Cloning and functional expression of CC CKR5, a human monocyte ...

Cloning

and functional

monocyte

expression

CC chemokine

of CC CKR5,

receptor

a human

selective

for MIP-la,

MIP-1 3, and RANTES Christophe

Combadiere,

The Laboratory ofHos: Bethesda, Maryland

Abstract: novel CC

cc

We have chemokine

CKR5

that

other CC constitutively in

primary

has

K. Ahuja,

National

CMI)

amino

Irutitwe

ofAllergy

cDNA for designated

human

(CC

H. Lee Tiffany,

acid

a

identity

to

CKR5 mRNA was detected adherent monocytes but not or eosinophils. Macrophage (MIP-la), MIP-13, and

protein-la

RANTES

a

48-75%

neutrophils

inflammatory

Defenses,

cloned receptor

CKRs. CC in primary

Sunil

were

transfected affinities

all potent agonists for CC CKR5 (EC50 nM) when calcium flux was measured in HEK 293 cells, yet the apparent binding of the corresponding iodinated chemokines

to intact

cells

3-30

=

expressing the receptor were low (1C50 nM). The calcium flux responses were comblocked by treatment of transfected cells with

o.olOO pletely

pertussis toxin. These a G1-coupled receptor responses

to

Leukoc.

Rio!.

data that

MIP-la,

suggest may

that mediate

MIP-1f,

60: 147-152;

CC

and

CKR5 monocyte

and

Infectious

with

Words:

chemotaxis

.

.

overlapping

nated CC monocytes.

J.

this

claim,

having

(MIP-ia), tractant

The CC branch macrophage

MIP-l, protein-i

fea-

of the chemokine superfainflammatory protein-la

23 and reported

eosinophils [1-9]. phils [10-12]. Specific RANTES,

Eotaxin

amino

acid

CKR5

selective

for

cells.

that

the

variant.

novel

90

on calcium We

later

cells

flux

retracted

thought

plasmid

and

eosino-

reported

a human MIP-i3, from

[24].

Here

This

work first

the

allelic

to have

were

inadver-

pertussis

variant

and

our

the

extends

AND

made

MIP-la, saline; Philip Room

April

22,

of Samson

at CC

et al.

by describing

that

its

a

ligand

in primary leukoit couples to a pathway.

cDNA

from

BSA,

blood

bovine

human serum

The

National

cDNAs

mononuclear

inflammatory HEK,

M. Murphy,

11N113,

receptor-like

peripheral

protein;

10,

only

transduction

macrophage

chemoattractant requests:

named our

by detailing

used to clone novel chemokine library

a

METHODS

of the CC CKR5

Abbreviations:

Reprint

signal

also

in detail

the work

CKR5,

of

receptor

sequence

for CC CKR5,

of CC

cloning

RANTES,

CC CKR5,

we characterize

cDNA

toxin-sensitive

a ?Lgtl 1 cDNA

the CC We later not other

CC chemokine

binding properties and RNA distribution cyte subtypes, and by demonstrating

Cloning

of

recently

MIP-ia,

by identifying

NLAID,Bldg.

from

cells

protein-la; embryonic

of

MCP,

kidney;

PBS,

albumin.

Laboratory

of Host

Institutes

ofHealth,

Defenses, Bethesda,

MD 20892.

monocyte MCP-i,

is highly

basophils,

based

293

CC CKR3

encodes

that differs

phosphate-buffered

lymphocytes,

lower levels in to be selective

[12].

have

that

for

tract

neutrophils,

a!.

gene

monocyte

activate

the

tested et

The methods

(aa) sequences, the first two

MCP-i,

correction]. This mistake occurred because plasmid DNA was mislabeled CC CKR3. that CC CKR3 is selective for eotaxin and

which are adjacent. All of these molecules except for eotaxin induce monocyte chemotaxis but they vary both in monocyte chemotactic potency and in their ability to atand

with

MATERIALS

RANTES, 1-309, monocyte chemoat(MCP-i), MCP-2, MCP-3, and eotaxin.

They have highly conserved amino acid including four conserved cysteine residues,

RANTES,

at much reported

RANTES HEK

discovered

transfected

human

G protein

ture of the inflammatory response to infectious and other noxious agents, however, the specific types of leukocytes that accumulate depend on the inciting agent and can vary widely. The factors determining this specificity are not fully known but probably include members of the chemokme superfamily. mily includes

and

in transfected

CC chemokines

is a characteristic

of Heallh,

for M1P-ia,

is expressed was originally

MIP-l,

responses

CKR5

INTRODUCTION of leukocytes

selectivity

for MIP-ia,

CC CKR5,

accumulation

Institutes

identified a family of distinct but in monocytes that encode receptors

CKR3 that CC CKR3

selective

Local

National

tently transfected with a related cDNA encoding a novel CC chemokine receptor that we have designated CC CKR5 [ref.

