Functional characterization of the alternatively spliced, placental ...

Vol. 268, No. 25,Issue of September 5, pp. 19025-19032,1993 Printed in U S A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Characterizationof the Alternatively Spliced, Placental Human Growth Hormone Receptor* (Received for publication, April 8, 1993, and in revised form, May 7, 1993)

Margrit UrbanekSg,J. Eric RussellSQlI, Nancy E. CookeglI, and Stephen A. Liebhaber$glIll From $The Howard Hughes Medical Institute, and the Departments of $Genetics and YMedicine, University of Pennsylvania, Philudelphia, Pennsylvania 19104

The human growth hormone family of peptide hor- hCS-A, hCS-B, and hGH-V are expressed exclusively by the mones is encoded byfive genes, pituitary growth hor- syncytiotrophoblasts of the placental villi (Cooke et al., 1988a; mone (hGH-N), and four placentally expressed genes, Liebhaber, 1989;Cooke et al., 1991). Incontrasttothis growth hormone variant (hGH-V), chorionic somato- highly restricted pattern of expression, hPrl isexpressed in a mammotropin A andB (hCS-A, hCS-B), and prolactin number of tissues, most prominently lactotrope cells bf the (hPrl). As part of aneffort to define the localeffects of anterior pituitary, endometrium, placental decidua, and lymthe placentally expressed members of the GH/Prl famphocytes (Cooke, 1993; Clements et al., 1983; and DiMattia et ily of hormoneson the placenta,we have identified an al., 1990). While the functions of hGH-N and hPrl are well isoform(hGHRd3)ofthe growth hormonereceptor established, the roles of the placentally expressed hCS, hGHexpressed in the placental villi. hGHRd3 mRNAdiffers V, and hPrl areless well understood. a from theliver GHR mRNA by the deletion of66-base The functions of GH and Prl are mediated by specific pair segment encodingexon 3. In this study we show binding to a set of cell surface receptors belonging to the that hGHRd3 mRNA encodes a stable and functional cytokine receptor superfamily (Cosman et al., 1990). Recepreceptor. hGHRd3 mRNA is efficiently translated and processed in a rabbit reticulocyte lysate translation tors in this superfamily, which encompass receptors for a system as well as in an in vivoXenopus laevis oocyte broad spectrum of growth factors and peptide hormones, are expression system. In Xenopus oocytes hGHRd3 is sta- characterized by a large amino-terminal extracellular domain bly integrated into the cell membrane and binds and which contains four highly conserved cysteine residues, a single transmembrane domain, and a cytoplasmic domain of internalizes ligand as efficiently as hGHR.hGHRd3 binds all three of the placentally expressed members variable size. Although no kinase or other enzyme activities of the GH/Prl gene family (hGH-V, hCS, and Prl) as have been assigned to these receptors, several, including the well as both the22 and 20 kDa isoforms of the pituitaryerythropoietin and GH receptors, are phosphorylated in rehGH-N. Theresults of the present studystrongly sup- sponse to ligand binding (Foster et al., 1988; Taga and Kishport theexpressionof a functional hGHRd3 isoreceptor imoto, 1992; and Linnekin et al., 1992).As a general rule these in the placenta which may serve in autocrine, para- receptors dimerize upon ligand binding, then internalize as a crine, and/or endocrineactivation. ligand-receptor complex. The mode of signal transduction for this superfamily of receptors remains undefined. The expression of certain GH/Prl genes by the placenta raises the question of whether theymight be acting locally on The human growth hormone/prolactin family of peptide this rapidly growing organ as well as on more distant tissues. hormones is encoded by five genes: growth hormone (hGHhGH-V, the most recently characterized of the hormones in N)’, hGH-variant (hGH-V), chorionic somatomammotropin this group, binds to both somatogen and lactogen receptors A and B (hCS-A, hCS-B), and prolactin (hPrl). The GH and with a binding profile that defines it as the most purely CS genes are clustered on chromosome 17 (George et al., 1981) while the single hPrl gene is located on chromosome 6 (Ow- somatogenic human hormone (Ray et aL., 1990). Considering the placental expression of this potent somatogen as well as erbach et al., 1981). The expression of each of these genes is tissue-specific: hGH-N is expressed exclusively by somato- the two locally expressed lactogens, hCS and hPrl, it is nectrope cells of the anterior pituitary (Whilhelmi, 1961) while essary to consider the potential for autocrine and/orparacrine activities in addition to their effects on distant tissues. Such * The costs of publication of this article were defrayed in part by local activities would necessitate the presence of a correspondthe payment of page charges. This article must therefore be hereby ing set of receptors on the surface of placental cells. marked “advertisement” in accordance with 18 U.S.C. Section 1734 As part of an effort to define the local effects of the GH/ solely to indicate this fact. Prl family of hormones on theplacenta, we recently screened (( To whom correspondence should be addressed Dept. of Genetics, a placental cDNA library to detect expression of the hGH University of Pennsylvania, Rm. 437A Clinical Research Bldg., 422 receptor (hGHR) mRNA. We found that the placental villi Curie Blvd., Philadelphia, PA 19104-6145. The abbreviations used are: hGH, human growth hormone; BSA, are enriched for hGHR mRNA. Structural analysis of the bovine serum albumin; CPMM, canine pancreaticmicrosomal mem- placental villous hGHR mRNA revealed that itdiffered from branes; hGH-V, human growth hormone-variant; hCS, human cho- the previously defined hepatic hGHR mRNA by the selective rionic somatomammotropin; hPrl, human prolactin; hGHR, human deletion of a 66 bp segment encoded by exon 3 (Urbanek et growth hormone receptor; hGHRd3, alternatively spliced human growth hormone receptor lacking exon 3; PBS, phosphate-buffered aL., 1992). This placental hGHR mRNA isoform, hGHRd3 saline; PCR, polymerase chain reaction; RT, reverse transcription; mRNA, predicts the expression of a corresponding hGHRd3 SOE, splice overlap extension; endo H, endoglycosidase H; 0,ovine; isoform that differs from the hepatic hGHR by a deletion of NTR, non-translated region. 22 amino acids within the extracellular domain of the recep-

