CD6—ligand interactions: a paradigm for SRCR domain function?

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CDG-ligand interactions: a paradigm for SRCR domain function? Alejandro

Aruffo, Michael A. Bowen, Dhavalkumar D. Pate& Barton Gary C. Starling, John A. Gebe and JOrgen Bajorath

n recent years, br\.eral cDNAs been cloned encoding proteins

Icsidues, while Group B domains contain, 64th a few exceptions, eight Cys residues.

have with

The SRCR rently consists

domains that are homologous to the scavenger nxeptor cysteine-rich SRCR) domain found he type I macmphage &se

are either

cell-surface or secreted one or more SRCRMany of these pro-

of immune

rcsponse~.

In 1994,

A and 8) based on the number and pattern residutin each SRCR domain. -110 residues

long.

Group

The SRCR

do-

coded by two exons”. Analysis of the genomit organization of the genes encoding CD5 and Cl%, which are two Group B pro. km, showed that each of the three SRCR domains found in these proteins is encoded by a single exon”‘. This suggests that SRCR


/ ’ ,f cysteine I tJomain is

B proteins.

mnms of the type 1 macrophage scavenger receptor and hllman complement factor I, which are two Group A proteins, are en-

eins have bcrn implicated in the devcl,pment of the immune system and the vgulation

superfamily (SRCRSF) curof 17 members: seven Group

A and ten Group

at the C-terminus of scavenger receptor’.

and contain I 1xoteins, ~~~rologous domains.

i *

F. Haynes,

Croup A and Group B genes also differ in their genomic organization. This article pro. vides an overview of the Croup B SRCRSF On average,

A domains

an SRCR

contain CRP-ductin

six Cys

which tncludes sevcml by cells of the immune

proteins system,

that are predominantly expressed I as well as a det&d review of thr

interaction between CD6 and activated leukocyte cell adhesior , molecule (ALCAM; CDlti), which constitutes the only SRCRl&and in&action that has been studied in detail at a molecular level

The Group B SRCR subfamily of proteins Eight

members

of the Group B SRCRSF

are shown

in Fig. 1 and al

members of the Group A and Group B SRCRSF are listed in Table 1 The ten currently known members of the Group B SRCRSF are CD! (Ref. 7), CD6 (Ref. 8), WC1 (T19) (Ref. 9), Ml30 (CD163) (Ref. 101 PemaSREG I Ebnerin i ,

Ml30 /

3

Cd,,.

.I

0 C T 0”B

.,i

,,1.

E R

Spa (Ref. II), Pema-SREG (Ref. 12), Ebnerin”, CRP-ductin” hensin15 and gall-bladder mucinlh. Figure 2 shows alignment of tht amino acid sequence of the Group B SRCRSF. While activities

the functions of some SRCRSF proteins are known, hove mrely been mapped to their SRCR domain(s).

thei Thor

although SRCR domains are well conserved across species bound artes, their functions remain elusive. To date, five protein-ligam interactions have been reported for members of the Group L SRCRSE These are: the interaction between CD5 and CD72

r.l,^,i,,,r~

I,,,,

1997

,,,ii,,

&IV,“‘,“i,‘,~,,,,“l

IMMUNOLOGY

T0DP.Y

(Ref. 17), which is a typ+z II membrane protein expressed by B cells with homology to C-type lectins; the interaction between CD5 and an as-yet-uncharacterized &and expressed by activated T and B

thymocytes, mature T cells and chrome B-cell lymphocytic leukemios. In addition, CD6 has been detected in various qions of the brain, including the basal ganglia and cerebral cortex. Among

cells named CDJL (Ref. 18); the interaction between CD5 and immunoglobulin (Ig) heavy-chain variable (V,,) framework regions’q;

SRCR proteins, the domain organization of CD6 is most similar to CDS. Furthermore, CD6 and CDS share a similar patlern of cellular

the binding

expression

of Spa to a molecule(s)

expressed

on peripheral

blood

monocytes”; and the interaction between CD6 and ALCAM (Ref. ZO), a member of the &gene superfamily @SF). Of thtse interactions, only the ClJ6-ALCAM interaction has been characterized in detail.