1996

inflammation

DLceases,

and MCP-3 [2, 13-22]; their deduced amino acid sequences have >45% aa identity to each other. Three of us recently reported the sequence ofa human eosinophil receptor desig-

Samson

Key

M. Murphy

molecular cloning has related genes expressed

been

is

RANTES.

and Philip

binding and

MCP-3

sites have

for

MIP-la,

been

identified,

MIP-13, and

Received

1996;

revised

May

13,

1996;

accepted

May

14,

1996.

Journal

of

Leukocyte

Biology

Volume

60,

July

1996

147

a patient

with

[20J. One

of the

sequence

eosinophilic

highly

to 813

of the

CC

CKR2B

from

ORF.

to

endotoxin-stimulated

scribed

matched

ment

of the

HI and subcloned

Jolla, was

clone

8.5

were

picked

create

HEK

of

293

expanded cell

as

lines

described

the

cells

used

as

a

monocytes

as

designated

KS

de-

clone

fragby Barn

polymerase,

(Stratagene,

San

)

(10

La

to

grown

CA).

log

fetal bovine G418-resistant

[23].

expressing

Diego,

CC

Hu-

phase

in

serum were colonies

The

methods

CKR1

and

used CC

are

to

CKR2B

[21].

required

C-terminal

zation

to

leukocyte

the

viously

RNA

indicated

was

cDNA

[20].

An

prepared

probes

using

antisense

not

bind

used

for

Northern

to CC

blot

CKR1,

CC

binding

analysis

analyzed

methods

30-mer

5’-GTCATAGATFGGACTFGACACTFGATAATC +33 of the clone 8.5 eDNA, where does

and

or CC

using

conditions

described

pre-

oligonucleotide (nucleotides adenine in codon

+ 1 is the

CKR2,

analysis

by hybridi-

CKR3

DNA

+4 to 1) that

or RNA

described

for

nM

HEK

293

125I-labeled

cific

activity

MA)

cells

varying

chemokines

with

incubated MIP-1,

Du Pont/New of Hill,

Rocky

1 mg/mL

in duplicate

MIP-la,

Ci/mmol,

(Peprotech,

[RPMI-1640

were

concentrations

-2200

and

(106) MCP-1,

England

unlabeled NJ)

bovine

in 200

serum

cells (PBS)

pelleting

[Ca2]j

a

10%

were

activation changes

previously

with [21].

measured

Fura-2

upon

Where

h at 37#{176}C, then saline

after

pertussis

indicated,

washed

solution.

twice Cell

toxin

with

(List,

separated

was

ATP

transfected

loaded

with

and

were

250

ng/mL

resuspended

-80%

was from Sigma

293 incufor 2

in Hanks’

by trypan

blue

of CC

CKR2B

known [28].

chemokine Like all other

CKR5

has

Cloning

AND

al.,

our

at

position

polyadenylated

sequence. is

flanked

consensus

148

Journal

ATG

by

sequence

rules

lacks a polyadenylation codon proposed to initiate

of

established

Leukocyte

that

conforms

consensus translation

favorably

by Kozak

Biology

of the

[25].

Volume

The

60,

July

with

ORF

1996

N-terminal that

a consensus

predicted

the sequence

third

of Samson

in place

sequences

same

of CC CKR5

et

al.

screened

found

of alanine

probably

derive

gene.

RNA several

RNA

promyeloblastic

for

cells

cultured

CC

[24].

which

probe

Northern

primary

leukocyte

CKR5

only

cell

in

KG-iA

Cross-hybridization

is similar

clearly

blot

adherent

recognized

samples

a

in

monocytes,

but

to

CC with

to the

RNA

from

2,

left).

RNA

of reCC band

made

from

primary We

CC

transcript

3.5-kb

total

not

(Fig.

a 30-mer antisense not cross-hybridize

probe the same probe, confirming 2, right).

in size

hybridization

or eosinophil

neutro-

next

synthe-

CKR5 oligonucleotide CC CKR2 and used

that it to

blot. The same band was identified by this monocyte expression of CC CKR5 (Fig.

for CC CKR5

described

MIP-ia, MCP-3

of CC CKR5

and

The

two

for

loop

has

in the

a leucine

The

alleles

and

Agonists

The clone 8.5 cDNA for CC CKR5 is 1370 bp in length. The 5’- and 3’-untranslated regions and ORF are 26, 288, and 1056 bp, respectively. The 3’-untranslated region is not

in the

CKR5

with

contains

in

the

previously published in CC CKR5-transfected

analysis

CC

Compared

whereas

extracellular

bond.

