bred with an H-2kb-tsA58 transgenic strain expressing temperaturesensitive SV40 T-antigen under the control of a γ-interferon inducible promoter [146]. These cells were propagated in serum-free defined medium consisting of a 1:1 mixture of Dulbecco’s-modified Eagle’s medium and Ham’s F-12 medium, supplemented with insulin (8.3 × 10-7 M), prostaglandin E1 (7.1 × 10-8 M), selenium (6.8 × 10-9 M), transferrin (6.2 × 10-8 M), triiodothyronine (2 × 10-9 M), dexamethasone (5.09 × 10-8 M), and recombinant γ-IFN (10 units/ml-GIBCO-BRL, Gaithersburg, MD) at permissive temperature (33ºC).

Confluent cultures were re-fed daily with γ-IFN-free medium

supplemented with 5% fetal bovine serum (FBS) for 4 to 6 days at non-permissive temperature (37ºC) to facilitate terminal differentiation. Canine MDCK and pig LLCPK1 renal epithelial cell models were maintained in minimal essential medium (MEM) or MEM-α, respectively, supplemented with 10% FBS and 2 mM glutamine. All renal cell lines were seeded on polycarbonate Transwell filter inserts (0.4 µm pore size; Costar Corp, Cambridge, MA) at high density to generate electrically resistant monolayers suitable for domain-specific assays approximately 4 days later.

Retroviral Gene Transfer

Recombinant retroviruses expressing wild-type and mutant human EGFRs were produced in HEK GP2-293 packaging cells using established methods. Cell populations with 46

stable human EGFR expression were generated by selection in 200 µg/ml G418 (Calbiochem; San Diego, CA) for 10 to 14 days followed by 2 to 3 rounds of human EGFR enrichment using sterile flow cytometry after cells were stained with EGFR1 antibody [188].

Microscopy

For scanning electron microscopy filter-grown cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, post-fixed in 2% osmium tetroxide, dehydrated in graded ethanol, and subjected to critical point drying in liquid CO2. Excised filters were mounted on aluminum stubs, sputter-coated with platinum, and viewed on a JEOL JSM840 Scanning Electron Microscope at × 3,000 magnification. For confocal microscopy, samples were prepared using a published method [197]. Briefly, cells were perforated with 0.5% β-escin in a solution of 80 mM PIPES, pH 6.8, supplemented with 5 mM EGTA and 1 mM MgCl2 for 5 min and fixed with 3% paraformaldehyde–PBS for 15 min. Cells were stained with primary or secondary antibodies overnight at 4°C or for 1 h at room temperature. Antibodies were diluted in a solution containing 0.5% β-escin and 3% radioimmunoassay-grade BSA and samples were blocked with a solution containing 1% normal serum from the host animal used to generate the secondary antibody between incubations with primary and secondary antibodies. Alternatively cells were fixed with 3% paraformaldehyde–PBS for 15 min and incubated with antibodies added to apical or basolateral surfaces to detect extracellular epitopes [188]. Cells were optically sectioned with a Zeiss LSM 510 Meta laser scanning microscope equipped with argon and helium-

47

neon lasers (Carl Zeiss MicroImaging, Jenna, Germany). Image resolution with a Zeiss 100 × Plan Apo, NA 1.4 oil immersion objective and Zeiss LSM software was 1024 × 1024 pixels.

Domain-Specific Biotinylation and Triton X-100 Extractability

Filter-grown cells were rinsed three times with PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 (PBS-CM) and then incubated for 30 min at 4ºC with EZ-Link™ SulfoNHS-LC-biotin (1 mg/ml) dissolved in borate buffer (85 mM NaCl, 4 mM KCl, 15 mM Na2B4O7, pH 9.0) or PBS-CM added to the apical or basolateral surface, respectively. The reaction was quenched with 50 mM NH4Cl and cells were lysed with a solution of 1% (w/v) Triton X-100 in 20 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA, 0.2% BSA, 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM leupeptin (immunoprecipitation buffer). Experiments to determine temperature sensitive Triton X-100 extractability were carried out as described in [198]. Briefly, cells were lysed with 0.5% (v/v) Triton TX100 in 10 mM Tris, pH 7.5, supplemented with 120 mM NaCl, 25 mM KCL, 2 mM EDTA, 2 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM leupeptin. Lysates were incubated on ice or at 37oC for 30 min and clarified by high-speed centrifugation.

48

Pulse-Chase Experiments

Filter-grown cells were pre-incubated in methionine and cysteine-free medium for 1 h. The amino acid-starved cells were pulse-labeled from the basolateral surface with

35

S-

Express Protein Labeling Mix (2.5 mCi/ml; PerkinElmer Life Sciences, Boston, MA) diluted in amino acid-deficient medium supplemented with 10% dialyzed FBS and 0.2% BSA. The radio-labeled cells were then incubated at 37ºC in medium supplemented with a 10-fold excess of non-radioactive methionine and cysteine (chase medium) for up to 3 h. In some experiments cells were pre-incubated for 2 h at 18ºC followed by a 37ºC recovery period. Cells were solubilized with immunoprecipitation buffer.

In Vitro Pull-down Assays

A synthetic peptide corresponding to human EGFR residues 645 to 677 (Peptide 1) was coupled to Sepharose beads using an NHS-activated bead kit (Pierce). GST fusion proteins were purified from BL21 cells transformed with pGEX-3X plasmids encoding wild-type (Peptide 2) and mutant (labeled 658-AA, T654A, and T654D in Fig. 4B) EGFR juxtamembrane sequences. Bacteria were cultured at 37°C until reaching OD600, induced with 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 16 h at room temperature, and collected by low-speed centrifugation. Cells were subjected to one freeze-thaw cycle and lysed with B-PER solution according to the manufacturer’s instructions (Pierce). Supernatants were adjusted to 3% Triton X-100 and incubated with glutathioneSepharose beads (Amersham-Pharmacia) for 20 min at 4°C with rotation. Beads with

49

attached fusion proteins were incubated with crude sub-cellular fractions enriched for clathrin adaptors according to published methods [199, 200]. Briefly, cells were scraped in PBS supplemented with 2 mM EDTA, 5 mM EGTA, and protease inhibitors, resuspended in 0.1 M MES, pH 6.5, 0.2 M EDTA, 0.5 mM MgCl2, 0.02% NaN3, 10 mg/ml BSA, and protease inhibitors, and lysed with 1% NP-40 for 5 min at room temperature. Post-nuclear supernatants were centrifuged at 60,000 × g for 30 min to collect a crude cytosol fraction. Membrane pellets were resuspended in the NP-40 lysis buffer and incubated with 0.5 M Na2CO3 for 5 min on ice to release peripheral membrane proteins. Beads were incubated with peripheral membrane proteins for 1 h at room temperature followed by extensive washing, and bound proteins were eluted with Laemmli sample buffer and resolved by SDS-PAGE.

Immunoprecipitation and Immunoblotting

Immunoprecipitations were carried out using antibodies adsorbed to protein A-Sepharose CL-4B beads (Sigma). Immune complexes containing biotinylated proteins were boiled for 5 min in 100 µl of 10% SDS and SDS-protein solutions were diluted with immunoprecipitation buffer and incubated with streptavidin-agarose beads for 16 h at 4°C. Affinity-purified protein complexes were eluted with Laemmli sample buffer and resolved by SDS-PAGE. Gels with radioactive proteins were treated with En3Hance (PerkinElmer-NEN, Wilmington, DE) for fluorography. Non-radioactive affinity-purified protein complexes or equal aliquots of total cellular protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes using standard methods. Nitrocellulose

50

filters were incubated with primary antibodies followed by isoform-specific HRPconjugated secondary antibodies for detection by enhanced chemiluminescence (Amersham-Pharmacia).