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Function of the Placental hGH Receptor (hGHRCl3)

tor. While others have shown that there is no substantial effect of this region on hGH-N binding by bacterially expressed extracellular domains of the hGHR (Bass et d.,1991), the significance of this finding with respect to the biological activity of the full-length, processed, transmembrane receptor has not been addressed. Determining whether the hGHRd3 mRNA can encode a stable membrane-bound receptor and establishing its ligand binding profile isof central importance to understanding the potential roles of the placentally expressed hormones. The studies reported in the present paper demonstrate that the full-length, transmembrane hGHRd3 isoform encodes a stable and functional GH receptor. These results establish the potential for a GH-GHR axis within the placenta.

template plasmid for 25 cycles using Vent DNA polymerase (New England Biolabs). The cycles consisted of 1 min denaturation at 95 ‘C, 1 min annealing at 50 “C, and 2 min extension at 72 “C with the exception of a 3-min denaturation interval for the first cycle. Each reaction was resolved on a 1.0% agarose gel, and the amplified fragments were excised and purified. The @-globin5’-NTR and two of the hGHR coding fragments (B-R and C, or B-Rd3 and C) were then joined by SOE/PCR to generate the final hGHR and hGHRd3 transcription templates, respectively. In each case 2% of each of the three fragments were mixed and amplified between primers 1and 4. The PCR conditions were the same as described above except that the extension step was increased to 3 min. The full-length products generated by each of the two reactions were gel purified, and 2% of the final products were then used as template for reamplification between primers 1 and 4 for 25cycles. This final amplification reaction was subsequently extracted with phenol-chloroform-isoamyl alcohol, ethanol precipitated, and used directly as a template for in vitro transcription. EXPERIMENTAL PROCEDURES I n Vitro Transcription and Translation-In vitro transcriptions Materials-Restriction and modification enzymes were purchased were carried out using 1 pg of linearized plasmid DNA or 0.5 pg of from New England Biolabs (Beverly, MA), Boehringer Mannheim, cDNA fragment as template. The 2 0 4 transcription reaction was U. S. Biochemicals (Cleveland, OH), and Promega (Madison, WI). carried out in the presence of0.10 p1 of [aSzP]CTPand 5 mM Avian myeloblastosis virus reverse transcriptase was purchased from ‘mG(5’)ppp(5’)G with all other reaction conditions as suggested by Life Sciences (St. Petersburg, FL), and Taq DNA polymerase from the manufacturer (MEGAscript SP6 RNA polymerase). After a 2-h Perkin-Elmer-Cetus. Each enzyme was used according to the manu- incubation, transcripts were desalted on Sephadex G-50 columns to facturer’s specifications. [-p3’P]ATP (>5000 Ci/mmol), [CY-’~P]~ATPremove unincorporated cap analogand nucleotides. An aliquot of the (400 and 3000 Ci/mmol), and [a-32P]dATP (>400 Ci/mmol) were final transcript was electrophoresed on a 6% acrylamide, 8 M urea gel purchased from Amersham Corp. Oligonucleotides were synthesized and analyzed by autoradiography to document ita quantity andintegby the DNA synthesis facility of the Howard Hughes Medical Insti- rity. tute at the University of Pennsylvania. ’%I-hGH-N was purchased I n vitro translations were carried out in rabbit reticulocyte lysate from New England Nuclear (Wilmington, DE). Recombinant 22-kDa in a 15-pl reaction volume at 30 “C for 2 h as detailed previously hGH-N was a gift from Eli Lilly. hCS and ovine ( 0 ) Prl (containing (Liebhaber et al., 1984). Where noted, translations were carried out less than 0.1% anterior pituitary hormone contamination) were gifts in the presence of 1.5 pl of canine pancreaticmicrosomal membranes of the National Hormone and Pituitary Program of the National (CPMM) (Promega Corporation) and translatedfor 2.5 h. N-Linked Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK). SP6 carbohydrate residues were removed by incubating the translation Megascript in vitro transcription kits were purchased from Ambion reaction with 2 units of endoglycosidase H (endo H) at 37 “C for 6 h Inc. (Austin, TX). Xenopus lnevis were obtained from Xenopus I in 0.1% SDS and 0.1 M sodium citrate, pH 5.5. Aliquots of each (Ann Arbor, MI). reaction (1 or 5 p l ) were analyzed directly or following immunopreGeneration of cDNA Templates-Templates for in vitro synthesis cipitation on 8% SDS-PAGE. of hGHR and hGHRd3 mRNAs were generated by joining