Selectivity and antagonism of chemokine receptors - CiteSeerX

Selectivity

and antagonism

of chemokine

receptors

Timothy N.C. Wells,* Christine A. Power, Manjula Lusti-Narasimhan, Arlene J. Hoogewerf, Robert M. Cooke,t Chun-wa Chung,t Manuel C. Peitsch, and Amanda E. I. Proudfoot *Glaxo

Instiuuefor

Stevenage,

Abstract:

Molecular

United

The

Geneva,

Biology,

Switzerland

and

G1axo

Weilcome

Medicines

Research

Centre,

Kingdom

chemokine

superfamily

can

be

INTRODUCTION

sub-

divided

into two groups based on their amino terminat cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human cloned. The CC chemokines chronic

inflammation,

a fourth

leukocyte

recently

receptor

Understanding range of agonists potent vestigate

and

what makes is important

by looking

to the at the

CXC role

ligand,

and

at why

one

CC

solved

have been involved in have

receptor if we are

bind its develop

We have started of this receptor

to inselec-

ways.

do

not

bind

First, we looked structure of the

three-dimensional

struc-

for the protein-1, interface

flammatory ferent dimer interleukin-8

(IL-8).

of

tures

all

the

CC

chemokines

the are

monomer

very

and

in

bind

CC-chemokine

receptor-i

(CC-CKR-1)

troduces monocyte chemoattractant other mutations in this region, we interaction we have flue

with the found that

of HANTES

N-terminus modification

by addition

it into

an antagonist

together, receptor

this

with

data activation

activity. can show

in-

Using a direct

of one

amino

acid

a two-site selectivity

have

for CC

Biol.

distress

gic

This in

men.sional

structures

interleukin-8

the

CC

chemokine

and

characterization

areas

the

for

which such

arthritis,

of the

work

taken

to

receptors;

carried

and

out

clone

which

novel leads

between

finally,

of antagonists

and

structural

chemokines,

of specificity

there as aller-

three-dimensional of

control

clinically from acute respiratory

diseases

approaches

mutagenesis

of

and

re-

Chemok-

range acute

dermatitis,

three the

receptors; into

fatal

atopic

reviews

and

CXC

the

to

and

identification

to CC

chemokine

re-

ceptors.

i996.

Abbreviations: netic

resonance;

acids;

IL-B,

A

and

melanoma tractant tory

resonance

spectroscopy

-

three-di-

CC-CKR,

CC-chemokine

ENA-78,

epithelial

interleukin-8;

-B;

NAP-2, growth

IL-8RA

and

-1 and

-3;

requests: chemin

PCR,

normal

N.C.

Aulx,

interleukin-8 and

MIP-1,

polymerase

T cell

Timothy des

MCP-1 and

Wells,

receptors-

-3, monocyte

chain and Glaxo

mag-

78 amino

MGSA/Gro-a,

macrophage

expressed

1228

nuclear

factor,

polypeptide-2;

MIP-la

-1;

on actvation

NMR,

activating

IL-8RB,

stimulatingactivity;

proteins

14

receptor,

neutropil

neutrophil-activating

polypeptide-laand Reprint

magnetic

physiological

a variety

is often

psoriasis,

chemokine

in

As

are highly receptors at

range.

to chronic

laboratories:

insights

their

diseases. These diseases such as which

article

our

exert

implicated

treatment,

asthma,

Biology,

nuclear

been

syndrome,

regulated

Words:

and

on neutrophils.

chemokines bind their

subnanomolar-concentration

important inflammatory neutrophil-mediated

activation

types in inflaminvolved in the

are less important in the such as monocytes. CC profile in terms of cellular

or no effect

concentrations, in the

selective

Taken

model between

Leukoc.