Image Preparation

Digital images were prepared using Adobe Photoshop® CS4 and Adobe Illustrator® CS4 software packages.

51

Chapter 3 Autosomal recessive polycystic kidney disease epithelial cell model reveals multiple basolateral EGF receptor sorting pathways

3.1 INTRODUCTION

The nephron is the basic structural and functional unit of the kidney [201]. Each nephron has an initial filtering component composed of a glomerulus and Bowman's capsule connected to a long convoluted tubule lined by transporting epithelia. Epithelial cell polarity is vitally important for correct function of different tubule segments [201]. In addition to the apico-basolateral axis, most renal epithelial cells exhibit planar polarity featuring primary cilia extending from the apical membrane [202]. Cell polarity defects have been linked to a number of hereditary kidney diseases including polycystic kidney diseases (PKDs) characterized by the accumulation of fluid-filled cysts in the cortex and medulla [195, 203, 204]. Approximately 50% of afflicted individuals develop end-stage renal disease requiring dialysis or kidney transplantation before the age of 60. Although presently incurable, improved understanding of disease mechanisms is uncovering new

52

prospects for effective pathophysiology-based therapies [205]. Cystic cells and tissues are also unique cell biological models for studying polarized sorting mechanisms. Human PKD susceptibility genes encode large membrane proteins. PKD1 and PKD2 are involved in the autosomal dominant form of the disease (ADPKD) and fibrocystin is defective in autosomal recessive PKD (ARPKD) [206, 207]. Multiple lines of evidence indicate these proteins are functionally related, and all three localize to primary cilia suggesting these organelles have a critical role in cyst formation [147, 148]. Model organisms have also advanced our current understanding of PKD pathophysiology [153, 208]. Some models involve targeted disruptions in PKD orthologs or genes that regulate ciliogenesis. Others such as the BPK (deficient in B-cell progenitor kinase) model arose by spontaneous mutations leading to identification of novel PKD susceptibility genes. The BPK allele encodes an mRNA silencing protein called Bicc-1 providing the first evidence linking translational regulation to PKD disease pathways [153, 162].

The BPK model has the same broad spectrum of renal and hepatic

involvement as ARPKD and played a major role in furthering our understanding of the human disease before fibrocystin was discovered [155, 209].

The BPK mouse also

provides novel insights to cyst formation during prenatal development, and is an important model for evaluating new PKD therapies [210, 211]. ErbB receptor tyrosine kinases (EGF receptor or EGFR, ErbB2, ErbB3, and ErbB4) and their respective ligands have important roles in kidney development and tubule repair [212]. EGFR is normally sorted to basolateral membranes in adult tubular epithelial cells [213, 214].

However, numerous primary PKD genetic defects perturb

EGFR polarity leading to increased apical expression [210]. EGF and EGF-like ligands

53

are also secreted into the apical medium of cultured cystic epithelial cells and detected in cyst fluid from ADPKD patients [215]. Importantly, EGFR tyrosine kinase inhibition significantly improves kidney function and reduces morbidity in PKD animal models [192, 210, 216]. These observations suggest chronic EGF signaling from the apical membrane is a common disease progression factor in multiple forms of PKD. Polarized sorting is mediated by highly specialized sub-cellular machinery that recognizes specific sorting signals in membrane protein cargo [217-221].

These

interactions target newly synthesized molecules from the trans-Golgi network (TGN) to the plasma membrane, recycle internalized cargo back to the same plasma membrane domain, and facilitate transcytosis from one pole of the cell to the other. Apical sorting is regulated by diverse signals including GPI anchors that partition into lipid rafts and Nglycans that are sorted by unknown mechanisms. Although both pathways have the same final destination, lipid-raft and N-glycan dependent cargoes traverse different endosome intermediates called apical sorting endosomes (ASE) and apical recycling endosomes (ARE), respectively [50, 52]. Basolateral sorting is usually regulated by short linear amino acid signals located in the cytosolic tails of protein cargoes [218-221]. The epithelial cell specific clathrin adaptor protein complex AP-1B recognizes some of these signals in common recycling endosomes (CRE) [68, 222, 223]. Relatively less is known about AP-1B independent pathways that may target the plasma membrane directly or via basolateral sorting endosome (BSE) intermediates [221].

Polarized sorting is also

regulated by the multi-protein exocyst complex involved in tethering, docking and fusion of post-Golgi transport vesicles with the plasma membrane [194]. In addition to the core machinery, some cargoes require additional specialized exocyst components [194].

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Our data provide new molecular insights into how EGFR membrane polarity is regulated by multiple hierarchical sorting pathways in renal epithelial cells. Not all the pathways are perturbed in the BPK model, which has important implications for understanding PKD pathogenesis and designing novel therapeutic approaches. Furthermore EGFR sorting plasticity explains why PKD perturbs trafficking of specific membrane cargo without disrupting renal epithelial cell barrier function.

3.2 RESULTS

Cell Culture Models Derived From the BPK Mouse

Although the BPK model has been studied for nearly two decades [224], the underlying Bicc1 gene defect was only recently identified [153]. The goal of these experiments was to characterize Bicc1 gene products in conditionally immortalized cell lines from cystic animals and normal age-matched controls. Endogenous protein expression was analyzed with a newly developed antibody to the Bicc1 amino terminus. The Bicc1 gene has 22 exons encoding two splice variants in mouse kidney (Fig. 3.1A). Exon 21 is spliced out of transcript A and transcript B encompasses the entire open reading frame [153]. However, a two-nucleotide frame-shift in the mutant allele produces an abnormal transcript A in cystic animals. Furthermore, a premature stop codon in exon 21 truncates transcript B. The Bicc1 antibody detected one major protein species from normal renal cells, in contrast to cystic cells that exhibited an additional higher molecular protein

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consistent with the presumptive size of disease-specific transcript A (Fig. 3.1B). Since the cystic cells were derived from a Bicc1 −/− homozygous animal [146], the protein species detected in both cell lines probably corresponds to the presumptive product of transcript B (Fig. 3.1A).

We also determined whether the disease allele impairs

molecules or organelles linked to other primary ARPKD genetic defects. The predicted structure for fibrocystin is shown in Fig. 3.1C [195].

In contrast to recombinant

fibrocystin that is post-translationally processed into multiple peptide fragments [225, 226], the bulk of the endogenous mouse protein is cleaved once in both cell lines (Fig. 3.1D).

Based on size and reactivity with two newly developed fibrocystin antibodies,

the endogenous protein is apparently digested at an extracellular domain site previously identified in the recombinant protein (Fig. 3.1C and D) [225]. The Oak Ridge Polycystic Kidney (ORPK) mouse model of ARPKD involves an intraflagellar transport protein necessary for ciliogenesis [227]. The ORPK defect causes loss or severe stunting of primary cilia [228]. In contrast, conditionally immortalized cell lines from the BPK model had morphologically similar primary cilia (Fig. 3.1E) ranging in length from 1 to 2.5 µm according to [229]. Altogether these data provide the first experimental evidence that Bicc1 proteins are produced in the BPK model. The cystic cells translate both transcripts in contrast to normal cells that express transcript B. Furthermore the disease allele does not perturb fibrocystin expression or ciliogenesis.

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Normal and Cystic Cells Exhibit Similar Distributions of Multiple Epithelial Cell Polarity Markers

The goal of these experiments was to determine whether conditionally immortalized normal cells exhibit properties of renal epithelial cells, and if the disease allele perturbs overall cell polarity. Cells were stained with antibodies to a number of epithelial cell markers, and Fig. 3.2A shows vertical optical sections exhibiting polarized expression for each molecule. Primary cilia were identified with antibodies to detyrosinated tubulin and fibrocystin that are both typically associated with this organelle [230, 231].