[24].

distinct

residues

glycosylation,

sequence

90

of zero,

MO).

DISCUSSION

and sequence

a

plasma

this domain is highly chemokine receptors, CC

predicted

N-linked

loop.

et

charge

receptors known

third

a disulfide for

a net

cysteine

the

form

from

has

conserved and

extracellular

As

RESULTS

be

to the

bal-

exclusion

(St. Louis,

could

domain

ORF

HEK

Fura-2

[26]. with

by

as described

CA)

receptors

this

as

receptor by analogy

CKR5

saline

chemokines

Campbell,

in PBS

viability

treatment.

2 million

cells

of B. per:u.ssis

in holotoxin

anced

using

phosphorylation

the 2-adrenergic residues that domain

main

sized does

stimulation

receptor

the

threonine

from

mM

assay

were

for

and

After

25

7.4].

sucrose/phosphate-buffered

1). receptors,

(Fig.

of senine

tethering

other acidic

phil

loaded

bated

through

chemokines

pH

sites

small

was not excluded in this study, and analysis peripheral blood mononuclear cell RNA was as negative. In our experiments, a full-length

cushion.

Receptor cells

1 h at 37#{176}C,unbound

for

by

acid,

be

content

CC

very

found, primary ported

medium

and

a few

alignment

48%

mRNA,

human

(BSA)

Only

and

CKR2

Boston,

jsL of binding

albumin

0.2 (spe-

Nuclear,

may

Distribution

with MCP-3

recombinant

N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic

incubation

or

51,

CKR2B,

membrane [27]. The net charge of the N-terminal extracellular segment of CC CKR5 is -1. The corresponding do-

lines

RANTES,

a high

palmitoylation,

Samson Transfected

75,

CC

was

previously

[21].

Ligand

optimal

seven-transmembrane

site

could

blood

has

that

sequence Peripheral

for

70,

CKR2A,

respectively.

they are in rhodopsin and It also contains cysteine other

is 57,

CC

seven-transmembrane-domain tail

residues

sequence

CKR1,

CC CKR4,

other

segment

RNA analysis

The

CC

and

Like

A 1.4-kb DNA

codons.

to

CKR3, gaps

(final prepared

352

identical

105

library

vector II

and 10% DNA, and

described

stably

bp

Pfu DNA

with

(Invitroen,

Eagle’s medium 20 jig of plasmid

and

from

then

63-2.

pBluescript

pREP9

(HEK)

a novel

conditions

clones,

from

tains

had

on both strands. The cDNA insert Barn HI and Hind III sites of the

the

vector

was

of clone

excised

site

only

blood

isolated

previously

63-2,

cDNA

blunt-ended

completely

kidney

293

been

of the

between

modified with

stringency

a ?.pCEV9

was

RV

the

embryonic

Dulbecco’s electroporated

One

cDNA

expression

cDNA

low peripheral

digestion,

subcloned

63-2

the sequence

Eco CA), and sequenced into

then

have

23].

extended

Xl double

Bst

mammalian

man

[20,

and

it extended

mm)

human

previously

8.5,

but

under

55#{176}C for 30

described

clone

The

screen

been

designated

to CC CKR2B,

probe

5x SSPE,

have

obtained,

related

hybridization wash:

leukemia

cDNAs

the

con-

MIP-i, were

son et al. RANTES,but allelic ured

study and

incorrectly

have

variant

not

Introduction,

Figures

RANTES

but

attributed

also reported that MCP-i or MCP-3

of CC

CKR5

3 and

showing calcium HEK 293 cells

when

not to

4 of our

flux responses stimulated with

MCP-i,

CC CKR3

MCP-2,

[23].

or Sam-

MIP-la, MIP-i3, and were agonists for their pH

changes

were

meas-

as the functional response in stably transfected CHOKi cells. We have extended these studies by showing that treatment of CC CKR5 transfectants with pertussis toxin completely abolished the calcium flux response to MIP-la, MIP-i, and RANTES (Fig. 3). In contrast, the calcium

I 20

cc

CKR2B

cc

CKR5

cc

ci

I

I

I II

II

.

1111 11111

II

I

II

I

III 120

.yax Ill I rn

:xr I I

I

r

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Ill

I I

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Ill I I

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I

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?VI

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II

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,

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1111 sXv

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Alignment

.

CKR5

sequence

boxes,

predicted

accession

flux The

potency

and the

I III

ES

three

variability

same

system.

tested

variant

MIP-la binding shown).