CD6 A cDNA clone encoding human (hKD6 was isolated using a mammaliao transient expression cloning system”. The extracellular parLion of the mature CD6 protein includes three SRCR domains (SRCR-Dl, -DZ and -D3), followed by a 33 amino acid membrane proximal ‘stalk’ region, a 26 amino acid hydrophobic transmembrane region, and a cytoplasmic domain. Clomng of additional cDNAs coding

encoding CW, murine (mKD6

along with analysis of genomic clones enand hCD6, demonstrated the existence of

multiple CD6 &forms that arise from alternahvc encoding its cytoplasmic domain”,“,” ?‘. CD6

splicing of exons is expressed by

and are genetically

linked

in humans

on chromosome

11

(Ref. 6) and in mice on chromosome 19 (Ref. 24). Although CD6 is expressed on nzstlng T cefls, its levels are upregulated following activation. Experiments with anti-CD6 monoclonal antibodies (mAbs) suggest that CD6 is a signaling molecule that may modulate T-cell receptor signalit@. It is presently not known how CD6 is linked to the intracellular signaling pathways. However, it has been shown that Tyr residues in the cytoplasmic domarn of CD6 are phosphorylated following T-cell activation*“. This finding sug$,est~ that CD6 could be linked to intracellular signahng p.lthwys in part by an interaclion with pmtems containing sr.-homology 2 (SH2) domdins. Furthermore, proline-rich motifs, which ; lay mediate the binding of cytoplasmic proteins SH3 domains, have been identified in the cytoplasmic

containing region of

some CD6 Isofom@. Additional information on the mlc of CD6 in modulating T-cell activation comes from studies with CD6 T celW. CD6 T cells are only weakiy stimulated by alloantigen compared wtth unfractionattd T cells. By contrast, the

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’ Ii

ALCAM Since antKD6 mAhs partially Mock the binding of thymocytcs to TE cellb, an assay :a5 developed for CD6 binding 16 TE cells in

OCTOBER

I997 a

using the hCD6-Ig fusion protein and mAb J4-81 (Ref. 20). Thii cDNA clone encoded a type I membrane pm&in whae extmcellular region has five Ig-like domains, comprising two N-terminal V-like

domains

followed

by

thaw

constant

W-like

domains,

a

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SGPWQKN.T.. HRPWQFH.0.. TSLRH

CD6-3 Ml30-1 Ml30-2

+

.

H.....SRWOM FRKIKPQKSGRV

AYLWO LTLSN SALWO

. . . . . . . .

Nl30-3 Nl30-4

. . . .

nno-5 ,4330-6 m30-7 n130-S

. . . . . . . . . .

Wcl-2

. . .

wd-3 wcl-4 wcl-5

. . . . . .

wcl-6 wcl-7 wcl-5 wcl-9 WCl-10

. . . .OAT.. . . . . . . . . . . . .

PGLP.GC2H.Y.. SWR.OWNSNi... KHOGWGKNSN.. Kt4QGWQKH.W..

. .

KHHEWGKH.Y.. KWWQWGG.LT..

. . .WAT.. GSANFGAGSQP.IWLOWL . .OSGTLNSSVALRRGFRPQWVORI

PSRGWGQH.N. PSOPWNYNS..

VSSFFGTGSGP.IWLOKV

PSWGWRQH.N. PRVP.CPOGT. PVTALGGP.0. PSRGWGRH.0. PSQPWKYSS..

SNRGWLSH.N. SNRGWLSW.N. RNPQWLVN.N. SNRGWLSH.N. - ~Yn”Yr.sY .. _ - .. SHRGWLSH.N.

ouct-5 I..

owe-4

. .

ouch-5 ouct-6

. . . .SAP.. . . . .SAP..

ouct--I

.

lydrophobic

. .

GSARFGQGTQP.IVHOOV GSARFGQGTGP.IVNOOV GNAYFGPGSQS.IVLOOV GNAWFGQGSGL.IVLOOV

.SAP.. .SAP..

bansmembrane

w;ion

and a short cytoplasmic

domain.