These

images confirm data in Fig. 3.1E indicating cystic cells have primary cilia and also demonstrate the disease allele does not impair fibrocystin localization. Normal and cystic cells both displayed GPI-anchored CD73 on apical membranes (Fig. 3.2A) (Strohmeier et al., 1997).

The punctate CD73 staining pattern is typical for proteins enriched in

microvilli on the apical surface (Strohmeier et al., 1997). Both cells also exhibited ZO-1positive tight junctions at the apex of the lateral membrane and β-catenin on the lateral cell membrane (Fig. 3.2A) [25, 232, 233]. Importantly, the transferrin receptor (TfR) that requires AP-1B for polarized membrane expression was localized to lateral membranes in both cell lines [222, 234] (Fig. 3.2A). Furthermore, both cell lines expressed similar protein levels of the AP-1B µ1B subunit as well as subunits from ubiquitous clathrin adaptor complexes AP-1A, AP-2, and AP-4 (Fig. 3.2B).

The domain-specific

distribution for several proteins was also determined by analyzing immune complexes from filter-grown cells biotinylated at apical or basolateral surfaces. CD73 was localized to apical membranes and also soluble in Triton X-100 at 37ºC (Fig. 3.2C) but not 4ºC

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(Fig. 3.2D) consistent with involvement of lipid raft carriers in CD73 trafficking [235, 236]. In addition to TfR (Fig. 3.2E), biochemical data show that the receptor tyrosine kinase c-MET localizes to basolateral membranes in normal and cystic cells (Fig. 3.2F) [237]. Altogether these data indicate both of the conditionally immortalized cell lines have expected distributions of typical epithelial cell polarity markers, have functional lipid raft- and AP-1B-dependent sorting machinery, and retain other receptor tyrosine kinases on the basolateral membrane.

EGFR Expression in Conditionally Immortalized Cell Lines

EGFR expression was analyzed using multiple experimental approaches. First, cells were stained with an antibody to endogenous mouse EGFR demonstrating basolateral expression in normal cells but non-polar membrane distribution in cystic cells (Fig. 3.3Α).

In contrast, both cell lines exhibited typical polarized distribution of the

microfilament membrane linking protein ezrin just beneath the cell apex, as well as Ecadherin along the lateral membrane (Fig. 3.3Α).

Recombinant human EGFR also

exhibited the same loss of membrane polarity in cystic cells as the endogenous receptor indicating the EGFR phenotype is an innate property of the cell and not an unrecognized mutation in the endogenous receptor (Fig. 3.3Α). Second, ERK1/ERK2 activity was monitored to assess domain-specific EGFR activation. Results indicate this pathway is induced by basolateral EGF in normal cells, but from both membranes in cystic cells (Fig. 3.3B).

Interestingly ERK1/ERK2 activation was significantly prolonged in

basolaterally stimulated cystic cells compared to normal cells (Fig. 3.3B) suggesting the

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disease allele perturbs EGFR cell signaling at both membrane domains. Third, newly synthesized receptors undergo similar molecular weight mobility shifts consistent with Nglycan maturation in normal and cystic cells (Fig. 3.3C) [238]. Furthermore both cell lines have equivalent levels of total EGFR protein (Fig. 3.3D). Taken together these results rule out incomplete glycosylation or insufficient basolateral sorting capacity as likely explanations for the cystic EGFR phenotype [239-241]. Fourth, EGFRs were soluble with Triton X-100 at different temperatures (Fig. 3.3E and F) suggesting apical EGFR missorting involves a lipid raft-independent mechanism [52].

Finally, we

examined domain-specific delivery of newly synthesized EGFR. These studies were carried out by synchronizing pulse-labeled proteins in the Golgi with an 18°C temperature block, followed by a 37°C recovery period to induce vesicle transport [242]. Results in Fig. 3G indicate the bulk of newly synthesized EGFRs were biotin-accessible at apical and basolateral surfaces within the first 30 min of the 37oC recovery period in cystic cells. Apical delivery was completely blocked at 18oC in cystic cells. In contrast, basolateral delivery was reduced but not eliminated in either cell line consistent with reported differences in temperature sensitivity for polarized sorting pathways [242]. The cystic cells also exhibited a modest reduction in radio-labeled apical EGFRs during the 37°C recovery period that is probably due to rapid equilibration of newly delivered apical molecules with biotin-inaccessible internal pools [52].

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LLCPK1 Cells Also Exhibit EGFR Sorting Abnormalities

Although both conditionally immortalized cell lines express functional AP-1B complexes, it is currently unclear whether this pathway contributes to polarized EGFR expression. This question was addressed in LLCPK1 cells that lack the endogenous AP1B µ1B subunit resulting in defective sorting of AP-1B-dependent basolateral cargo [222]. LLCPK1 cells expressing recombinant human EGFR were grown on permeable filter supports and stained with EGFR1 monoclonal antibody added to the apical or basolateral surface (Fig. 3.4A) [188].

EGFR1 recognizes a peptide epitope in the

extracellular domain of the receptor and is human-specific [243, 244]. Human EGFRs were detectable on both membrane domains (Fig. 3.4B) indicating they follow multiple pathways to the basolateral surface including one regulated by AP-1B. We have reported previously that EGFR polarity is regulated by multiple hierarchical signals including two conserved basolateral sorting motifs located in the juxtamembrane region of the receptor (Fig. 3.4C) [65, 188]. Using in vitro pull-down assays, we demonstrated that EGFR peptides encompassing the dileucine-based 658-LL consensus basolateral sorting motif specifically interacted with AP-1 but not AP-2 involved in clathrin dependent sorting at the level of the plasma membrane (Fig. 3.4D) [245].

Furthermore this sequence

interacted with AP-1B and not AP-1A and in vitro AP-1B binding was greatly reduced by a 658-AA substitution (Fig. 3.4E). Similar dileucine signals often require an acidic residue at the −4 position [i.e., D/ExxxL(L/I)] [63] for full biological activity. Although the 658-LL motif lacks an acidic residue, Thr654 at the −4 position is a known serinethreonine kinase substrate that could serve a similar role by introducing a negative charge

60

in its phosphorylated state [246, 247]. Surprisingly, introduction of a T654D phosphomimetic substitution significantly reduced in vitro AP-1B binding in contrast to T654A that essentially had no effect (Fig. 3.4E). It is conceivable that the T654D substitution enhances 658-LL facilitated binding to another clathrin adaptor involved in basolateral sorting such as AP-4 [73]. However all wild-type and mutant juxtamembrane fusion proteins were negative for AP-4 binding ruling out this possibility (Fig. 3.8A). Introduction of the 658-AA substitution to full-length human EGFR did not eliminate basolateral expression in LLCPK1 cells confirming the existence of a second AP-1Bindependent constitutive pathway (Fig. 3.4F). Unexpectedly, the T654D substitution abolished apical EGFR expression in LLCPK1 cells suggesting this mutation offsets µ1B deficiency (Fig. 3.4G). These data indicate EGFR is sorted by at least three mechanisms: AP-1B-dependent and AP-1B-independent constitutive pathways and an inducible AP1B-independent pathway regulated by a latent signal involving Thr654.

EGFRs Follow Multiple Pathways to the Basolateral Membrane in MDCK Cells

The hypothesis that different EGFR sorting signals mediate trafficking in distinct pathways was further evaluated in MDCK cells which express µ1B constitutively [68, 223]. Permanent MDCK cell lines encoding recombinant human EGFR proteins (Fig. 3.5A) were subjected to domain-specific EGFR1 staining demonstrating that wild-type human EGFR and human EGFR with a T654D substitution are enriched on basolateral membranes (Fig. 3.5B and C). In contrast human EGFR with a 658-AA substitution was detectable on both membrane domains (Fig. 3.5D).