MIP-i, CC CKR5

II

I

V 22

C

RI

fl(V]

I

Ill

III

TLXXVX1

C

RI

V1IV

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C

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P’IERTCBLEPPIZBLU

from

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*I

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inserted

IRYRX

are noted.

to optimize

CPV7YRZTVDQVI

the

I

and

I

XAXRYC

RQL

CC CKR2B,

I-Vu

340

tXTXRYC

I IlIl II III IIIIIIII

C VNPVXTJ

for CC CKR1, segments

gaps

BVY

I II II III I I II II

TRVXATI

cDNAs

320

YRR

C XRPXXTJ

Ill

DLM

deduced

probably

acts

was

by

largely

by Samson

differs using

et al.,

RANTES responses

>

study

agonists

and

binding

to

unaffected. MIP-la

for the

MIP-i.

>

I

alignment.

II I I I

ILYYL8VVRLIRV

Arabic

?$P-I?0rW41

numerals has

r

correspond

between

sequence

I

ZXS

II I

identities

The

]IVSJ

I 1I1TTJ

I

CC CKR5. lines,

I

CBXVQQIRPflM

IRRVAVRLV

Vertical

I

TNTI

adjacent

been

to the

CC

residues;

deposited

open

in GenBank,

of Samson

between

from

experiment

CHO-Kl

did not bind

or CC CKR5 to CC CKR1,

HEK

293

transfectants

two

studies

transfectants

when

binding

CC CKR1 specifically 22], but not

M

in

using

28S

0...’

18S

....i

the

I

4#{176}C[24].

expressing

However, specific binding and RANTES was more

to either

NME

to CC CKR5

at

specifically

as expected

‘25I-MCP-3 bound previous reports [21,

study than

and could result in the materials

activity, we observed low levels binding at 4#{176}C, whereas much were found for cells expressing

out at 37#{176}C (Fig. specifically to CKR2B-transfected

transfectants; confirming

‘251-MCP-3,

the dose-

to experiment

cells

and

were very simithe differences

the

are quite small of differences

125IMCP..i

‘25I-MCP-i

was

In our potent

et al.,

bound both not shown).

The

to MIP-ia

studies. less

latter ( data

>

slightly from that reported a calcium flux assay as

in both threefold

in the

transfected

CC CKR5

I I

I?XINXZ

300 C

TITZ

II I

dashes,

reported

of CC chemokines

our

Q

IllIllIl IIIIIIIII

In the description of their CC CKR5 variant, Samson were unable to show specific binding of ‘25I-MIP-ia stably

I

I

20’

)avYvcopTYpp.G----

used, including the CC CKR5 variant tested, of expression achieved, differences between and HEK 293 cells, the source of chemokines,

and

Binding

DQAI

glycosylation;

which

potency and efficacy any one of a number

CHO-Ki

1111

i (.TTBEcIQW

curves for RANTES and MIP-i 24]. It should be appreciated that

methods levels

I

membrane-spanning

receptors,

order

whereas,

the

250 TZTLII

DQM

Putative

iO nM, respectively, was approximately

response lar [23,

VGLBZ8TS(

i

sequence

response: MIP-ia for half-maximal

RANTES,

both from

acid

for N-linked

MIP-l,

CKR5

‘-3 and MIP-1

among

amino

to ATP,

functional concentration

Ill raN’!

t?J

purinergic =

I III

U57840.

response

RANTES our CC

II Evi

are left-justified.

sites

endogenous

I

I

ofthe and

number

cJrl

II 1111

Fig.

I 111111

II

VII 260

Ev1

1111 I

IllIlI

II I I

11111

111111

150 2

VI

r

I Ill

LVIr1

t.k

Ill EDI

240

lEPkVL

II

160

t’V’l

II

FY80

I

IIIIII

llllllllll

-

140 L..1

rm

ILS

Ill

so

60

Iv

100 Ii

II

40

When

high

levels

were

Probe

: cDNA

oligo

we

of

of specific 125I higher levels of CC CKR1 (not

of 125I-labeled readily detected assays

et al. to

MIP-ia, on our carried

4). The same radioligands did not bind untransfected HEK 293 cells or CC HEK 293 cells at 37#{176}C,whereas the

2.

Fig.

CC

CKR5

mRNA

distribution

in

blot containing 10 tg total RNA neutrophils (N), adherent monocytes

Nytran

human hybridized

with

the

clone

at 65#{176}C in 0.2x

washed

an intensifying

screen

SSPE

with

A separate

lane containing

a 30-mer

was

washed

IL8RB antisense

in

and

for CC

(M),

1 h, then

CKR3

and

exposed Lanes

eosinophils

ei al.