together X. lnevis Oocyte Injections-Experiments utilizing X. lneuis were three overlapping cDNA segments by splice-overlap extension (SOE) reviewed and approved by the Institutional Animal Care and Use polymerase chain reaction (PCR) (Horton et al., 1989 ,1990). Frag- Committee of the University of Pennsylvania. Care of X. lnevis and ment A, common to both templates, contains the SP6 RNA polym- procedures for oocyte harvesting followed standard protocol (Coleerase promoter, the entire 5”nontranslated (5’-NTR) region and man, 1984; Gurdon and Wakefield, 1986; Huez and Marbaix, 1986). initiation codon of human @-globin,and thefirst 20 bp of the common After anesthesia in 0.1% ethyl m-aminobenzoate (Tricaine, Sigma), hGHR/hGHRd3 coding sequence. The @-globincDNA template for ovaries from female X. lnevis were removed and treated with 2 mg/ fragment A amplification, pSPk@cA,was amplified between a sense ml collagenase until individual oocytes were free from their membraprimer that contains a BamHI cloning site at its 5’ end followed by nous coverings. Released oocytes were thoroughly washed in modified the SP6 promoter sequence (primer 1: 5’-TATAGGATCCATTT- Barth’s saline (MBSH 88 mM NaCl, 1.0 mM KCI, 2.4 mM NaHC03, AGGTGACACTATA-3’) and an antisense primer that contains the 15 mM HEPES-NaOH, pH7.6, 0.3 D M CaN03, 0.41 mMCaC12,0.82 last 16 bases of the @-globin5’-NTR, the adjacent initiation codon, mM MgSO,). Individual oocytes were injected with approximately 40 and thefirst 17 bases of the coding sequence common to hGHR and nl of desalted transcription reaction. Oocytes were incubated overnight at room temperature in MBSH supplemented with 100 units/ hGHRd3 mRNA (primer 2: 5’-AGCAGCTGCCAGAGATCCATGGTGTCTGTTTGAGGT-3’). The unique middle fragments, B-R and ml penicillin and 100 mg/ml streptomycin sulfate (Life Technology B-Rd3, encompass the first 700 or 634 bp of the hGHR orhGHRd3 Inc.) before being used in subsequent experiments. Oocytes which mRNA coding sequences, respectively. These fragments were ampli- were nonviable by visual inspection were discarded prior to analysis. Analysis of Injected mRNA-To assess the stability of the injected fied from either an hGHR (258~-13)or an hGHRd3 (258b-9) cDNA template. Both of these plasmids contain two separate single base- RNAs, transcripts were labeled to a high specific activity, injected, pair deletions at nucleotides 719 and 1200 (the A of the AUG is and incubated as detailed above. At the noted times oocytes were designated +1) which lie outside the region of amplification. These snap-frozen ondry ice and stored at -70 “C until processed. Oocytes amplifications utilize a sense primer which contains 16 bases of 5’- were subsequently homogenized and incubated at room temperature NTR and theAUG of @-globinmRNA and the first 17 bases of the for 45 min in 75 pl of RNA extraction buffer/oocyte (2% SDS, 20 mM NaCl, 5 mM MgCl2,0.2 M Tris, pH 7.5, and 1 mg/ml proteinase common hGHR/hGHRd3 coding segment (primer 2’: 5”ACCTCK). NaCl (final concentration of200mM) and carrier tRNA were AAACAGACACCATGGATCTCTGGCAGCTGCT-3’) and an antisense primer which contains 18 bases of central coding region (bases subsequently added to samples that were extracted twice with phenol683-700 encoded in exon 7) common to hGHR and hGHRd3 (primer chloroform-isoamyl alcohol, then precipitated with 2 volumes of 3: 5’-GTTTGGATCTCACACGCA-3’). Fragment C, common to the ethanol. RNAs were analyzed by electrophoresis on a 6% acrylamide, two receptor mRNAs, extending from thecentral coding region 8 M urea gel with subsequent autoradiography. Receptor Binding Assays-Oocytes injected with hGHR mRNA, through 85 bp of the 3’-NTR region was amplified from the hGHR cDNA plasmid 258-21 template. The sense primer corresponds to hGHRd3 mRNA, or uninjected controls were incubated overnight at bases 612-631of the coding region (primer 3’: 5”GAAAATG- room temperature and washed with fresh MBSH. An experimental ATGGACCCTATAT-3’) and the antisense primer is located in the sample consisted of five identically injected oocytes incubated for an 3’-NTR (primer 4: 5’-GCGGGATCCTTTAAACATTGTTTTGG- additional 4 h at 4 “C in 50 pl of MBSH containing 2 mg/ml BSA, CTT-3’). Plasmid 258-21 contains three A to G point mutations at approximately 25,000 counts/min ‘“I-hGH-N, and theindicated connucleotides 57,153, and 351, all of which are excluded from the centrations of unlabeled hormones. Unbound ‘“I-hGH was subsefragment prepared by PCR. Fragments were amplified from 20 ng of quently removed as described with a few modifications (Peacock et