Key

in the

variety of cell are principally

little

into

spacing of cysteine and on overall se-

makes

potency.

chemokines, with an initial receptor contact by the main body of the chemokine, and provided by the amino terminal region. J. 53-60;

a role

and

have

on the region,

molecular divided

.

and CXC provided activation

59:

play

of a large chemokines

are

ofCC-CKR1 Third, of the amino termi-

nanomolar

suggests and for

and

They

selectivity

studies

tion

based terminal

with

They

well as this selectivity of response, potent molecules, the majority

tations in IL-8,

kDa.

of neutrophils, and of other leukocytes, have the opposite

atheroma.

region, Leu-25 at this posimutant IL-8 to

of proteins

10

activation activation chemokines

physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Muof one of the residues in this to tyrosine (which is conserved CC chemokines) enables the

family

8 and

identity.

and recruitment mation. CXC

is no effective

at

a large

between

CC chemokines in the amino

residues

ines

struc-

similar,

and

CXC

sponse

chemokine macrophage inand it has a completely difto that of the CXC chemokine

However,

of

nanomolar

hire of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined

are

masses

quence

of ligands.

group

chemokines

the

cloned

to

receptors in several of the three-dimensional have

we

this

for

selective antagonists. the molecular basis

tivity

receptors tend to be

Chemokines

chemoatinflamma-

reaction;

RANTES,

secreted.

Institute

Plan-les-Ouates,

for

Molecular

Geneva,

Switzer-

land. Received

August

Journal

of

19,

Leukocyte

1995;

accepted

Biology

September

Volume

29,

59,

1995.

January

1996

53

CHEMOKINE RECEPTORS AS THE TARGETS OF ANTI-INFLAMMATORY Chemokines were ability to recruit inflammatory

Clinically

to be present at elevated diseases. Based on this hypothesis that interfering

specificities, adds to the role in disease, and thus least

27

human

chemokine-like

sequences

that more than for the recruitment

one

key

may

However, complexity

complexity identifying To

chemokines:

cloned genes, sequence

chemokine

have

been

there

are

me

the in

some

from

purified

1).

chemokine is going of any individual cell vary

from

disease

antagonism result

of

of inflam-

were

cloned,

These

were

as many

In 1991, and

two

shown

called

the

of the

chemok-

receptors

for CXC

to be present

interleukin-8

on neureceptor-A

own,

receptors,

have

used

egy, designing gions

to be responsible type, and that the to disease. degree of Our ulti-

of groups,’ chain

This initially produced for which ligands still

our

(PCR)

strat-

in studying

a large number of orphan have to be identified [2].

The

difficulty

due

to the

and The

the availability of labeled chemokines CC-CKR-1 originally isolated from

cells

including

reaction

degenerate primers based on conserved rein chemokine and chemotactic factor

found

receptors. receptors

likely

a variety

a polymerase

lack

these

receptors

of reproducibility

[2, 3] has

been

shown

has

in ligand

been

largely

binding for

HL6O

to be activated

assays analysis. or U937

by macrophage

Chemokines

IL- 8

SAKELRCQCIICTYSKPFHPKFIKELRVIESGPHCANTEI

T3 976 5 NAP - 2 ENA-

IVKLSD

TLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEI AELR#{216}4CIKTFSG . IHPKNIQSLEVIGKGTHCNQVEVIATLKD

78

. SIVCVDPQAEWIQR*IEVLRKRSSSTLPV . GRKICLDPDAPRIKJCIVQKKLAGDESAD

. VHPKMISNLQVFAIGPQCSKVEVVASLKN

. GKEICLDPEAPFLKKVIQICILDGGNKEN

ASVATELRCQCLQTLQG

. IHPKNIQSVNVKSPGPNCAQTEVIATLKN

. GRKACLNPASPIVXKI

GRO-b

APLATELRCQCLQTLQG ASVVTELRCQCLQTLQG

. IHLKNIQSVKVKSPGPHCAQTEVIATLKN

. GQXACLNPASIk4VXXI

Gro-

AGPAAAVLRELRCVCLQTTQG

. GRELCLDPKENWVQRWEKFLKRAENS

IVWKKNK

GRO- a c

I P- 1 0 GCP NAP-

a particular

in a reduction

to characterize

as possible.

chemokine

proteins,

point of view, this not be a problem.