Monocyte

The

CC

chemokine

(E)

was

blot

was

(not

with

positively shown).

RNA was hybridized

specific

poaition

A

film

E were

respectively

5x SSPE at 50#{176}C for 15 mm. The result film.

The

to XAR-2 N and

monocyte probe

subsets. blood-derived

CKR5.

CC

probes,

oligonucleotide

leukocyte

peripheral

encoding

10 tg adherent

weeka of exposure to XAR-2 indicated at the left.

Cornbadiere

cDNA

at -80#{176}Cfor 2 days.

controlled with

8.5

human from

for

CC CKRS

shown

of ribogomal

receptor

and

is after

2

bands

ia

149

MIP-la

RANTES

MIP.1

binding of chemokine agonists to CC CKR5 is due to structural differences from iodination or differences in G pro-

AlP

5) U C

teins 5) 0

is

between

and both

monocytes

important

to

that

note

HEK 293 cells. However, the calcium flux assay

it and

-P

the

U. 0 +PTX

-

_____

5) 0

0

A

A

100

2000

100

100

2000

C protein

relative

fluorescence

CC CKR5, Each million

stimulated

agonist and

indicated

bottom

(-PTX)

and

tested

at 100

at

results the

above

rows

(+PTX),

measured

stably

the

containing

arrow

column to cells

with

toxin or vehicle.

cuvette by

as the

transfected

with pertussis

corresponding

tested

was

was

cells

correspond

toxin

nM; ATP

293

indicated

the

of tracings

pertussis

[Ca2},

of a separate

time

same

applied

with

and

CKR5-trans-

specificity,

RANTES,

negative

CC

are

positive

for

other

tested.

they

are

least

related

CKR5 receptors,

in function,

the most struchaving 75% aa having

no corn-

mon agonists. CKR2B and

In contrast, CC CKR4,

MCP-i is an agonist for both CC and MCP-3 is an agonist for CC

CKR2B

CC

even

and

CKR1,

though

these

pairs

of recep-

CC

the

oftracings.

treated

respectively.

2

to CC

chemokine

Although CC CKR2B and turally similar CC chemokine

CKR5.

and treated

the

assays

the

MIP-i,

chemokines

200

(sec)

by HEK

with Fura-2,

represents

cells

CKR5

to CC

emitted

loaded

tracing

top

coupling

gave

MIP-ia,

identity, 3.

Fig.

binding

cells

for

A

100

2000

Time

radioligand

fected

The

with

vehicle

Chemokines

were

A

125

I2S4jp.

-.--

at 5 j.tM. 100

CCCKRI

s*i\cKcKRs

75

to the CC CKR5 transfectants (data not shown). These results are consistent with the agonist specificity of the two receptors. The binding of ‘25I-MIP-ia and ‘25I-MIP-i to

CC CKR1be easily

and CC distinguished

CKR5-transfected in two ways.

so 25

cells at 37#{176}C could First, binding of both

0

#{149} o

(MIP-la]

‘25I-MIP-la

and

‘25I-MIP-i

peted effectively at 500-fold molar

to CC

CKR1

could

by unlabeled MCP-3 (>75% excess), whereas MCP-3 did

be com-

C

CKR1

tory concentrations and unlabeled more effectively

than

to CC

CKR5

(half-maximal

substantially

lower

affinity

to CC

CKR5

than

75

..T.I1.... I so 25

0

)

1

10

[MIP-1J

C

125

Fig. with

to CC

CKRS

0

inhibi-

and 200 nM, respectively; suggest that MIP-ia binds

CCCKRI

cc

-.---

100

-5 and iOO nM, respectively), MIP-i competed approximately twofold for ‘25I-MIP-i binding to CC CKR5 than (ICsos iOO The results

‘25I-MIP-.--

C

[ICso]

to CC CKR1 4, A and B).

B

125

#{149}0

binding of either chemokine to CC CKR5 when tested at 500-fold molar excess. This pattern is consistent with the agonists for CC CKR1 and CC CKR5. Second, MIP-ia competed -20-fold more effectively for ‘251-MIP-ia bindto CC

woo

(nM)

at

competition not compete

for

ing

I

0

100

1000

(nM)

‘251-RANTES

100

-0--

CC OCRI

-0--

CC

0(55

CKR1, 75

whereas

MIP-i

receptors.