Function of the Placental hGH Receptor (hGHRd3) al., 1988). Oocytes were rinsed briefly in ice-cold MBSH containing 2 mg/ml BSA, layered over 400 p1of a 1:1.5 mixture of dibutyl phthalate-dioctyl phthalate in a 400-p1 microfuge tube, and spun for 10 s at 6,000 revolutions/min. The overlaying oil and aqueous layer were discarded, and the radioactivity from pelleted oocytes quantitated in a gamma counter. Each data point represents an average value from four separate experimental samples containing five oocytes each. Reported 50% maximal binding values represent the average of at least two independent determinations. Preparation of ConditionedMedin-The generation of stably transfected mouse fibroblast C127 cell lines which express hGH-N (Cooke et al., 1988b), 22 or 20-kDa hGH-N (Estes et al., 1992), or hGH-V (Cooke et al., 1988b) has been previously described. Each of these cell lines was grown to 90% confluence in minimal essentialmedia (JRH Biosciences, Lenexa, KS) supplemented with 10% fetalbovine serum, 2 mM glutamine (Sigma), 100 unita/ml penicillin, and 100 mg/ml streptomycin sulfate, rinsed with PBS, andincubated overnight with serum-free minimal essential media. These conditioned media were concentrated using Amicon Centriprep-10concentrators (Amicon Division, W. R. Grace & Co, Beverly, MA), and the amount of GH was quantitated using an enzyme-linked immunosorbant assay prior to use. For enzyme-linked immunosorbantassay,serialdilutions (1:40, 1230, 1:160, 1:320, and 1:640) of each sample were made using media conditioned by nontransfected C127 cells as diluent.Each sample volume was then expanded with PBS to1.25 ml and applied to a single wellof a 96-well Linbro Titertek plate and incubated overnight at 4 ‘C. The fluid in each well was subsequently removed and the wells blocked with 400 pl of PBS, 0.5% gelatin for 1 h at room temperature. Wells were emptied and washed twice with PBS, 0.05% Tween 20, 50 pl of 1:lOO dilution of rabbit anti-hGH antibody (Liebhaber et al., 1986) in PBS, 0.5% gelatin, 0.05%Tween 20 were added to each well, and incubated 1 h at room temperature. Plates were then washed three times with PBS, 0.05%Tween 20 followed by the addition of 50 p1 of a 1:2000 dilution of peroxidase-conjugated goat anti-rabbit IgG in PBS, 0.5% gelatin, 0.05% Tween 20. After 1 h of incubation at room temperature, the secondary antibody was removed, wells were washed five times with PBS, 0.05%Tween 20, 100 pl of fresh OPD color reagent was added (0.4 mg/ml o-phenylene diamine and 0.4 pl of HzOZin citrate buffer), and the absorbance at 450nmwas measured in a Titertek Multiskan Plus (ICN Flow, Irvine, CA). Western Blotting-The integrity of hormones in the conditioned media was determined by Western immunoblotting. The volume of conditioned media which contained 8 ng of hormone as determined by enzyme-linked immunosorbant assay was assayed on an 8% SDSPAGE (Sambrook et al., 1989). Protein was electrotransferred from the gel to a nitrocellulose sheet for 2 h at 0.4 amps in Tris-glycine transfer buffer (39 mM glycine, 48 mM Tris base, and 20% methanol). The hormones were detected using a rabbit anti-hGH antibody (Liebhaber etal., 1986)and theantigen-antibody complexes were visualized by an enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Corp.). Primary antibodywas used at a 1:lOO dilution in 10 ml of blocking buffer (PBS, 0.05%(v/v) Tween 20, and 5% (w/v) nonfat dry milk), and the second antibody was used at a 1:500 dilution in 10 ml of blocking buffer. Analysis of Receptor Internalization-Oocytes injected with receptor mRNAs were incubated overnight at room temperature in MBSH. Intact oocytes were incubated for 2 additional h at 4 “C with approximately 25,000 counts/min ”‘1-hGH-N with or without an excess of cold hGH-N (2 mg/ml). The oocytes were then washed twice with ice-cold MBSH containing 2 mg/ml BSA and rapidly brought to room temperature to allow internalization of the bound hormone. At the indicated times three samples of five oocytes each were removed and assessed for ligand internalization. Surface-bound hormone was released by incubating the ice-cold oocytes twice for 5 min each with 1 ml of glycine buffer (50 mM glycine, 100 mM NaC1, pH 2.5) (Rodriguez et al., 1992). These acid washes were pooled and designated the surface-bound fraction. The radioactivity associated with the acidtreated oocytes was designated as the internalized fraction. Specific binding to the injected oocytes was defined as total binding in the absence of unlabeled hGH-N competitor minus total binding in the presence of an excess of unlabeled competitor. RESULTS

Synthesis of hGHR and hGHRd3 mRNA-We have previously demonstrated that hGHR gene transcripts are alterna-