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All the permanent cell lines

displayed normal patterns of ZO-1 and E-cadherin staining, ruling out the possibility that non-polar human EGFR (658-AA) expression was due to loss of cell-cell junctional contacts. Despite similarities in membrane domain targeting, our data indicate wild-type human EGFR and human EGFR (T654D) traverse distinct basolateral pathways. Receptors with a T654D mutation co-localized with Rab11 sub-apical compartments (Fig. 3.5E). Interestingly, sub-apical Rab11 is a marker for ARE involved in lipid raftindependent apical trafficking as well as basolateral-to-apical transcytosis [248, 249]. In contrast, wild-type EGFR was largely excluded from this compartment in MDCK cells (Fig. 3.5F). Altogether these data confirm results obtained in µ1B-null LLCPK1 cells that EGFRs are constitutively transported to the basolateral membrane by multiple pathways.

They also indicate receptors in the inducible Thr654-dependent pathway

traverse Rab11-positive sub-apical compartments en route to the basolateral membrane.

Recovery of Basolateral EGFR Expression in Cystic Cells by Activation of Latent Thr654-Dependent Signal

Studies to this point have uncovered multiple routes for polarized EGFR trafficking. These findings raise the possibility that the EGFR cellular phenotype associated with PKD could be overcome by activating an alternative basolateral sorting pathway.

This

hypothesis was tested by analyzing the distribution of human EGFRs with sorting signal mutations in permanent cystic cell lines. Non-permeabilized cells were stained with domain-specific EGFR1 (see Fig. 3.4A). Cells on replicate filters were stained for CD73 to verify that ectopic human EGFR expression does not perturb lipid raft-dependent

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apical sorting. Cells were also stained for ZO-1 and E-cadherin as indicators of cell polarity.

Wild-type human EGFR was detectable on both membrane surfaces (Fig.

3.6A) confirming results in Fig. 3.3A obtained with permeabilized cells. Similar to the wild-type receptor, human EGFR with a 658-AA substitution was also present on both membrane surfaces in cystic cells (Fig. 3.6B). However, human EGFR with the T654D substitution was targeted exclusively to basolateral membranes (Fig. 3.6C). In addition to microscopy studies, human EGFR membrane domain delivery was examined in pulsechase experiments (Fig. 3.6D). In contrast to Fig. 3.3G, these studies were carried out without synchronizing newly synthesized molecules in the Golgi. Although wild-type human EGFR and human EGFR (658-AA) both exhibited non-polar delivery, human EGFR (658-AA) appeared on the apical surface approximately 30 min before wild-type human EGFR consistent with subtle differences in delivery pathways. In contrast to these two receptor molecules, human EGFR (T654D) was delivered directly to the basolateral surface in cystic cells. EGFR residue Thr654 is a known PKC substrate [246] suggesting PKC might activate the latent signal and restore basolateral localization of wild-type EGFR in cystic cells. Cystic cells were treated with phorbol 12-myristate 13acetate (PMA) that is known to activate the major PKC isoforms expressed in adult renal collecting duct to test this hypothesis [250]. Although PMA rapidly induced Thr654 phosphorylation in cells expressing wild-type EGFR but not EGFR (T654D) (Fig. 3.8B), this treatment had no discernable effect on apical EGFR expression in cystic cells (Fig. 3.8C). Altogether these data indicate wild-type EGFR and EGFR (658-AA) arrive at the apical membrane via kinetically distinct pathways. They also suggest that receptors with

63

a T654D substitution traverse a novel PMA-insensitive basolateral sorting pathway that is unaffected by the disease allele.

EGFR co-localizes with Rab11-positive sub-apical compartments in cystic cells

Normal and cystic cells expressing human EGFRs were co-stained with antibodies to Rab11 and the human receptor.

Our data indicate that wild-type human EGFR is

excluded from Rab11-positive sub-apical compartments in normal cells (Fig. 3.7A, 3.7D), in contrast to cystic cells which exhibit substantial overlap of these two markers (Fig. 3.7B, 3.7E). Whereas these compartments had a tubulovesicular appearance in normal cells similar to the structures identified in MDCK cells (Fig. 3.5E and F), the Rab11-positive sub-apical compartments appeared more punctuate in cystic cells. Similar to data from MDCK cells (Fig. 3.5E), human EGFR (T654D) also co-localized with Rab11-positive sub-apical compartments in cystic cells (Fig. 3.7C, 3.7F).

E-

cadherin, which requires Rab11 and functional ARE for normal basolateral trafficking (Desclozeaux et al., 2008) localized to similar Rab11-positive sub-apical compartments in all three of the cell lines (Fig. 3.7D, 3.7E, 3.7F). Altogether these data indicate wildtype human EGFR and human EGFR (T654D) transit Rab11-positive sub-apical compartments resembling E-cadherin ARE sorting intermediates in the cystic cells. However, in contrast to wild-type EGFR that missorts to the apical membrane, EGFR (T654D) trafficking to the basolateral membrane is unaffected by the disease allele.

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3.3 FIGURES

Figure 3.1 Conditionally immortalized cell lines from ARPKD mouse model

(A) The Bicc1 gene has 22 exons encoding two alternatively spliced transcripts (A and B) in mouse kidney. Predicted structures for normal and disease-specific protein products shown beneath each transcript highlight RNA-binding KH (K homology) domains, exon 21 and exon 22-encoded sequences (dark grey and light grey, respectively), diseasespecific carboxyl-terminal extension (black), and predicted molecular weights (in parenthesis). Adapted from [153]. (B) Equal protein aliquots were immunoblotted with Bicc1 antibody.

(C) Predicted fibrocystin domain structure highlighting extracellular

immunoglobulin-like TIG repeats (light grey), transmembrane domain (diagonal lines), FCY-Exo and FCY-Cyt peptide antibody sequences (black), and approximate cleavage site generating major endogenous protein species (dashed arrow). (D) Equal protein aliquots were immunoblotted with fibrocystin antibodies. scanning electron microscopy.

(E) Cells were examined by

Arrow = central primary cilia.

magnification × 3000.

65

Size bar = 1 µm,

Figure 3.1 Conditionally immortalized cell lines from ARPKD mouse model

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Figure 3.2

Epithelial cell markers distributed normally in conditionally

immortalized cell lines

(A) Selected vertical X-Y confocal optical sections (see schematic) from permeabilized cells stained with antibodies listed in figure.

Size bar = 5 µM. (B) Equal protein

aliquots were immunoblotted with antibodies to clathrin AP-specific subunits highlighted in grey in schematics above individual panels. (C and D) Cells were harvested with Triton X-100 (TX-100) buffer at 37ºC (C) or 4ºC (D) after domain-specific biotinylation. Ap = apical, BL = basolateral.

(E and F)

Biotinylated cells were harvested with

immunoprecipitation buffer at 4ºC. (C to F) Protein-specific immune complexes were immunoblotted for biotin.

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Figure 3.2

Epithelial cell markers distributed normally in conditionally

immortalized cell lines

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Figure 3.3 EGFR expression in conditionally immortalized cell lines

(A) Horizontal X-Z optical sections of permeabilized cells stained with antibodies to proteins listed listed in the figure.

Cells in bottom panels were transduced with a

recombinant human EGFR retrovirus. Size bar = 5 µM. (B) Cells were stimulated with domain-specific EGF (50 ng/ml) for times indicated.