In this

similar

MIP-1 CKR1

binds

potency

as

it is interesting for

both

effective

could

and

demonstrating

CC CKR5

using

excess

22], whereas binding. The

CC CKR5

The high

ICo

values

transduce be needed

Journal

also bind transfectants the specificity

unlabeled

unlabeled and CC

MIP-la

receptors,

both

whereas

agonist

MIP-ia CKR5,

signals is very efficient. to determine whether

Leukocyte

25

for CC

as

have

others

[16,

for ‘251-RANTES at CC CKRi and

by heterologous

compe-

(IC,os -2O respectively;

and Fig.

100 4C).

values for binding compared with the agonist for CC CKR5 suggest that its ability to

of

so

has

specifically to both CC (Fig. 4C). We had difof ‘251-RANTES bind-

RANTES,

be distinguished

tition with excess nM for CC CKR1

to

00

MIP-ia clearly competed ‘251-RANTES binding sites

could

affinity

that

and

ficulty

150

low

an agonist

CKR1

ECso

regard,

similar

is a much less potent than for CC CKR5.

‘25IRANTES

ing

with

Biology

Additional the apparent

Volume

60,

studies will low-affinity

July

1996

(MIP-la]

Fig.

4.

Radioligand

293

cells

stably

0.2

nM

box.

of the

(A-C)

binding

ofunlabeled

binding

is defined absence

of the

unlabeled

CC

CKR1

follows:

6%; for

CC

and (C).

CKR5,

C) or MIP-1 between and

indicated

on the

respectively,

(A)

and

(B),

and

in the

Maximal

presence

abscissa

of each specific

Data

counts of 1000

nM

panel.

For

of each

binding

‘25I-MIP-1, from

top

of increasing

cell-associated

nonspecific

31 ± 6%;

HEK

at 37#{176}C with

at the (B).

total

chemokine

CC CKR5.

as a function

65 ± 2 and 58 ± 2%. for

and incubated

indicated

plotted

(A and

3 ± 2 and

‘251-RANTES, experiments

were

cpm)

difference

chemokine

CKR1

CKRs

binding

MIP-lu

of unlabeled

and

ofCC

CC

(200,000 specific

as the

‘251-MIP-la,

separate

with

radioligands

amounts in the

properties

transfected

% maximal

(nM)

was

37 ± 5 and are

pooled

2 separate

as

22 ±

from

experiments

3-5

tors have only [16-19, 21, 22, ships

have

47 30].

and 57% Surprising

aa identity, structure-function

Exp.

7.

two human interleukin-8 (IL-8) receptors identical in amino acid sequence

previously

been

identified

for

the

CXC chemokine receptors, A and B, which are 78% [31, 32]. IL8RB is selective for IL-8 and at least two other related CXC chemokines, whereas IL8RA is monoselective for IL-8. The N-terminal segment, and the second and third extracellular determinants

loops of IL8RB, are all when tested separately

chimeric may also for

receptors be useful

[33]. Analysis in identifying

CC CKR2B and CC CKR5. CC CKR5 is the first receptor

is a potent

agonist.

RANTES

are

which

are

also

In addition

also

agonists

8.

9.

10.

of chimeric receptors selectivity determinants

CC

CKR1

in monocytes

and

CC

[16,

11.

CKR4,

17,

19,

12.

20].

monocyte-directed actions of MIP-ia and RANTES. Monocytes are long-lived cells capable of further differentiation as they move from the blood to establish residence in the tissues as macrophages. The functional properties oftissue macrophages can differ in different organs, and in the same organ depending on the presence of priming agents. In this regard, it will be imporbution

of each

of these

tant to determine tially expressed macrophages

CKRs will

to the

receptors

whether monocyte on subsets of from

different

CC CKRs monocytes

organs,

and

are

or

whether

14.

15.

shown

other

17.

to act as suppressors of replication for certain strains making CC CKR5 the best-known candidate to this activity [36].

HIV-i,

mediate

18.

19.

20.

added

strains

of

Kennedy,

inproofi CC CKR5 is a fusion HIV-1 (Alkhatib, G., Combadiere, P. E.,

Murphy,

P.

M.,

and

cofactor

Berger,

C.,

for macrophage-tropic Broder,

C. C.,

E. A. (1996)

Feng,

Y.,

Science.

In

press.

22.

REFERENCES 23. 1.

2.

3.

4. 5.

6.

Baggiolini, M., Dewald, B., Moser, B. (1994) Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines. Adv. Immunol. 55, 97-179. Wang, J. M., Sherry, B., Fivash, M. J., Kelvin, D. J., Oppenheim, J. J. (1993) Human recombinant macrophage inflammatory protein-la and 3, and monocyte chemotactic and activating factor utilize common and unique receptors on human monocytes. J. Immunol. 150, 3022-3029. Schall, 1. J., Bacon, K., by, K. J., Goeddel, D. V. (1990) Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347, 669-671. Miller, M. D., Krangel, M. S. (1992)The human cytokinel-309 is a monocyte chemoattractant. Proc. Nail. Aced. Sci. USA 89, 950-953. Yoshimura, 1., Robinson, E. A., Tanaka, S., Appella, E., Leonard, E. J. (1989) Purification and amino acid analysis of two human monocyte chemoattractants mononuclear

produced leukocytes.