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tively spliced in the placental villi to exclude exon 3. This hGHRd3 mRNA predicts the expression of an hGH receptor isoform with a 22-amino-acid deletion inits extracellular domain. In the presentstudy we attemptto express this putative hGHRd3 receptor in a membrane-associated form and determine whether it can bind GH or GH-related hormones. Thesestudies were carriedout by microinjecting hGHR and hGHRd3 mRNAs into Xenopus oocytes. Initial attempts to clone full-length hGHR and hGHRd3 cDNAs for use as templates for in vitro transcription were unsuccessful. Numerous full-length GHR cDNAs were isolated either by screening a placental cDNA library (Urbanek et al., 1992) or by RT-PCR cloning from placental RNA. In each case fulllength inserts contained one or more rearrangements, missense mutations, or nonsense mutations (data not shown). After screening a large number of such clones, we concluded that thebacterial strains that we were using (Escherichia coli HBlOl and DH5a) could not support the expression of an intact hGHR cDNA. To circumvent this problem full-length transcription templateswere generated by direct transcription of amplified full-length cDNAs using a SOE/PCR strategy (see “Experimental Procedures”). In these templates the 5’NTR of the hGHR cDNA was replaced by the full 5’-NTR of the human @globin cDNA to maximize translational efficiency in the Xenopus oocyte.2For convenience these mRNAs are referred to as hGHRandhGHRd3 mRNAs with the understanding that the 5’-NTRs are @-globinin origin. The receptors encoded by each chimeric mRNA contain only hGHR sequences. The full-length hGHR and hGHRd3transcripts generated from the SOE/PCR cDNA templates contain only trace amountsof prematurely terminated transcripts (Fig. 1A). The expected 66 base difference in their sizes can be clearly discerned on agarose gel analysis (data not shown). I n Vitro Translation of hGHR and hGHRd3 Transcr@tsTo establish that theSOE/PCR-generated transcripts canbe translated, the synthetic hGHR and hGHRd3 mRNAs were incubated in rabbit reticulocyte lysate in the presence of [3sS] methionine.Incubationduration was prolonged to 2 h to obtain maximal levels of synthesis (Fig. 1B). An 84-kDa product was specifically generated by translation of the hGHRd3 and hGHR mRNAs. Although the predicted size for the unprocessed GHR is 70 kDa, previous studies indicate that deglycosylated GHR from IM9 cells migrates at 95 kDa on SDS-PAGEgels, significantly slower than predicted (Kelly et al., 1991). To demonstrate appropriate processing of the encoded proteins by the endoplasmic reticulum, a stepcritical to membrane receptor biogenesis, the in vitro translations were carried out in the presence of CPMM (Fig. 1C). hGHR and hGHRd3 have identical NH2 terminiencompassing their signal peptide cleavage sites and they both contain N-linked glycosylation consensus sites (four and five sites, respectively). Translation of the hGHRd3 mRNA in the presence of CPMM results inan apparent net increase of 10 kDa (8494 kDa). A parallel increase is seen in the hGHR mRNA translated under identical conditions (Fig. IC). Treatment with endo H, which cleaves the unmodified N-linked sugar residues, decreases the size of the translation products to the unprocessed preproteins. These results are consistent with in vitro expression and processing of the predicted hGHR and hGHRd3 by their respective synthetic RNAs. Stability of the hGHRd3 and hGHRmRNAs in X. laevis Oocytes-To study the receptors on an intact cell surface, the two synthetic mRNAs were separately injected into the cytoplasm of Xenopus oocytes. To assess the utility of this J. E. Russell, unpublished observations.

Function of the Placental hGH Receptor (hGHRd3)

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A.

C.

B. (3

RNA

‘0 (3

A

Endo H CPMM

hGHRd3

- -

+

+

+

-

hGHR

g+’

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+

+ s +glycosylated hGHR

+- prehGHRlhGHR

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FIG.1. In vitro transcription and translation of hGHR and hGHRd3 cDNA templates. A, in vitro transcribed hGHRand hGHRd3 mRNA. The hGHR and hGHRd3 templates prepared by the SOE/PCK reactions see “Experimental Procedures” were transcribed by SP6 polymerase in the presence of [cY-~*P]CTP, resolved on a denaturing acrylamide gel, and autoradiographed. hGHR and hGHRd3 transcripts migrate at theirpredicted sizes and areindicated by the arrow (DNA molecular weight markers not shown). B , in uitro translation of hGHR and hGHRd3 transcripts. Aliquots of hGHRd3 or hGHR transcription reactions (1 or 5 pl) were translated in a rabbit reticulocyte lysate in uitro translation system in the presence of [35S]methionine.Aliquots of the translation reaction were assayed by SDS-PAGE after a 2-h incubation. The positions of protein molecular size standards areindicated on the left of the autoradiograph, and thelocation of the hGHR/ hGHRd3 specific bands is shown on the right by the double arrows. The identity and quantityof transcript used in each reaction is indicated a t the top. C, effects of microsomes and endo H onthe sizes of the hGHRd3 (left panel) and hGHR (right panel)in uitro translation products. hGHR and hGHRd3 transcripts were translated in the rabbit reticulocyte lysate system in the presence (+) or absence (-) of CPMM. An aliquot of each sample translated in the presence of CPMM was subsequently incubated with endo H. The locations of molecular size standards are indicated between the two autoradiographs. Treatment with CPMM or endo H is indicated above the respective lanes. The locations of each preprotein (prehGHRd3 or prehGHR), the glycosylated isoforms (glycosylated hGHRd3 or glycosylated hGHR), and the deglycosylated, mature proteins (hGHRd3or hGHR) areindicated. (data not shown). Each transcript was coinjected with the highly stable human P-globin mRNA as an internal control for the efficiency of RNA harvest. The absolute and relative levels of hGHR and hGHRd3 mRNAs did not change substantially over 30h of incubation. The stability of the hGHR and hGHRd3 mRNAs suggestedthat anovernight incubation of the injected oocytes would be a reasonable time for their expression and the assembly of their encoded receptors on the oocyte membrane. X . laeuis Oocyte Binding Assays-A competition binding I ” ” assay was established to measure the interaction of ligands 0.1 1.0 10 0 10 0.1 1.0 0 with the hGH receptors assembled on the surface of intact 22 kD hGH-N (nM) 20 kD hGH-N (nM) microinjected oocytes. The sensitivity and accuracy of the FIG.2. Specific binding of hGH-N to Xenopus oocytes ex- assay was established by determining the binding of recompressing hGHR and hGHRd3.A , binding of recombinant 22-kDa binant GH to oocytes injected with the mRNA forthe hGHR hGH-N to hGHR and hGHRd3. The percent ’T-hGH-N bound as a function of quantity of unlabeled competitor present in the incubation the binding affinity for this interaction is well established in is illustrated. 100% binding is defined as the amount of lZ5I-hGH-N a number of systems (Lesniak et al., 1977; Hocquette et al., bound in the absence of cold competitor. Binding to hGHR is indi- 1989; Fuh et al., 1990; Spencer et al., 1990; and Dusquesnoy cated by the open boxes and binding to hGHRd3 by the closed boxes. et al., 1991). Oocytes injected with hGHR mRNA and incuThe amount of binding observed with uninjected oocytes is shown by bated overnight to allow expression of the hGHR were subthe closed circles. The average values and standard deviations from three experiments are shown. B, binding of recombinant 20-kDa sequently incubated with lz5I-hGH-N along with known hGH-N to hGHR and hGHRd3. The axes and symbols are the same amounts of competing unlabelled hGH. The relationship beas in panel A. The average values and standarddeviations from three tween lZ5I-hGHbinding to the amount of cold competitor experiments are shown. establishes the competition binding curve (Fig. 2). 100%binding, defined as the amount of binding which occurs in the system for expressionand comparison of the hGHRd3 relative absence of cold competitor, is between 5-10% of the total1251to thehGHR mRNA and to optimize the incubation time, the hGH-N added to the incubation, and the amount of binding 5-10% of the relative stabilities of these two mRNAs were assessed post- to uninjected oocytesisgenerallybetween injection. Internally 32P-labeledtranscripts were injected into maximal specificbinding seen in oocytes injected with recepXenopus oocytes, and mRNA was extracted 0, 10,20, and 30 tor mRNA. 50% maximal binding or EC, of hGH for the h post-injection and directly analyzed by gel electrophoresis hGHR expressed on the surface of the injected oocytes is 14 A.