Equal protein aliquots were

immunoblotted with activation-specific phospho-MAPK antibody followed by panERK1/2 antibody to check protein loading. (C) Cells were pulse-labeled with 35S-labeled amino acids and switched to chase medium for various times. Radio-labeled EGFR immune complexes were detected by fluorography after SDS-PAGE. P = precursor EGFR; M = mature EGFR.

(D) Equal protein aliquots were immunoblotted for

endogenous EGFR or β-actin. (E and F) Biotinylated cells were harvested with TX-100 buffer at 37ºC (E) or 4ºC (F), and mouse EGFR immune complexes were immunoblotted for biotin. (G) Pulse-labeled cells incubated in chase medium at 18oC for 2 h were biotinylated immediately or following a 37oC recovery period. Streptavidin affinitypurified mouse EGFR immune complexes were detected by fluorography.

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Figure 3.3 EGFR expression in conditionally immortalized cell lines

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Figure 3.4 EGFR 658-LL basolateral sorting signal interacts with AP-1B

(A) Human-specific EGFR expression was evaluated by domain-specific EGFR1 staining of non-permeabilized cells with well-formed tight junctions (TJ). (B) Horizontal X-Z optical sections from LLCPK1 cells expressing wild-type human EGFR following domain-specific EGFR1 staining. (C) EGFR protein schematic highlights extracellular region with four sub-domains (I – IV) and multiple N-glycosylation sites (

),

transmembrane (TM) domain, and cytoplasmic region with juxtamembrane (Jx), kinase catalytic, and carboxyl terminal (C-term) domains. Amino acid sequences of wild-type (Peptide 1 and Peptide 2) and mutant (658-AA, T654A, and T654D) peptides used in pull-down assays are shown beneath the schematic.

Critical residues involved in

basolateral sorting are highlighted in bold [65, 188]. Peptide sequences derived from human EGFR (Swiss-Prot: P00533.2) are precisely conserved in mouse EGFR (GenBank: AAA17899.1). (D) Peptides conjugated to Sepharose beads (Peptide 1) or GST fusion protein attached to glutathione beads (Peptide 2) were incubated with cell fractions enriched for AP complexes, and bound proteins were immunoblotted with APspecific antibodies. Cell fractions were also incubated with empty Sepharose beads (Control 1) or affinity purified GST protein (Control 2). (E) Top panel: Coomassiestained gel of affinity-purified GST fusion proteins. Middle and bottom panels: GST fusion protein pull-down assays analyzed with AP-1 µ-subunit specific antibodies.

(F

and G) Horizontal X-Z optical sections from LLCPK1 cells expressing human EGFRs with 658-AA (F) or T654D (G) substitutions stained with domain-specific EGFR1. (B, F, and G) Size bars = 5 µM.

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Figure 3.4 EGFR 658-LL basolateral sorting signal interacts with AP-1B

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Figure 3.5 EGFR follows multiple basolateral sorting pathways in MDCK cells

(A) Human-specific EGFR immune complexes from permanent MDCK cell lines expressing recombinant human EGFR proteins listed in the figure were immunoblotted with a second EGFR antibody. We also attempted to produce stable cell lines expressing human EGFR (T654A), but this recombinant protein was biosynthetically unstable (data not shown). (B, C, and D) Horizontal X-Z optical sections from non-permeabilized MDCK cells expressing wild-type human EGFR (B), human EGFR with a T654D substitution (C), or human EGFR with a 658-AA substitution (D) stained with domainspecific EGFR1 (see Figure 4A). Permeabilized cells were also stained with ZO-1 and Ecadherin antibodies. (E and F) Vertical X-Y optical sections from representative MDCK cells expressing wild-type human EGFR (E) or EGFR with a T654D substitution (F) costained for Rab11 (red channel) and human EGFR (green channel). Unmerged and merged images from Z-sections indicated by dotted lines are shown to the right. Arrows = Rab11-positive sub-apical compartments. (B to F) Size bars = 5 µM.

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Figure 3.5 EGFR follows multiple basolateral sorting pathways in MDCK cells

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Figure 3.6

T654D mutation reconstitutes basolateral EGFR expression in cystic

cells

(A, B, and C) Horizontal X-Z confocal optical sections from cystic cells expressing wildtype human EGFR (A) or human EGFR with 658-AA (B) or T654D (C) substitutions stained with EGFR1 according to Figure 4A, or antibodies to epithelial cell markers. Size bars = 5 µM. (D) Cells were pulse-labeled with 35S-Express Protein Labeling Mix for 15 min and biotinylated immediately or following a 37oC incubation period in chase media. Radio-labeled human EGFR immune complexes purified by streptavidin affinity chromatography were detected by fluorography.

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Figure 3.6

T654D mutation reconstitutes basolateral EGFR expression in cystic

cells

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Figure 3.7

EGFR co-localizes with Rab11-positive sub-apical compartments in

cystic cells

(A, B, and C) Vertical X-Y confocal optical sections from the mid-lateral (left panels) or sub-apical (middle panels) regions of normal cells expressing wild-type human EGFR (A), cystic cells expressing wild-type human EGFR (B), or cystic cells expressing human EGFR with a T654D substitution (C) co-stained for Rab11 (green channel) and human EGFR (red channel). Panels to far right are magnified images of individual and merged channels from boxed areas in sub-apical sections. Arrows denote Rab11-positive subapical compartments. Asterisks in mid-lateral sections denote cells with Rab11-positive sub-apical compartments. (D, E, and F) Individual and merged channels of horizontal X-Z confocal sections from normal cells expressing wild-type human EGFR (D), cystic cells expressing wild-type human EGFR (E), or cystic cells expressing human EGFR with a T654D substitution (F) co-stained for Rab11 (green channels in both sets of panels) and either human EGFR or E-cadherin (red channels in top and bottom panels, respectively). Arrows denote Rab11-positive sub-apical compartments. Size bars = 5 µM.

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Figure 3.7

EGFR co-localizes with Rab11-positive sub-apical compartments in

cystic cells

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Figure 3.8

The T654D substitution does not enhance in vitro AP-4 binding and

PMA does not alter EGFR localization in cystic cells

(A) Wild-type EGFR and EGFR (T654D) juxtamembrane peptides shown in Figure 4C were attached to glutathione beads and incubated with cell fractions enriched for AP complexes. Cell fractions were also incubated with affinity purified GST protein as a negative control. Bound proteins were immunoblotted with AP-4 µ-subunit specific antibody. (B) Cystic cells expressing wild-type human EGFR or human EGFR (T654D) were incubated with DMSO vehicle or PMA (20 nM) for 30 min. Human-specific EGFR immune complexes were immunoblotted for EGFR (phospho-Thr654). stripped and re-probed for EGFR.

(C)

Filters were

Horizontal X-Z optical sections from non-

permeabilized cystic cells expressing wild-type human EGFR stained with EGFR1 added to the apical surface following a 30-min incubation with DMSO vehicle or PMA (20 nM).

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Figure 3.8

The T654D substitution does not enhance in vitro AP-4 binding and

PMA does not alter EGFR localization in cystic cells

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Chapter 4 Discussion and Future Directions

4.1 DISCUSSION

EGFR can sort through multiple pathways in polarized epithelia

Our studies indicate that regulation of EGFR polarity in renal epithelial cells is more complex than previously appreciated. Newly synthesized EGFRs follow at least two constitutive basolateral sorting pathways (summarized in Fig. 4.1). One pathway is mediated by a direct interaction between EGFR residues 658-LL and AP-1B. Similar to other AP-1B dependent cargoes, EGFRs are probably targeted from TGN to CRE that are the primary sites of action for clathrin facilitated sorting in this pathway (routes A to B in Fig. 4.1). The CRE is a major sorting nexus for proteins delivered to both the apical and basolateral surfaces [251]. Basolateral cargo that traverses the CRE from the TGN typically is directed to the basolateral surface via interaction with the exocyst complex and the clathrin adaptor AP-1B [57, 67].