that

would

(IL-8R-A), which is specific for IL-8; and IL-8R-B which binds IL-8 and other CXC chemokines such as neutrophilactivating polypeptide-2 (NAP-2), epithelial neutrophil activating factor, 78 amino acids (ENA-78), and melanoma growth stimulating activity (MGSA/Gro-a) [1]. To find CC

of at

It is therefore

show

important

receptors

trophils.

and more recently a large number tag sequences that correspond to (Fig.

to

The molecular basis of selectivity is far more likely at the level of the various receptor types. It was

chemokines

their are

reports

is

receptor

therefore

shown

of understanding which chemokines

date

from a therapeutic and pleiotropy may

Cxc

mation. to lie

in a reduction in the level of the sheer number of chemokiso far, and their overlapping

targets.

some from of expressed

they

goal

chemokine

levels in a variety of inflammatory type of data, we can suggest with the chemokine cascade

diseased tissues will result inflammation. In this light, nes that have been discovered

therapeutic

mate

initially discovered on the basis of their and activate a variety of cell types in

conditions.

good

POTENTIAL THERAPY

-

2 4

SDF-

. IHLKNIQSVNVRSPGPHCAQTEVIATLKN

VPLSRTVRCTCISISNQPVNPRSLEKLEI GPVSAVLTELRCTCLRVTLR EAELQDLQV

1

PF4

. GKXACLNPASPMVQKI

IPASQPCPRVEI

IAThKXXGEKRCLNPESKAIKNLLKAVSXEMSKRSP

. VNPKTIGKLQVFPAGPQCSKVEVVASLKN

. GKQVCLDPEAPFLKXVIQKILDSGNK

. . . KTVXQVSPVHITSLEVDKAGR

. TPNCALQIVARLXNNN

GKPVSLSYRCPCRFFESH

. VAR.ANVKHLKILN

EAEEDGDLQCLCVKTTSQ

. VRPRHITSLEVIKAGPHCPTAQLIATLXN

MIG

IEXMLNSDKSN IEXMLKNGKSN IEKILNXGSTN

TPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEI

IATLXN

sssss

ssssssss

sssss

ssssssss

. RQVCIDPKLKWIQEYLEKALNK

IK.XLLBS

. GRKICLDLQAPLYIUCI

. GVQTCLNPDSADVXELIKICWEKQVSQ

sssssss

HHHHHHIi14HHflffi

CC Chemokines RANTES

SPYSSDT

I 3 09 MIP - la

SKSMQVPFSRC ASLAADTPTAC

R83 915

. TPC

SFHFAADC

. RQVCANPBKXWVREYINSLEMS

. CFSFAEQEIPLRAILCYRNTSSI

. . CSNEGLI

. KEACALDTVGWVQRHRKMLRNCPSXRK

. CFSYTSRQI

. . CSKPGVI

PQNFIADYFETSSQ

TESSSRGPYHPSEC

. CPTY’I’TYKIPRQRIMDYYETNSQ

T58 84 7 MIPlb

QPKVPEVGEHPSTC APMGSDPPTAC

. CLXYYEKVLPRRLWGYRXALN

CSKPGVI

.

. . . CHLPAI

-

1

QPDAINAPVTC

. CYNFTNRKISVQRLASYRRITSSK.