B.


*

9 x lo-'' M. This value compares quite well with the ECm of hGH-N for hGHR measured in a number of other systems (Lesniak et al., 1977; Hocquette et al., 1989; Fuh et al., 1990; Spencer et al., 1990; and Dusquesnoy et al., 1991). Having established that the Xenopus oocyte system is a suitable system in which to study the hGH-N and hGHR interaction, we next investigated whether hGH-N would show specific binding to oocytes injected with hGHRd3 mRNA. AS seen in Fig. 2.4, the competition binding profile of hGH-N to oocytes injected with hGHRd3 mRNA is indistinguishable from the profile of oocytes injected with the hGHR mRNA; EC5o of the hGHRd3 injected oocytes is 19 f 7 X 10"' M, essentially identical to the hGHR value of 14 f 9 X 10"' M. Circulating pituitary hGH is present intwo isoforms, 90% as a 22-kDa protein and the remaining 10% as a 20-kDa protein lacking an internal 15-amino-acid segment. Previous studies by others have revealed that the 20 kDa form has a unique receptor binding profile (McCarter et al., 1990; Kostyo et al., 1986; Smal etal., 1985,1987;Baumann and Shaw, 1990; Siege1 et al., 1981; Spencer et al., 1981; Mosier and Lewis, 1982; and Closset et al., 1983). We analyzed the ability of recombinant 20-kDa hGH-N to bind tothe hGHR and hGHRd3 receptors (Fig. 2B). The 20-kDa hGH-N was generated by a stably transformed cell line expressing only this alternatively spliced product (Estes etal., 1992). The 20-kDa hGH-N isoform has a %fold weaker affinity for hGHR and hGHRd3 than does the 22-kDa hGH-N isoform, and ECW occurs at 39 f 15 x 10"' M for hGHRd3 and 64 f 9 X 10"' M for hGHR. Although the hGHRd3 has a slightly higher affinity for 20-kDahGH-N than does hGHR in each of three independent binding experiments, the degree of variability in our system makes it difficult to state with certainty whether this difference is significant. The binding studies in Fig. 2 clearly demonstrate that thehGHRd3 mRNA encodesa stable receptor protein in Xenopus oocytes that binds the two isoforms of pituitary hGH-N. The hGHRd3 therefore has the potential to actas a functional hGH receptor. Placental Hormone Binding Assays-The alternatively spliced hGHRd3 mRNA is selectivelyexpressed in placental syncytial cells. To assess its ability to bind and potentially mediate the action of placentally expressed hormones of the GH gene cluster, we carried out a series of competition binding studies with each of the placentally expressed members of the family (hCS, hGH-V, and Prl). In these studies the binding was measured by the ability of the placental hormones to compete forbinding with '251-hGH-N.In each case the hGHR and the hGHRd3 were compared. These studies are summarized in Fig. 3. Conditioned media from a mouse fibroblast cell line stably transfected with the hGH-V gene was usedas the source of hGH-V. The results of the binding studies using the hGH-V media demonstrate that hGH-V binds hGHRd3 and hGHR with the same affinity (Fig. 3A). The ECso occurs at 10 f 1 X 10"' M for hGHRd3 and 12 f 2 X 10"' M for hGHR. Thus the binding affinities of hGH-V for the two receptors are quite similar to the affinities of hGH-N. We next assessed the ability of the most abundant protein expressed by the placental villi, hCS, to bind to hGHRd3. Fig. 3B is a competition binding curve in which hCS competes with labeled hGH-N for binding to hGHR or hGHRd3 expressed in Xenopus oocytes.hCS binds equally to hGHR and hGHRd3, but its affinity is approximately 1000-fold lower than that of the 22-kDa hGH-N. ECw occurs at 11 f 1.7 x M for hGHRd3 and 17 2 4.2 X M for hGHR. The prolactin gene is expressed in both the pituitary and the placental decidua. To further assess the ability of lacto-