Van Damme, and functional

J., Proost, P., Lenaerts, J. P., Opdenakker, identification of two human, tumor-derived

J.

by phytohemagglutinin-stimulated Immunol. 142, 1956-1962.

human

24.

25.

binding

and

functional

properties

provide

a

by homologous

chemotactic

cytokines.

J.

Exp.

145,292-297.

Neote, K., DiGregorio, D., Mak, J. Y., Honuk, R., Schall, T. j. (1993) Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72, 415-425. Gao, J.-L, Kuhns, D. B., Tiffany, H. L, McDermott, D., Li, X., Francke, U., Murphy, P. M. (1993) Structure and functional expression of the human macrophage inflammatory protein-la/RANTES receptor. J. Exp. Med. 177, 1421-1427. Charo, 1. F., Myers, S. J., Herman, A., Franci, C., Connolly, A. J., Coughlin, S. R. (1994) Molecular cloning and functional expression of two monocyte chemoattractant boxyl-terminal

21.

Note

receptor

Van Riper, G., Siciliano, S., Fischer, P. A., Meurer, R., Springer, M. S., Rosen, H. (1993) Characterization and species distribution of high affinity GTP-coupled receptors for human RANTES and monocyte chemoattractant protein 1. J. Exp. Med. 177, 851-856. Yoshimura, 1., Leonard, E. J. (1990) Identification ofhigh affinity receptors for human monocyte chemoattractant protein-i on human monocytes. J. Immured.

exist that are expressed in these cells. Additional work also be needed to address the role of each of the CC

in other biological phenomena such as lymphocyte chemotaxis and hematopoiesis [34, 35] that are regulated by their ligands. Finally, it is important to note that the ligands for CC CKR5 are the same three molecules that have been

Expression,

mechanism for the selective recruitment of eosinophils. J. Clin. Invest. 97, 604-612. Kitaura, M., Nakajima, T., Imai, T., Harada, S., Combadiere, C., Tiffany, H. L, Murphy, P. M., Yoshie, 0. (1996) Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification ofa specific eosinophil eotaxin receptor, CC chemokine receptor 3. J. Biol. Chem. 271, 7725-7730. Wang, J. M., McVicar, D. W., Oppenheim, J. j., Kelvin, D. J. (1993) Identification of RANTES receptors on human monocytic cells: competition for binding and desensitization Med. 177,699-705.

16.

CKRs

of

13.

differenin tissue CC

jose, P. J., Griffiths-Johnson, D. A., Collins, P. D., Walsh, D. T., Moqbel, R., Totty, N. F., Truong, 0., Hsuan, J. J., Williams, T. J. (1994) Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J. Exp. Med. 179, 881-887. Ponath, P. D., Qin, S., Ringler, 1., Clark-Lewis, I., Wang, J., Kassam, N., Smith, H., Shi, X., Gonzalo, J.-A., Newman, W., Gutierrez-Ramos, J.-C., Mackay, C. R. (1996) Cloning of the human eosinophil chemoattractant, eotaxin.

blocking reagents specific for mdiwill be needed to define the contri-

Additional studies using vidual receptor subtypes

proteins

Med. 176,

64-68.

dominant selectivity in IL8RA-IL8RB

identified forwhich MIP-i to CC CKR5, MIP-ia and

for

expressed

(MCP-2 and MCP-3) belonging to the chemokine family. J. 59-65. Dahinden, C. A., Geiser, T., Brunner, T., von Tschamer, V., Caput, D., Ferrara, P., Minty, A., Baggiolini, M. (1994) Monocyte chemotactic protein 3 is a most effective basophiland eosinophil-activating chemokine. J. Exp. Med. 179,751-756. I.Aetscher, P., Seitz, M., Clark-Lewis, I., Baggiolini, M., Moser, B. (1994) Monocyte chemotacti proteins MCP-1, MCP-2, and MCP-3 are majorattractants for human CD4 and CD8 T lymphocytes. FI4SEBJ. 8, 1055-1060. Uguccioni, M., D’Apuzzo, M., Loestcher, M., Dewald, B., Baggiolini, M. (1995) Actions ofthe chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-la, and MIP-13 on human monocytes. Eur. J. Immunol. 25, tactic

respectively relation-

protein 1 receptors reveals tails. Proc. Nail. Acad. Sci.