MCP

-

2

QPDSVSI

MCP-

3

T642

62

. RQICADPNKKWVQICYISDLKLNA

RQVCAXPSGPGVQDQ4X1LKPYSI

. MSVCTNPSDKWVQDYIXIt4KEN

I FVTKRGTEEVCTNPNDDWQPTPACLPGNFVHG . KQVCADPSESWVQEYVYDLELN

. . CSQPAWFQTXRS

CLGYTDRILHPKFIVGFTRQLANEGCDINAI

MCP

. RQVCADPSEEWVQKYVSDLELSA

FLTKXG

. . CSKPGIVFITKRG

. CFSYTARKLPRNFVVDYYETSSL

SEAASNFDC

FKLKRG FLTKR.S

. . CPKLGVILLTKRG

. CTSYISQSIPCSLF4KSYFETSSB

HCC1

D3 1 06 5

HHHHHHHIIHHHNHH

. . CSNPAVVFVTRKN

. CLVYTXXQIPQKFIVDYSETSPQ

QVGTNXELC

R9 17 3 3

sssssss

. CPAYIARPLPRAHIKETFYTSGK

IFHTKXK

. LSVCANPXQTWVXYIVRLLRKICVKNt4

CPKEAVIFKTIVA

. KEICADPKQKWVQDSMDHLDKQTQTPKT

. CFNVINRKIPIQRLESYTRITNIQ

. CPKEAVIFXTKRG

. KEVCADPKERWVRDSMKHLDQIFQNLKP

QPVGINTS7I’C

. CYRFINKXI

. CPREAVIPXTXLD

. KEICADPTQKWVQDF4KHLDKKTQTPKL

QPDALNVPSTC

. CFTFSSKKISLQRLKSYVITTSR

PITC

PKQRLESYRRTFSSH

. . CPQKAVIFRTKLGQGDLCADPKEKWVQNYMXHLGAGKPT

C Chemokine LTN

Fig.

1

group

II- sheet a search

54

.

GVEVSDKRT

Multiple

sequence

corresponding and ofthe

Journal

alignment

to Leu-25

H representing NCBI

of

database

Leukocyte

in

of human

IL-8

a-helix.

Included

in September

Biology

is also

. CVSLTTQP.LPVSRIKTYTITEG

CXC,

CC,

shown in the

and

in bold. alignment

C chemokines. The are

1995.

Volume

59,

January

1996

secondary several

. .

The

. AVI

SLR

conserved

structure sequences

.

FITKRGLK

. VCADPQATWVRDVVRSMDRKSNTRNNMIQT

cysteines of the

from

are

shown

chemokines

expressed

sequence

The

iii bold.

is indicated, tag

...

conserved

hydrophobic

S representing

databases,

which

residues were

found

in a from

inflammatory

polypeptide-la

(MIP-lu)

though

is no

binding

there

published

and

RANTES

data

for

(al-

tivity

nd more recently to bind monocyte chemoattractant protein-3 (MCP-3) (B. Dougherty, unpublished observations). which was cloned from Monomac6 to bind MCP-1 and MCP-3, and

CC-CKR-2, been shown to be important

in monocyte

a third

receptor,

monocyte nophils

library has been and gives calcium

extravasation

CC-CKR-3, found fluxes

from

One

specificity gross differences ligands.

The

MIP-13, and RANTES [7]. No binding data has been shown to date for the recombinant receptor. Ourwork has identified a fourth CC-chemokine receptor in

three-dimensional

try-related anti-parallel 3). We have recently of the CC chemokine [13]. The overallfold CXC

C-terminal

helices

1 over35#{243}amino

ever,

topologies

360

shows

high

levels

of expression

in the thymus

and in peripheral blood leukocytes. Using FACS sorted cell populations we could also show that mRNA for CC-CKR-4 specifically

was

expressed

in T-cells,

cytes, as well as in platelets. Human detectable CC-CKR-4 expression. tion

for 15 mm

up-regulation CC-CKR-4

with

IL-5

(10

of receptor were initially

B-cells,

basophils However,

ng/ml)

there

and

mono-

ologically supported variant

a significant

mRNA expression. The ligands for determined to be MCP-1, MIP-la,

and RANTES from measurements ofCa2+activated currents in Xenopus laevLc oocytes injected with CC-CKR-4. confirmed lines(C.

The results by binding

of the genome. cluster

me

for MIP-la experiments

A. PowerandA.J.

The two groups

to chromosome receptors cluster

quence

comparisons,

unpublished

are located

four human

CC chemokine

the Duffy

antigen

that

binding or vice

or erythrocyte

and versa.

the

binding.