19029

gens to bind to the hGHRs, oPrl was used as a prolactin source due to its availability in pure form. As seen in Fig. 3C, oPrl competes effectively with 22-kDa hGH-N for binding to hGHR or hGHRd3. There is no difference in the ability of oPrl to bind to the two forms of the hGHR. ECm occurs at 214 f 162 x 10"' M for hGHRd3 and 237 e 4 X 10"' for hGHR. The affinity of oPrl for the hGHRs is, therefore, 1020-fold weakerthan thatof hGH-N. Internalization of HormoneReceptorComplexes-Ligand binding to the GHR is followed byits internalization (Roupas and Herrington, 1989).To determine whether this post-binding internalization also occurs after ligand binding to hGHRd3, we established an internalization assay using the Xenopus oocytesbased on protocols of previous investigators (Rodriguez et al., 1992; Peacock et al., 1988). Oocytes were injected with hGHRd3 or hGHR mRNA and incubated overnight to permit receptor synthesis and assembly on the cell surface. The receptors were then loaded with '*'I-hGH-N at 4 "C, and internalization was measured over time by determining the proportion of radioactive tracer that remained on the surface of the oocyte after shifting the cells to RT. Oocytes injected with hGHRd3 or hGHR mRNAswere studied in parallel. The rates of internalization of labeled ligand-bound to the hGHR and hGHRd3 are shown in Fig. 4. The ligandbound hGHRd3 internalized with the same kinetics as the hGHR. The internalization of the receptor-ligand complex as assayed by the increasing resistance of hormone to release with acid started almost immediately after shifting the oocytes to RT and was complete after approximately 50 min. Essentially the same results were generated in two additional experiments. These data suggest that the initial post-ligand binding steps mediating receptor internalization are identical for hGHRd3 and hGHR. DISCUSSION

The importance of the exon 3-encoded regionof the hGHR is unknown. All sequenced GHRs in mammalian species conserve this regionwhile it is specifically absent in Prl receptor mRNAs (Boutin et al., 1988; Davis and Linzer, 1989; Smith etal., 1989; Adams et al., 1990; Cioffiet al., 1990;Edery et al., 1989; Hauser et al., 1990; and Urbanek et al., 1992). This pattern of evolutionary conservation suggests that the loss or retention of exon 3 by alternative splicing could have a major impact on receptor expression and/or function. In addition to affecting hGH-N ligand binding, deletion of exon 3-encoded sequences might affect receptor processing, transport, stability, binding to other related ligands, and/or signal transduction. In this study we show that the exon 3-deleted isoform of hGHR encodes a stable and functional receptor. We demonstrate that hGHRd3 mRNA can be efficiently translated in an in uitro rabbit reticulocyte lysate translation system as well as in an in uiuoX. h u i s oocyteexpressionsystem. Furthermore, the latter system is demonstrated to be appropriate for ligand binding studies. In Xenopusoocytes we demonstrate that hGHRd3 is stably integrated into the cell membrane and efficiently binds and internalizes ligand. Our data indicate that hGHRd3 is as efficient as hGHR at the level of ligand binding or internalization. hGHRd3 binds all three of the placentally expressed members of the GH/Prl gene family, hGH-V, hCS, and Prl, aswell as the 22- and 20kDa isoforms of the pituitary hGH-N. Of special significance is the finding that hGHRd3 is expressed in placental tissue as aretwo potential ligands, hGHV and hCS. The high affinity of hGH-V for the receptor

19030

-


A.

B.

n

7

J

0

10

hGH-V (nM)

100

hCS (nM)

1000

0

,

I

1.0

10

100

1000

oPrl (nM)

FIG.3. Binding of placentally expressed members of the GH/Prl gene cluster to hGHR and hGHRd3. A , binding of recombinant hGH-V to injected oocytes. The axes and symbols are the same as inFig. 2. Each point representsthe average and standarddeviation of four experimental samples containing five oocytes each. For each hormone one representative binding study is shown, although at least two independent experiments for each yielded identical results. B , binding of purified hCS in parallel experiments. C, binding of purified oPrl in parallel experiments.

0

0

20

40

60

80

100

120

140

Time (min) FIG.4. Internalization of hGH-N and receptor in a complex. Oocytes injected with hGHR or hGHRd3 mRNA were incubated at 4 “C with ‘%I-hGH. The oocytes were then brought to room temper-

ature and the receptor-hormone complex allowed to internalize for increasing amounts of time at room temperature. The ratio of acid releasable (surface-bound) lZ61-hGH-Nto the total ’T-hGH-N bound expressed as a percent is shown as a function of time. Each point represents the average and standard deviation of three data points utilizing five oocytes each. Internalization of the hGHRd3 complex is indicated by closed boxes and thatof hGHR by open boxes.

makes its potential for local interaction quite evident. Although hCS has only 1/1000 the affinity for hGHRd3 compared to hGH-V, a case can also be made for its potential significance as a ligand since hCS is found in the sera of pregnant women during the second and third trimester at levels that areapproximately 1000-foldthat of normal serum hGH-N and 300-fold that of hGH-V at term (Frankenne et al., 1988; Phillips and Vnencak-Jones, 1989; Walker et al., 1991). The fact that hGHRd3 may serve as a receptor for both hGH-V and hCS supports their potential to act in an autocrine fashion in the syncytial cells of the placental villi. What effect this autocrine stimulation might mediate is open to speculation, but the rapid growth of the placenta presents an obvious possibility. The validity of the Xenopus oocyte expression system for the study of GHR and ligand interactions is supported by several lines of evidence. The ECso values that we obtained