4LCAM displays significan* sequence homology to the chicken tewal adhesion molecule BEU (DMGRASP; 93)” and 8s rat homeog KGCAM (Ref. 32). These proteins shalr equwalent extrac~llular lomain organization and belong to a small subset of IgSF members \at includes Analysis

MUC 18 (CD146) of the expnzssion

load mononuclear

(Ref. 33) and B-CAM (Ref. WI. of ALCAM by activated peripheral

cells indicates

that ALCAM

is only

transiently

xpressed on the surface of activated leukocytes. On T cells. rLCAM expression can be detected 24-48 h following activation, caking three days post-activation, and can no longer be detecttul ight days post-activation. It is not yet known if ALCAM itself has igwling pmperttes. The regulated expression of ALCAM indicates hat the CD6-ALCAM

interaction

is controlled

in a simdar

way

D that of other receptor-l&and pairs that play important mlts in nodulating the immune msponse, such as the interaction between ID26 and its two ligands, CDftO and CDt36. In this case, the expwion of CD28 is constitotive while the expxssion of CD80 and CD86 F tightly

- __ . .w . .

.

regulated.

Cell bmdmg the rxtract4lular

and chmmatographic region of ALCAM

studies have suggtited is capable of homotyprc

that inter-

actions’\, which appear tube predominantly mediated by theC-like Ig domains of ALCAM. Recent reports have also implicated ALCAM in hetemtypic interactions that are distinct from Its CD6binding properties. In the brain, ALCAM IS capable of bmding neuron-$1 identified mtrractwn

cell adhesion

molecule

(Ng-CAM),

as well

as other un-

proteins”. However, the significance of the CD6-ALCAM in the nervous system has not yet been e~plowd

CD64LCAM interactions Bmding studies with domain-specific Ig fuswn pmtems of CD6 and ALCAM have shown that the membrane-proximal CD6 SRCR domain (D.1) and the ALCAM N-terminal V-like Ig domam (Dl) are sutficwnt to mediate specific CLMALCAM bmding (Fig 3)“‘-. Thew hndmgs are also suppOrted by the observahon that antGSRCR-D3 mAbs. but r?t anti-CD6 mAbs nvognizmg SRCR domains, bloLk the CD6-ALCAM interactIon’”

OCTOBER

I997

some clthrr The

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+.-+j ;) ,v ., *\-:: L.

CD6

c

iI II

CD6

ZV“

!

:’c)--+ 1 k)

ALCAM

i

ALCAM

was examined.Nine ALCAM residueswere identified that, when mutated, reduced01’abolishedCD6 but not mAb binding. These residuesform acluster on thepredictedA’GFCC’C”faceof theCD6binding domain of hALCAM (Fig.4). In other IgSFcell-surfaceproteins, including CDZ,vascular celladhesionmolecule1 (VCAM-1). CD80,CD22andsialoadhesin,the A’GFCC’C”facehasalsobeenimplicatedin the bindingof theseproteinsto their respectiveligands. ADother chaiacleristic of the CD6-ALCAM interaction is its

OCTOBER

I997

IMMUNOLOGY

Proml” hCc6 mw-3 hCD6 mCD6 hCDS mCD5 *c-o5 hCDS rnCD5 rCDS hCDS mCD5 tCDS

Lbm 3 3 2 2 I I I 2 2 2 3 3 3

ER AR aA KA SL AL AL XH TN TH

-EEL

hcD5 mw5 hCtX mw8 hCD5 mCD5 lCD5 LCDS mW5 rW5 LCD5 mw5

block

TODAY

t? 2 2 3 3

PRGLPHSLDG-.--RMYY LPQLHFTPQRQP, LAGLHFTPGDGP, GPFLVTY--TP. QPFPSLN-. . RPQHLTLWN-. . RPLPETEAQRADDPQEPREH LSJTEAAQTPAPAELRDP LSRIETAELDLCSELRDP SVNSYRVLDAQDPTSRQL . DL t SFHTVDADKTSPGF . NL I SFHVMDADRTSPGV

theCD6-ALCAM

intwxtion.