When this AP-1B-dependent pathway is

suppressed via mutation of the 658-LL or lack of the u1B subunit of AP-1B, the EGFR exhibits a non-polar sorting phenotype. However, not all receptor is directed to the apical surface in this case, which indicates there is still basolateral sorting information on the receptor in the absence of this AP-1B-dependent pathway. Although the molecular basis 81

for this AP-1B-independent biosynthetic route is not yet known, it may involve a conserved proline-dependent EGFR basolateral signal 667-PLTP that binds SH3-domains in vitro [65, 252]. Current models postulate AP-1B independent trafficking is mediated by an unknown clathrin adaptor acting at the level of the TGN to sort cargo directly to the plasma membrane (route D in Fig. 4.1). Alternatively, cargo may be sorted independent of clathrin via BSE intermediates (routes E or C to F in Fig. 4.1) [253]. We found that AP-1A, AP-2, nor AP-4 bound to an EGFR juxtamembrane fusion protein containing the 658-LL and 667-PLTP during in vitro pulldown assays, suggesting these adaptors are not involved in 667-PLTP-mediated basolateral sorting [221, 253]. We have also elucidated a latent basolateral pathway that was unmasked by replacing EGFR residue Thr654 with a negatively charged aspartic acid residue. This pathway transits Rab11-positive subapical compartments independent of AP-1B (route I in Fig. 4.1) [248, 249]. Based on sub-cellular localization, Rab11 staining, and the presence of E-cadherin in similar structures, these compartments are probably ARE.

At present it is unclear if

EGFR(T654D) receptors are targeted to ARE directly (route H to I in Fig. 4.1) or via a CRE intermediate (route A to G to I in Fig. 4.1). Interestingly, the EGFR T654D substitution inhibits AP-1B binding in vitro. This finding raises the possibility that T654D silences the AP-1B dependent signal in addition to activating a novel AP-1B independent signal.

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Apical EGFR is not sorted via transcytosis in cystic cells

One potential hypothesis was that the EGFR is sorted to the basolateral surface first in cystic cells and then undergo transcytosis to the apical membrane upon internalization from the basolateral membrane. Our data in cystic cells do not support the transcytosis model for apical EGFR. Though we see a slight delay in apical delivery of the wild-type EGFR in cystic cells that we might expect with transcytosis, we do not see a shift in receptor from the basolateral surface to the apical surface. One reason that transcytosis is not supported in our model is because the EGFR is not ligand stimulated in our delivery experiments. Upon stimulation via ligand, the EGFR is activated and is endocytosed in the endosomal pathway where it can be recycled back to the basolateral surface or to the lysosome for degradation.

In the absence of ligand the receptor

internalizes relatively slowly from the plasma membrane.

Cystic BPK cells lack co-factor required for EGFR-specific AP-1B-dependent sorting

While the dialanine mutation at 658-LL inhibits AP-1B binding in vitro, there was no observed exacerbation of the abnormal EGFR phenotype in cystic cells. This suggests that the BPK mutant allele is interfering with the constitutive AP-1B pathway. However cystic cells contain functional AP-1B machinery as other AP-1B cargoes sort correctly.

83

This indicates that there is an EGFR-specific co-factor required for AP-1B-dependent sorting that is missing or defective in the cystic cells. One example of this is the Rho GTPase TC10 which is required for exocyst-mediated membrane fusion of specific protein cargo in developing neurons [254-256]. Without this co-factor, EGFR which is delivered to the CRE could be recognized by the sorting machinery which targets Nglycan dependent cargo to the apical surface via the ARE [188]. Slight differences in delivery kinetics between the wild-type and 658-AA mutant receptors suggest that the 658-AA mutation may affect AP-1B-dependent sorting at the level of the TGN or perhaps even the ER. The ER has emerged as a common target for PKD susceptibility gene products. PKD1 has been shown to influence the shape and localization of the microtubule network and ER, while PKD2 has been shown to localize to the ER and acts as an intracellular calcium release channel [167, 257].

EGFR(T654D) follows a novel AP-1B-independent pathway

While we show here that the EGFR can sort to the basolateral surface via an AP1B-dependent mechanism which is perturbed in cystic cells, we have also discovered an alternate basolateral sorting pathway for the receptor. When Thr654 of the human EGFR was mutated to an aspartic acid (T654D) and expressed in the cystic BPK cells, this receptor was found to sort to the basolateral surface. Initially we believed that this mutation was enhancing a site for AP-1B binding in the cystic cells based on previous studies that indicated that negatively charged amino acids upstream of a dileucine motif 84

act as a binding site for clathrin adaptor proteins[258].

When tested, however, the

aspartic acid residue at Thr654 actually decreased AP-1B affinity of the EGFR in vitro. This suggested that the EGFR(T654D) was not taking an AP-1B-dependent pathway to the basolateral surface. This mutation was confirmed to recover basolateral sorting even in LLC-PK1 cells which lack endogenous mu1B, further supporting the hypothesis that the EGFR(T654D) was acting via an AP-1B-independent pathway. While the primary pathways for basolaterally sorted proteins have been shown to involve either the direct sorting from the TGN or first through the CRE, another pathway has recently emerged involving basolateral sorting from the ARE. E-cadherin has been shown to sort to the basolateral surface from the ARE in polarized MDCK cells and this sorting is facilitated by Rab11, a member of the Rab GTPase family[70, 71].

We found that the

EGFR(T654D) does co-localize with Rab11 similar to E-cadherin in endosomes typical of AREs both in cystic cells and MDCK cells while the wild-type receptor does not. We hypothesize that the EGFR(T654D) could be taking this Rab11-dependent pathway as opposed to Rab8 which controls basolateral transport of post-Golgi vesicles [259]. It is unclear whether the EGFR(T654D) pathway can be activated by phosphorylation of Thr654 of the wild-type receptor. Preliminary experiments have ruled out PMA-sensitive PKC isoforms from diverting the wild-type receptor in cystic cells to this pathway, but this pathway could be modulated by another serine/threonine kinase. Studies have shown that the EGFR juxtamembrane which includes Thr654 forms an amphiphilic helix which binds strongly to membrane-mimetic micelles in vitro, but a negative charge can disrupt the helical structure of the entire juxtamembrane region [260]. In addition to AP-1B, the EGFR juxtamembrane has been shown to interact with

85

number of other binding partners including calmodulin and phosphoinositide kinases [261, 262]. Either of these could activate a latent signal by inducing a conformation change in the juxtamembrane in the absence of Thr654 phosphorylation.

EGFR signaling is dysfunctional in cystic cells

EGFR-dependent signaling is also perturbed at both membrane surfaces in cystic cells.

The BPK allele could be affecting EGFR signaling by creating an alternate

distribution of receptor on the basolateral membrane subdomains through reduced incorporation into AP-1B-dependent basolateral transport vesicles.

This hypothesis

would be consistent with the idea that overlapping but separate routes to the same membrane domain allow different epithelial cell types to carry out unique cell signaling profiles.

Also, AP-1B-independent routes for some cargo develop after cells have

polarized, suggesting that AP-1B-dependent and independent pathways may have distinct roles during development or differentiation [253]. ERK1/ERK2 is hyper-activated in cystic kidneys and inhibition of this pathway slows disease progression in PKD mouse models [263, 264]. Our data suggests that the hyperactive ERK1/ERK2 signaling seen in PKD is the result of imbalanced EGFR signaling from both apical and basolateral plasma membranes.