alternative splicing USA 91,2752-2756.

of the

car-

Power, C. A., Meyer, A., Nemeth, K., Bacon, K. B., Hoogewcrf. A. J., Proudfoot, A. E. E., Wells, T. N. C. (1995) Molecular cloning and functional expression ofa novel CC chemokine receptorcDNA from a human basophilic cell line. J. Biol. Chem. 270, 19495-19500. Combadiere, C., Ahuja, S. K., Murphy, P. M. (1995) Cloning, chromosomal localization and RNA expression ofa novel human chemokine receptor-like gene. DNA Cell Biol. 14, 673-680. Combadiere, C., Ahuja, S. K., Van Damme, J., Tiffany, H. L. Gao, J.-L, Murphy P. M. (1995) Monocyte chemoattractant protein-3 is a functional ligand for CC chemokine receptors 1 and 2B. J. Biol. C/tern. 270, 29671-29675. Ben-Baruch, A., Xu, L., Young, P. R., Bengali, K., Oppenheim, J. J., Wang J.-M. (1995) Monocyte chemotactic protein-3 (MCP-3) interacts with multiplc leukocyte receptors. C-C CKR1, a receptor for macrophage inflammatory protein-i alpha/Rantes, is also a functional receptor for MCP-3. J. fbi. Chem. 270, 22123-22i28. Combadiere, C., Ahuja, S. K., Murphy, P. M. (1995) Cloning and functional expression of a human eosinophil CC chemokine receptor. J. fbi. C/tern. 270, 16491-16494 [Correction: J. Biol. C/tern. 270, 302351. Samson, M., [abbe, 0., Mollereau, C., Vassart, G., Parmentier, M. (19%) Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry 35, 3362-3367. Kozak, M. (1987) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 15, 8125-8148.

26. 27.

28.

blood

29.

C. (1992) Stroctural monocyte chemo-

30.

Lefkowitz, R. J. (1993) G protein-coupled receptor kinases. Cell 74, 409-411. Bouvier, M., Moffett, S., Loisel, T. P., Mouillac, B., Hebert, T., Chidiac, P. (1995) Palmitoylation of G-protein-coupled receptors: a dynamic modification with functional consequences. Biochern. Soc. Trans. 23, 116-120. Murphy, P. M. (1994) The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Jrnrnunol. 12, 593-633. Wu, D., LaRosa, G. J., Simon, M. 1. (1993) G protein-coupled signal transduction pathways for interleukin-8. Science 261, 101-103. Franci, C., Wong, L M., Van Damme, J., Proost, P., Charo, 1. F. (1995) Monocyte chemoattractant protein-3, but not monocyte chemoattractant pro-

Combadiere

et al. Monocyte

CC

chemokine

receptor

151

tein-2,

31.

32. 33.

of the human monocyte chemoattractant pro154, 65i 1-6517. Kuang, W.-J., Rice, G. C., Wood, W. 1. (1991) expression of a human interleukin-8 receptor.

ligand

L. (1991) Cloning of complementary DNA encoding a functional interleukin-8 receptor. Science 253, 1280-1283. Ahuja, S. K., Lee, J., Murphy, P. M. (1996) CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on mutiple domains of human interleukin-8 receptor B. Determinants of high affinity binding and receptor activation are distinct. J.

Biol.

152

is a functional

tein-1 receptor. J. Immunol. Holmes, W. E., Lee, J., Structure and functional Science 253, 1278-1280. Murphy, P. M., Tiffany,

C/tern.

Journal

Leukocyte

Taub, D. D., Conlon, Preferential migration MIP-la and MIP-1.

35.

Dunlop,

Biology

Volume

60,

July

1996

K., Lloyd, A. R., Openheim, JJ., Kelvin, D. J. (1993) of activated CD4 and CD8 T cells in response to Science 260, 355-358.

D. J., Wright, E. G., Lorimore, S., Graham, G. J., Holyoake, 1., Kerr, S. D., Pragnell, 1. B. (1992) Demonstration of stem cell

D. J., Wolpe,

H.

271,225-232.

of

34.

inhibition

and

myeloprotective

effects

of SCI/rhMIPla

in vivo.

Blood

79,

2221-2225. 36.

Cocchi, F., DeVico, A. L, Garzino-Demo, P. (1995) Identification of RANTES, H1V-suppressive factors produced 1811-1815.

A., Arya,

S. K., Gallo,

MIP-la,and

by

CD8

MIP-i

T

cells.

R. C., Lusso, as the Science

major

270,