LIAR.

_

-aEP HRDD” HRDOV -QSSI -QNQV -KNDI

However,

-AEA -TEA

EDAQ.

Q.........

-aLe

_.

a as as

_

s......... s......... , KPGKSGRVL TTAOECQDAL TTVGEGSOAL I( . . . . . ...” R . . . . . ...” R... . ...”

. SPW . PPW I awbc I I RWEA 1 RWEA . .a . ..E . . . E

CD6 also specifically

\\‘rn.

,mmu,,~~t~r”r,p,t.~t~‘i

\r,th

hCDh-lg

II, ., d,\alcnt-catwn-

binds BEN although, in this cast, the btndmg faces an’ not tully conserved”. Incontrast to IRSFmoltades, no threedimcnstonal structural in-

dcpcndcnt ,nmner”, sugg’stmg the ~wh,hty that CD6 may bmd to I,g.,,& other tlren ALCAM. Indcui, molcculcs other than ALCAM m.ly zwtribw to the ,ntw.w,w oi CD6 wth wll=~ .md !D

formation is currently available on any SRCR domain. Thus, multipie sequence compnsons between CD5 and CD6 SRCR domains

signal tr.lnsduct,on through may wpprt thla formatron

from different specie wcrc used to aid in tire xientification of CD6 residues that are important for ALCAM bmding (FIB. 5). The Ntenninal half of thaw domains shows significant wqucncc couwr-

ALC‘AM

vation, while the C-terminal half is m,uv variahlr. Single rniducz in both conserved and nonconwrvc~i sqwncc wgmc’nts of hCDh

Conclusion Stud,cs on thr CD&ALCAM

D3 wert hALCAM

mutated”. The binding of the CD6 mutant protein3 and a panel of conformationally sensitivr ant,-CD+D3

mAbs was tested. Mutation of residues ,n the C-trrmm.ll CD6 D3, which is the least conserved rq+m of the moltuulc,

to

half ot affect

CDh Altemat,vciv, dwalmt catwns of d cwnplcx ot pmtc,n\ mrlud,ng

that ha a higher affimty

tail4 wcw franr,%wl

:ntcract,o,,

,>t .I,, SRCR-ligand t<>r tuture rcwarcb

tunrtwn of other

tow md hws r~yarrimg

the function

the f,nt

drd

membtn of the SRCRSF. Swcral qoeof SRCR Jomam:. ,n gcnc’ml. and CD6

cations”. However, early stud,cs with hCD6-Ig showed that the ability of this fusion protein to hind to crlls was enhanced ,1pproximately twofold by divalent catkx&“‘. The divalcnt-catwn-

r~,‘rC7t~,r-ll#a,,J pain ,n\ olw SRCRSF-IgSF ,,ltcr.x+ons The 1 rrwt,I,ty of Ig dom.lins ,n med,.lt,ng II v.lr,cty of pmtcm-protcln and Fn~t~,,l-c.irhohydr.ltr intcrxt,on\ ha\ hwn well dthcr ,‘cr..,t,,r ,7l.ltt,I~,,, tar nlcd,atIll~ d,ffUVnt ty,V- Of ~lOIccl,I.1r

dependent to ALCAM

port,o,, of IGX-Is :wxl~,,): w.,, not inlubltcd by mAbs (Ref. 29). and at least two pmt‘?“s ot ‘Xl hD.1 .md 41 kD.1

C lX-+ALCAM

he

F”‘\‘&i

ALCAM but not mAb binding (Fig. 5). This suggest?. than th,s region of the Group B SRCR domain ,nctudt% ws,uue nnporrant ior mediating ligand-binding spwificities. CL%-ALCAM interactions occur in the abwnce of divakwt

I\ tlw

t‘,

haw

,ntrract,on, t!.crchy ehtabl,sh,,lg rffortz dtwg,wd to 4udy the euc-

111 ~~WtlC”h,.

t,mvr

T~,118,,,

for CIX

addw%‘d

mtcrxtmn

FOr

~‘\
tw

otlwr

h11\\.