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BicC and EGFR mislocalization in cystic cells

Though it is unclear how the bicc1 mutation causes the EGFR phenotype in the cystic BPK cells, there are other PKD-related mutation models which also display this phenotype. This begs the question of how the genes related to human PKD and those of the animal models all cause a similar EGFR phenotype. There must be some interrelated pathway that links the genetic defects in all of these models to cause EGFR mislocalization to the apical surface. Both the polycystin-1 and fibrocystin C-terminal tails have been shown to undergo proteolytic cleavage and translocation to the nucleus [225, 265-267]. This suggests that these cleavage products may have some role in either expression or repression of genes in the nucleus and mutations in polycystin-1 or fibrocystin seen in human PKD may result in some defect in either cleavage or proper nuclear translocation.

In PKD-related models which have defective primary cilia

formation such as the ORPK mouse model, the lack of this mechanosensory organelle could lead to loss of this regulation via lack of flow-induced cleavage products of the polycystin-1 and fibrocystin. BicC is an RNA-binding protein that has been shown to regulate poly(A)-tail length of mRNAs by activation of the CCR4-NOT deadenylation complex, and can regulate its own expression in this process [162]. BicC has also been shown to interact with RNA-processing bodies (P-bodies) through its SAM domain and inhibit Dvl signaling which can lead to hyperactivity of Wnt/β-catenin pathways [163]. Hyperactivity of β-catenin has been show to induce cystic growth in transgenic mice

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[164, 165]. The BPK mutation may be acting further downstream in the Wnt signaling pathway by regulating mRNA levels of either Dvl or another inhibitor of its downstream target GSK3β, leading to hyperactivity of β-catenin signaling. Overexpression of PKD1 was shown to prevent invasion and also increase E-cadherin and β-catenin expression in cancer cells [268]. Studies have also shown that EGF stimulation can cause ERK2mediated transactivation of β-catenin via protein kinase CK2 [269]. Upon damage to the renal epithelia in the collecting duct of the kindey by external sources such as infection, the EGF in the lumen can leech into the lateral membrane space and activate basolateral EGFRs. While transient transactivation of β-catenin would be normal during epithelial repair, cells with PKD-related defects may hamper the ability of the cell to regulate βcatenin and the Wnt pathway properly. This would lead to hyperactivity of β-catenin/TCF and promote a cystic phenotype. TCF transcriptional activity could to lead to expression or repression of genes which are regulating the co-factor required in AP-1B-dependent EGFR sorting, which could be a normal occurrence during the EMT. The lack of regulation of β-catenin signaling may be preventing the gene responsible for this cofactor to be turned back on in the PKD cells leading defective AP-1B-dependent sorting of the EGFR. If the EGFR mislocalization we see in cystic BPK cells and other PKD models is a lingering remnant of an EMT repair pathway, there must be a recovery mechanism to remove the apically localized receptors in normal epithelial cells. This may explain the EGFR(T654D) AP-1B-independent pathway seen in the cystic and LLC-PK1 cells. The re-establishment of polarity in epithelial cells may lead to activation of a serine/threonine kinase which can lead to phosphorylation of Thr654 of the EGFR and once internalized

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into the ARE it would be routed to the basolateral surface. Protein kinase CK2 has been shown to be involved in positive regulation of the Wnt/β-catenin signaling pathway [270]. The regulatory subunit of protein kinase CK2, CK2β, has also been shown to interact with a number of other serine/threonine kinases, including the aPKC isoform PKCζ [271-277]. Knockdown of the CK2β subunit has been show to induce an EMTlike phenotype in mammary epithelial cells [278].

One potential hypothesis is that

protein kinase CK2 may be regulating the activity of another kinase such as aPKC that is phosphorylating Thr654 of the EGFR during this EMT repair pathway.

4.2 FUTURE DIRECTIONS

Co-factors for AP-1B-dependent EGFR sorting

As mentioned before, other AP-1B-dependent cargoes traffic correctly in the cystic cells, indicating that there must be some EGFR-specific deficiency such as a missing or defective co-factor. One hypothesis is that there is EGFR-specific recruitment of a Rho, Rab or Ral GTPase which helps to localize the receptor to AP-1B positive subdomains in the CRE. One study has shown that during cell migration Ral GTPases are required for exocyst association with paxillin, a focal adhesion-associated protein, and that the exocyst is required for biosynthetic delivery of α5-integrin [279]. Also, in developing neurons it has been shown that the Rho GTPase TC10 is necessary for

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exocyst-mediated membrane fusion of certain cargoes as well as membrane expansion [254-256]. This supports the hypothesis that a membrane-associated GTPase could be directing the EGFR to the basolateral surface by targeting it into AP-1B-positive subdomains. One potential experiment that may yield possible co-factors would be a yeast-two hybrid screening. Utilizing the EGFR juxtamembrane as the binding domain and a library of putative Ral, Rho, and Rab GTPase-interacting proteins, potential binding partners can be established that may be involved in AP-1B-dependent sorting of the EGFR. The weakness in this assay is that it is unknown whether AP-1B binds the EGFR first and then the putative co-factor or vice versa. An additional yeast-two hybrid assay could be performed using μ1B as the binding domain and compared to EGFR binding partners to look for overlap, however, if the full AP-1B complex is needed for effective binding this would prove ineffective. Once potential co-factors are determined, we could then use RNAi knock-down to observe if these proteins create the abnormal EGFR phenotype in either the normal mouse or MDCK cells.

Role of BicC in EGFR sorting

As previously stated, the mutation the cystic BPK model is in the Bicc1 gene which encodes the BicC protein. While we have demonstrated the EGFR phenotype in the cystic cells, we have not elucidated the exact BicC-related mechanism that causes the mislocalization.

Recent data indicate the Drosophila protein regulates ER exit site

homeostasis necessary for normal protein sorting, and that specific proteins become 90

trapped in aberrant exit site compartments leading to defective exocytosis in Bicc-1-null embryos [280]. Although the EGFR sorting defect involves inaccurate incorporation into post-Golgi transport vesicles, a growing number of studies indicate protein cargo may be pre-sorted in the ER or by-pass the Golgi altogether [240, 281]. The ER is emerging as a common target for PKD susceptibility gene products.

Recent data indicate PKD1

influences the shape and localization of both the microtubule network and the ER, and PKD2 is an abundant ER protein that functions as a novel intracellular calcium release channel [139, 166, 167]. If the EGFR is being missorted at the level of the ER in the cystic BPK cells, one potential experiment that would determine if this pathway exists would be to treat cystic cells with brefeldin A (BFA). BFA slows ER-to-TGN transport of proteins and inhibits transport apical and basolateral surfaces from the TGN at specific concentrations. If cells were treated with BFA it could be determined if EGFR still could reach the apical membrane in a TGN-independent manner in cystic or LLC-PK1 cells [281].

Machinery involved in EGFR(T654D) basolateral sorting

As previously discussed, the EGFR(T654D) mutant receptor sorts to the basolateral surface in both cystic and LLC-PK1 cells via an AP-1B-independent manner. While it is clear that this receptor is transported through the Rab11-positive ARE, the exact mechanism behind this pathway is uncertain.