In our

initial

CC chemokine

suchas

receptor,

to understand

(Fig. CC

which this on the

we therefore studied chemokine, IL-8, and a

RANTES.

has

been

used

in these beta sheet

experiments, and with two symme-

are shorter

the low level although the

for the CC chemokines.

ofboth

RANTES

and

How-

MIP-1f

[14] are

relevant

not be important, since that IL-8 is monomeric

concentrations

[16,

by experiments with of IL-8, N-Methyl-Leu-25

17].

a chemically IL-8. This

functional selectivity

there is at physi-

This

is further

synthesised is unable

to

in a variety of cell- based is more likely, therefore, to

be due to the distribution of hydrophobic monomer surface. A theoretical analysis

sequences on the of hydrophobicity

shows that the distribution of hydrophobic regions is preserved in CC or CXC chemokines, providing some explanationforthelack of receptorcross-binding [19]. We therefore examined the sequences of CC and CXC for

regions

involved

One region that stood leucine, corresponding invariably 1). This

in

out contains to leucine

replaced is at the

receptor

selectivity.

a highly 25 in IL-8,

by a tyrosine dimer interface

conserved which

in CC chemokines in IL-8, but

is its

equivalent residue in CC chemokines is far away from the interface. To investigate the role of this residue in controlling cell and receptor specificity, we have made the mutant Leu25-*Tyr in IL-8 [10]. The resultant protein shows a 100-fold reduction in binding affinity to neutrophils, and this reduction is also seen in binding studies using recombinant IL-8R-A and -B expressed in HEK293 cells. The mutant calcium the

is also less mobilization

Leu25-Tyr

active in neutrophil assays. However,

mutation

introduces

chemotaxis and most importantly, monocyte

chemotac-

tic activity into IL-8, which is not present in the wild type protein (Fig. 4A). In addition, the mutant protein will compete with [‘25I]MIP-lcx for recombinant CC-CKR-1

STRUCTURE AND FUNCTION OF CHEMOKINES: MAKING CXC CHEMOKINES BIND TO A CC-CHEMOKINE RECEPTOR One ofthe initial chemokine-receptor

of IL-8

alpha helices over the top (see Fig. solved the three- dimensional structure RANTES by use of NMR spectroscopy ofthe monomers ofCC chemokines and

dimerise, but is fully assays [18]. Receptor

almost (Fig.

chemokine

identity of human

[9]. So far, however, a signaling response

attempts

molecular basis of receptor selectivity, the difference between a typical CXC

CC

structure

structure may data showing

chemokines all

signaling using CXC The exception to this is

chemokine

binds both CC and CXC chemokines receptor has not been shown to induce ligand

regions

receptors

3p2l-22, whereas the CXC chemokon chromosome 2q34-35. From seit is clear

receptors as ligands

been cell

results).

on different

receptors cluster together in terms ofsequence 2). However, there have been no reports chemokine chemokines

chloride cRNA for

and RANTES have using transfected

Hoogewerf,

ofreceptors The

quaternary experimental

showed barely after stimulawas

of

completely different from those ofthe CXC chemokines [11, 15]. For the CC chemokines, the dimer interface is formed by the N-terminal n-sheet of the protein (3O), rather than the second beta strand (termed p1). However, this difference in

can be clearly seen Northern blot analysis

of CC-CKR-4

dimer

their

is conferred by structure of the

chemokines appears to be similar, despite of sequence identity between the two proteins,

acids; 46% identity to the CC-CKR-2 (form amino acids; and 45% identity with CC-CKR-3

with

the molecularbasis

nuclearmagnetic resonance (NMR) and X-ray methods [1 1, 12]. The protein is dimeric at

the human basophilic cell line KU-812 [8]. We have called this K5.5,orCC-CKR-4 based on the otherrecently identified sequences. This receptor shows 49% identity with CC-CKRb) over

receptors

to explain

the millimolar concentrations forms a six-stranded antiparallel

activated

to be expressed on eosiin response to MIP-la,

over 356 amino acids. The similarities from the alignment in Figure 2 [8].

of chemokine

hypothesis

is that the receptor selectivity in the three-dimensional

solved by both crystallographic

Recently,

an

interaction

[10].

this

cells, has is presumed

[4-6].

cloned

of the

ligands

RANTES),

(RANTES/MIP-la with an affinity (Fig. 4B). To study

questions that we asked when we examined interactions was what controls the selec-

this

that

receptor) is only

effect

on selectivity

the Leu25-Cys mutation. to leucine so only a small

Wells

et al.