for hGHR using this approach correspond quite closely with those obtained in other systems (Lesniak et al., 1977; Hocquette et al., 1989; Fuh et al., 1990; Spencer et al., 1990; Dusquesnoy et al., 1991). In our system, only oPrlhasa significantly different binding affinity for the GHR than has been reported in other studies. Monkey kidney COS cells transfected with the rabbit GHR have an affinity for oPrl that is approximately 50-fold weaker than that for hGH-N (Spenser et al., 1990). In IM9 cells, a human lymphoblast cell line that has hGH binding activity, oPrl competes approximately 1000-fold less efficiently for receptor binding than does hGH-N (Lesniak et al., 1977). The reasons for discrepancy among these results are notclear. Our results are reproducible with several different batches of hormone. Each oPrl sample that we used contained less than 0.1% contamination with otheranteriorpituitary hormones as determined by radioimmunoassay (see “Experimental Procedures”). The strong competition observed with oPrl cannot, therefore, be due to contamination with oGH. The differences in results may reflect details of each experimental model. For example, IM9 lymphocytes may express a multitude of yet uncharacterized receptors that could compete for oPrl binding and shift the binding curve to the right. Lymphocytes have recently been characterized as possessing Prl receptors, and the structure of the ratNb2 lymphoma cell Prl receptor is unique, containing a shortcytoplasmic tail (Pellegrini et al., 1992; Ali et al., 1992). In theCOS cell experimental model, competition studies were carried out by binding to therabbit GHR which may have a different affinity for hGH than does the hGHR. Our findings using the full-length, transmembrane hGHR to study ligand binding complements several other studies. First, using a bacterially expressed soluble hGH-binding protein, Bass et al. (1991) demonstrated that this extracellular fragment of the hGHR binds to hGH equally well with and without the exon 3-encoded segment. Second, the recently established crystal structure of the hGH/hGH-binding protein complex has revealed that one molecule of hormone binds to two molecules of hormone-binding protein (De VOSet al., 1992). Therefore, sequences encoded by exon 3 are notimplicated in eitherthe ligand-receptor or receptor-receptor interaction. However, although hGHR and hGHRd3 have the same ligand binding and internalization profile, the tissue-specific splicing patterns of hGHRd3 and hGHR imply that hGHR and hGHRd3 may be biologicallydistinct. Therefore, if there are any functional differences between hGHR and hGHRd3, they would have to be at the level of signal transduction.

Function of the Placental hGH Receptor (hGHRd.3) Alternative splicing has been shown to impart different signal transduction activities to receptors (Pin et al., 1992; Nakamura et al., 1992; Doherty et al., 1992; Sokka et al., 1992; Danoff et ul., 1991; and Sommer et aL, 1990); however, in all these cases the alternatively spliced domain has been localized to theintracellular region. Morerecently, studies by Chiba et al. (1993) using chimeric cytokine receptors showed that the specificity of the signal transduction pathway whichwas activated in response to ligand binding was determined by the extracellular domain of the receptor. It is believed that the activation of the signaling pathway may be mediated via an accessory protein which recognizes specific extracellular domains. I t is therefore possible that thepresence or absence of exon 3 may allow the interaction of different accessory proteins which may activate different signal transduction pathways. Another possible difference betweenthe biological activity of hGHRd3 and hGHR in vivo may be at the level of expression via translational efficiency. In this study we used the 6globin 5'-NTR instead of the endogenous GH receptor 5'NTR and therefore did not address the significance of different endogenous 5'-NTRs of the hGHR. Evidence suggests that there is extensive variation in the 5'-NTRof the hGHR mRNA (Leung et al., 1987;Godowski et al., 1989). Such differences, possibly reflecting utilization of alternative promoters, might affect the subsequent splicing as well as translational efficiency of the processed mRNA (Moldave, 1985; Ratner et al.,1987; Kozak, 1987;London et al., 1987; Kozak, 1988). Finally, immunohistochemical studies directly support the expression of GHRs in the fetus and placenta. These studies have shown that GHRs are expressed at significant levels in fetal liver, pancreas, cerebral cortex, and epidermis as well as second trimester placenta (Hill et al.,1992). Immunoreactive GHR was also detected in the syncytial cells of the placental villi as early as 8 weeks of gestation, and strong staining for the GHR was detected at term (Hill et al., 1992). Decidual cells also stain for the GHR while cytotrophoblast, chorionic trophoblast, and amnion do not. This would indicate that hGHRd3 protein, which our earlier studies have shown isthe only GHR mRNA isoform expressed in the placental villi (Urbanek et al.,1992), is translatedat significant levels in the placenta. Taken together, the results of the presentstudies in the context of these previous reports strongly support the expression of a functional hGHRd3 isoreceptor in the placenta. The function(s) that this receptor may serve in autocrine, paracrine, and/or endocrine activation can nowbe addressed. Note Added in Proof-Subsequent to completion of this manuscript, a paper by Sobrier et al. (Sobrier, M.-L., Duquesnoy, P., Duriez, B., Anselm, S., and Gossens, M. (1993) FEBS-Lett. 319,16-20) was published, the results of which are consistent with our findings. REFERENCES Adams, T. E., Baker, L., Fiddes, R. J., and Brandon, M. R. (1990)Mol. Cell. Endocrinol. 72,135-145 Ali, S., Edery, M., Pelleyini, I., Lesueur, L., Paly, J., Djiane, J., and Kelly, P. A. (1992)Mol. Endocrmnol. 6,1242-1248 Bass, S. H., Mulkerrin, M. G., and Wells, J. A. (1991)Proc. Natl. Acad. Sci.

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19032

Function of the PlacentalhGH Receptor (hGHRd3)

Ray, J., Okamura, H., Kelly, P. A., Liebhaber, S. A., and Cooke, N. E. (1990)

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~