C;rwp H SSC‘RSF? Will CD6. l,kc CDS, havr the, ah,Iitv \vlth ml,It,?I~ Iigand4? Will Other ,mmu,lotog,ra~~~

OCTOBER

rcF’c,c’,l-

mc,nbrr<

of tlw

to ,ntcract ,~l~~,rt~l,lt

I997

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interactwn+, or whether It I$ hm&xi to a mare narrow lange of btndq speciftcttu and functton\ Answcri to these and many

20 Bowun, M A, Patd, D D, LI, X ct n’ (1995) / Et’?. Mnf 1%. 2213-2220 21 Whttney, G , Bowcn, M , Neubauer, M and Aruffo, A (1994) Mu’

other questtons domams awalt

‘~~ww~~‘~ 32.89-92 22 Robmson, W.I 1, Prohaska, 5 S , Santoro, JS , Robinson, H L and Parries. ] R. (1995) I ‘~rrrrw~ol 155, 4739-4748 23 Robmson, W H , de Vegvar, H.E N., Pwhaska, S S, Rhee, J.W and

mrdmted

that address the structure and function of SRCR the detailed analysis of .jther SRCR-domain-

mtcractwn~.

L Kodama, T, Freeman, L., Rohrer, J,, Zsbrecky, J, Matwdatra, E and hqer, M (1990) Nnttrr,‘343,531-535 t Resuck, D , Pearson, A and Krwger, M (1994) Trtwb Bmc’wrr,. Sc,. 19, S-B 5 Eml, M , A5aoka. H , Matsumoto, A ~‘1n’. (1993) / Bw’ Clwr. 26% 1120-212s 4 Vysc, TJ , B&r, C I’, Walport, M J and Mor’cy, B J (1994) Go~err~rr?; 24, WR 5 Tarakhovsky, A , Muller, W. and Ralavsky, K. (1994) Eur. 1, la~nr~rm~l. 24, 1678-16&l 5 Bowen, M A, Whrtncy, C 5, Neubawr, M r, o’ (1997) 1. I, ,,,,,,,,,, d. 158, 1149-1156 7 Jones, N.H , Clabby, M L., D~alynas, D.P. Huang, H J,, I lerzenberg, L.A. and Stmmmgvr, J L ‘IYUb) N,,,,ar~323, .346-349 B Amffo, A, Mehnck, M B, L~n+y, PS and Seed, B. (,99,), U,,‘, Mn,. 174.949-952

Wqng.,ard, PL, Metrclaar. M I MacHugh,

9

Cleven,

H C l19Y212)

/ ‘/~~~wo/o/

10 Law, SK A , Mlcklcm,

149,

N.D,

Morrwn,

WI and

3273-3.277

K J., Shaw, J M. rl nl. (1993) EIII.

1

Irrwtr~~,~l. 23,

232&2?25

11 Gcbr, I, Kwner, PA, Rmg, H , L,. X , Franckc, U and Aruffo, J. BIO’

Clw~u.

272,

A

(1997)

b151~lSB

12 Mayer, WE and T~chy, H (1995) Grrw 16-4.267-271 13 LI, X. and Snyder, S H. (1995) \ Bnd C’wrr 270,,7674-17679 14 Chen,e,, H., Blerknes, M and Chen, H. (1996) Amt. RN 244,3’7-343 15 Takrhr J, Hzkrta, C and Al-Awqat,, Q f,YY6,, Cl,,, ,,,a., YU, 2324-2331

16 Nunes. D P, Keates, A C , Afdhal, 6loc’tnrl

/ 310,

N H. and Otfwr,

C D (1995)

41-48

Van de Vuldc, H , YO” Hor~en, 1, Luo, W, f’arnes, J R. and Thi~lcmans, K (1991) Nutwr 351, 662-5 18 Btanconc, L, Bc>r\fcn, M , Amffo, A, Andra, G and Stampnkwic, (1996) I CI’I Mel 184, Xll-si9 19 “orp~sr’, R, Rth M G and Mdgr: R.G. 1’996) ,, Eq,. Mr, ‘84, 17

1279-1284

1.