Selectivity

and

expressed 12-fold less

Antagonism

in HEK293 than MIP-lu

further,

we have

Cysteine is very similar change in the biological

of Chemokine

Receptors

cells, itself made in size activity

55

50 IL-8R-B

MESDSFEDFW

KGEDLSNYSY

IL-8R-A

MSNITDPQMW

DFDDLN.

CC-CKR-3

MTTS

CC-CKR-1

METP

CC-CKR-2

cc

-

-

NTTE.DYD IRNTNESGEE

MNPTDI

4

...

LDTVE.TFG

MLSTSRSRF

CKR

SSTLPPFLLD

ADTTLDES

IY

AAPCEPESLE

INKYFVVIIY

FTGMPPADED

YSPMLETET

LNKYVVIIAY

TTSYYD

LLCEK.ADTRA

LMAQFVPPLY

T1’TEFDYGDA

TPCQKVNERA

FGAQLLPPLY

V’VFFFDYDYG

APCHKFDVKQ

IGAQLLPPLY

SNYYLYESIP

KPCTKEGIKA

FGELFLPPLY

. DVG




151

200

IL-8R-B

LTQKRY.LVX

PICLSIWGLS

LLLALPVLLP

RRWYSSNVS

IL-8R-A

LTQKRH.LVX

FVCLGCWGLS

)OILSLPFFL?

RQAYHPNNSS

PVCYEVLGND

LRARTVTFGV

ITSIVTWGLA

VLAALPIFIF

YETEELFEET

LCSALYPEDT

CC-CKR-3 CC-CKR-1

cc cc

PACYEDMGNN

LRARTVTFGV

ITSIIDIALA

ILASMPGLYF

SKTQWEFTHH

TCSLHFPHES

-

CKR

-

2

LKAR’IVTFGV

VTSVITWLVA

VFASVPGIIF

TKCQKEDSVY

VCGPYFP

-

CKR

-

4

LRARTLTYGV

ITSLATWSVA TMIV

VFASLPGFLF

STCYTERNHT

YCKTKYSLNS

C

...

201

250

IL-8R-B

TANWRIILLRI

LPQSFGFIVP

LLICJFCYGF

TLRTLFKA}iM

GQK.HRAMRV

IL-8R-A

TAKWRXVLRI

LPHTFGFIVP

LFVMLFCYGF

TLRTLFKAHM

GQK.HRAMRV

CC-CKR-3

VYSWRHFHTL

RMTIFCLVLP

LLVMAICYTG

IIKTLLRCPS

KKK.YKAIRL

CC-CKR-1

LREWXLFQAL

KLNLFGLVLP

LLVMIICYTG

IIKILLRRPN

EKK.SKAVRL

. RGWNNFHTI

MRNILGLVLP

LLIMVICYSG

ILKTLLRCRN

EKKRHRAVRV

T.TWXVLSSL

EINILGLVIP

LGIMLFCYSM

IIRTLQHCKN

EKK.NKAVKJI

cc

-

CKR

-

2

CC-CKR-4

I’MV




300

251 IL-8R-B

IFAVVLIFLL

CWLPYNLVLL

ADTLMRTQVI

QETCERRNHI

DRALDATIIL

IL-8R-A

IFAVVLIFLL

QETCERRNNI

GRALDATEIL

IFVIMAVFFI

CWLPYNLVLL FWTPYNVAIL

ADTLMRTQVI

CC-CKR-3

LSSYQSILFG

ND.CERSKHL

DLVMLVTIVI

CC-CKR-1

IFVIMIIFFL

FWTPYNLTIL

ISVFQDFLFT

HE.CEQSRHL

DLAVQVTIVI

CC-CKR-2

IFTIMIVYFL

FWTPYNIVIL

LNTFQEFFGL

SN.CESTSQL

DQATQVTITL

CC-CKR-4

IFAVVVLFLG

FWTPYNIVLF

LITLVELEVL

QD.CTFERYL

DYAIQATKTL

TMVI