One hypothesis is that the

EGFR(T654D) mutation could be changing the conformation of the juxtamembrane 91

region and in the process creating a favorable binding site for Rab11 or one of the Rab11interacting proteins. This is supported by recent data which suggest that the EGFR can associate with a Rab interacting protein, but only in concert with an integrin [282]. An alternate hypothesis is that the EGFR(T654D) mutation mimics a rescue pathway that is normally initiated via phosphorylation of Thr654 by an unknown serine/threonine kinase during development or repair. This would create a route for internalized receptors on the nascent apical surface to be rerouted to the basolateral surface as the cell becomes polarized. One study has shown that the transferrin receptor sorts to the basolateral membrane via an AP-1B-dependent pathway as MDCK cells are polarizing (1-3 days), but after 4.5 days sorting of the transferrin receptor is sorted by an AP-1B-independent mechanism[283]. If this T654D-dependent pathway is used in early development of polarity and differentiation, a key component of the Par protein complex, aPKC, could be involved [284]. Protein kinase D mutants have been shown to block basolateral transport in polarized MDCK cells [285]. Neither of these kinases has been shown to modify any specific membrane protein cargo, however. One experiment that may determine the role of aPKC in phosphorylation of the Thr654 of the EGFR is to express and/or overexpress constitutively active and dominant negative versions of both aPKC isoforms, PKCζ and PKCι, in cystic and LLC-PK1 cells. While this may indicate that an aPKC is involved in Thr654 phosphorylation, this may not be a direct interaction and could be happening via an intermediate serine/threonine kinase. Similar experiments could be performed on protein kinase D to determine if there is a link between Thr654 phosphorylation and this kinase. One potential problem with this experiment is that aPKC is a prominent member of the Par protein complex that helps to regulate polarity. By using inducible promoters,

92

such as the tet on/off system, we could first allow the cells to polarize and then induce expression of the mutant aPKC isoforms.

AP-1B-independent sorting pathways

While we have found some EGFR do require AP-1B to reach the basolateral surface, we do not see a complete reversal in receptor sorting from the basolateral to the apical surface in cystic or LLC-PK1 cells.

This indicates that there are AP-1B-

independent EGFR sorting pathways in polarized epithelial cells. Previously, it was shown that in MDCK cells, a proline rich PLTP motif at residues 667-670 of the EGFR was important for biosynthetic delivery of the receptor to the basolateral surface [65]. One hypothesis is that the 667-PLTP motif binds some unknown adaptor protein in the TGN and is sorted directly to the basolateral surface, bypassing the AP-1B-dependent pathway in the CRE. PxxP motifs have been shown to bind a variety of SH3-domaincontaining proteins including Crk and Nck adaptor proteins [252, 286]. A PxxP containing protein, c-Abl, and Crk and Nck have been shown to modulate Rac1 activity and cell spreading in NIH-3T3 cells [286]. The membrane scaffold protein Bem1p has been shown to bind PxxP motifs in yeast and aids in the development of bud formation and site-directed sorting to the bud via Cdc42 activation, however no known homologue of Bem1p exists in mammals [287]. The threonine at residue 669 of the PLTP has been shown to become phosphorylated in response to EGF by a member of the MAPK family [288]. Phosphorylation of Threonine 669 could affect binding of proteins to the PLTP 93

positively or negatively. Recently, the PxxP motif of the Ral GTPase GEF RalGPS2 was shown to act as a dominant negative for RalA GTPase [289]. RalGPS2 has also been shown to interact with Nck and RalA GTPase has been shown to promote basolateral trafficking of proteins through interactions with the exocyst complex (Fig. 1.6) [290, 291]. These data support the hypothesis that the 667-PLTP is acting as a binding site for some specific adaptor protein, RalA GTPase, and the exocyst complex independent of AP-1B. One potential experiment would be to inhibit RalA GTPase by expressing the PxxP motif of RalGPS2 and observing how this affects AP-1B-independent delivery of the EGFR in polarized cells such as the LLC-PK1 cells. By using either LLC-PK1 or cystic cells which lack the AP-1B-dependent EGFR sorting, we only have AP-1Bindependent basolateral delivery of receptors. If AP-1B-independent delivery of the EGFR is dependent on RalA GTPase we would expect increased sorting of the EGFR to the apical surface in these cells.

4.3 CONCLUSIONS

We have determined that the mislocalized EGFR exhibited by the cystic BPK mouse model cells is the result of a defect in the EGFR-specific sorting machinery in these cells. We have also shown that AP-1B mediates basolateral sorting of some EGFR through the CRE in polarized epithelial cells. This was accomplished by: 1) mutating an AP-1B binding site at 658-LL of the human EGFR juxtamembrane and expressing 94

these receptors in normal mouse kidney cells as well as MDCK cells; and 2) expressing wild-type human EGFR in LLC-PK1 cells which lack the endogenous μ1B subunit of AP-1B. Cystic cells contain AP-1B, so there must some additional co-factor which is necessary for AP-1B-dependent EGFR sorting in these cells. Also, EGFR still reaches the basolateral membrane in cystic and LLC-PK1 cells, indicating that AP-1Bindependent pathways for EGFR sorting are present in polarized cells. The LLC-PK1 and cystic cells could be further used as a model for analyzing AP-1B-independent EGFR sorting. Furthermore, we have discovered another mutation the in EGFR juxtamembrane at Thr654, EGFR(T654D), which allows for recovery of basolateral sorting in both cystic cells and LLC-PK1 cells, but does so through an AP-1B-independent mechanism. We found that the EGFR(T654D) sorts through a Rab11-positive compartment, most likely the ARE, en route to the basolateral surface. We hypothesize that the EGFR(T654D) creates a novel sorting signal which first inhibits AP-1B binding leading to sorting of the receptor to the ARE and then binds to either Rab11 or a Rab11-interacting protein before being sorted to the basolateral surface. This hypothesis is supported by co-localization between the EGFR(T654D) and Rab11 in what appear to be AREs in both cystic cells and MDCK cells. Rab11 was also shown to co-localize with E-cadherin in similar compartments in cystic cells. Studies which demonstrated that E-cadherin sorts to the basolateral surface in polarized epithelial cells in a Rab11-dependent manner further support the role of Rab11 in basolateral sorting of the EGFR(T654D) [70, 71]. The EGFR(T654D) could be used as another model to explore this new Rab11-dependent sorting pathway from the ARE in polarized epithelia and the sorting machinery involved.

95

In summary, we have found that mislocalization of the EGFR to the apical surface in cystic cells is due to a defect in the EGFR-specific AP-1B sorting machinery, possibly due to lack of a receptor-specific co-factor. Multiple models for PKD have been shown to exhibit the same EGFR sorting phenotype, suggesting that the proteins involved in each model share a common pathway. Further understanding of the defect in EGFR in PKD could lead to this common pathway and a better understanding of how these genetic defects contribute to PKD.

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4.4 FIGURES

Figure 4.1 EGFR sorting pathways in polarized epithelial cells

The model depicts a variety of routes followed by newly synthesized EGFR in polarized renal cells. EGFRs are transported to the CRE where they undergo AP-1B-dependent transport to the basolateral membrane via route A to B. EGFR also follows an AP-1Bindependent pathway that may bypass CRE using a direct route from TGN to basolateral membrane (route D). Alternatively this pathway may involve a hypothetical route from CRE to BSE (routes C to F, E to F). Introduction of an aspartic acid residue for Thr654 unmasks a third route that traverses ARE en route to the basolateral membrane (route I). Receptors in this pathway may reach ARE by a direct route from the TGN (route H) or via a CRE intermediate (route A to G). The B pathway is impaired in µ1B-null LLCPK1 cells as well as cystic cells from the BPK model with constitutive µ1B expression. The I pathway bypasses AP-1B and reconstitutes polar EGFR expression in both cell types. Apically missorted EGFR probably follows the N-glycan dependent J pathway rather than the lipid raft dependent pathway that involves ASE intermediates (not shown). AJ, adherens junction; TJ, tight junction.

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Figure 4.1 EGFR sorting pathways in polarized epithelial cells

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