The sticking point: how integrins bind to their ligands

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lntegrins mediate dynamic adhesion events during many developmental processes’. They also have an essential role in the behaviour of leukocytes, for example during inflammatory responses and in ‘homing2s3.lntegrins exist in different ligand-binding states, depending on the presence of intracellular factors* and extracehular divalent cations. Recently, isolated integrin domains have been shown to contain binding sites for both ligand and divalent cation. Using the integrins LFA-1(oL#~)and GPlIbllla (oL,,#~)as the principal examples, we discuss here how the study of these domains has contributed to new understanding of ligand binding, and speculate about the role of divalent cations in integrin function. S

/or Ca2+to bind their ligands. For example, the leukocyte integrins LFA-1 and VLA-2(0~~9~) both bind ligand in a Mg2+-dependent fashion, whereas ligand binding by the fibronectin receptor VLA-5(a@r) and the platelet integrin GPllbllla is Ca2+dependent. The first direct proof that integrins are metalloproteins came from an affinity labelling experiment with the vitronectin receptor @,P3), using cobalt ions in place of Ca2+. This study revealed that an average of 3.5 ions of s8C03+ cou’!dbe covalently coupled to each receptors. The binding of metal ions by integrins was also indicated indirectly by the fact that certain sites on integrins are recognized by monoclonal antibodies only in the presence of divalent cations: examples ndent 24 epitope6 and epitoper for LFA-1, and t CERMl/S cpitope for

The intefl’n adhesion receptors are ap heterodimers that exist in different ligand-binding states. Because of their large size and conformational lability, it has been d!f??cultto determine how they interact with their ligands. Ligand-binding sites have been identified in the p subunit, and now more recently in the ‘I’ domain and EF-hand-like domains V and VI of the cysubunit. We speculate here about how these various sites might operate together to bind ligand in a stable manner.

might not be needed for the weak (FMto mM)metalion-binding affinities of many integrins. A recombinant fragment of GPllbllIa OLsubunit comprising domains IV-VII bound four moles of Ca2+ per mole of protein, indicating that all four domains can bind metal ion@, Whether this holds true for the intact molecule or for all integrins remains to be seen. Recently, binding sites for the LFA-1HgandICAM1 have been mapped to this metal-ion-bindingregion on in&grins are part of a stretch of seven short of LFA-1”‘.Two ligand-binding sequences were repeated domains (I-VII) at the N-terminus of the a located in domains V-VI by experiments employing subunit (Fig. la). Domains V-VII (or IV-VII,depend- recombinant ICAM- to precipitate a series of LFA-1 ing on the in&grin) possess sequences similar to u subunit deletion fragments, followed by syntheticthe helix-loophelix divalent-cation-binding motif peptide blocking studies. The domain V site (residues known as the ‘EF-hand’g.We have recently proposed 458-467) is located on the C-terminal side of the a model of LFA-1 domains V-VI in which each metal-ion-binding sequence and corresponds to a --domain contains a pair of EF-hand-likemotifs, with highly variable region in integrins (Fig.3). This could The authors are at only one member of the pair retaining the appropri- account in part for the specificity of ligand recog- the Imperial ate metal coordination geometryrO (Fig. 2). This is nition by individual integrins. In contrast, the Cancer Research also a feature of some ‘classical’ EF-hands and is ligand-binding site in domain VI (residues497-516) Fund, Lincoln’s thought to maximize the binding affinity of the overlaps the highly conserved metal-ion-binding site Inn Fields, ‘active’ partner 1l. Our model must be viewed as a in this domain, Interestingly, when domains V and London, UK WC2A ‘best guess’, however, because the putative integrin VI of LFA-1are modelled as EF-hands,the two ICAM- 3PX: Nancy Hogg, EF-hand consensus sequence is missing the critical l-binding sites lie along one face and may therefore Clive Landis, Paula glutamate residue at position 12, which is replaced compose discontinuous parts of a single ligand-bind- Stanley and Anna by a hydrophobic residue (Fig.3). A popular idea was ing regionlo (Fig. 2; red loops). In addition, domain Randi are in the that the aspartate residues present in the well-charac- V of GPllbIlla has been found to contain d binding Leukocyte Alignment of Adhesion terized recognition motifs of in&grin ligands (e.g. site for the y chain of fibrinogenr4q15. the Arg-Gly-Aspor RGDsequence) might supply this domain V sequences indicates that the GPllblIIa Laboratory, and missing coordination group. Alternatively, the miss- ligand-binding site overlaps the metal-ion-binding Paul Batesis in the ing residue might be provided by other parts of the motif but not the LFA-1ligand-binding site, which is iliomolecular integrin molecule itself, by analogy to the Ca2+-bind- positioned nearer to the C-terminal end of this ?&ddling Laboratory. ing site of galactose-binding protein*2, or simply domain (Fig.3).

TRENDS IN CELL BIOLOGY VOL. 4 NOVEMBER 1994

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(a) T domain

Metal-binding domains

TM

I I II

lU IV V VlVll TM

,I (b)

IIII

I I

Conserved region

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+

+ + +

Cell membrane

P subunit

c~

subunit

FIGURE 1 (a) Schematic representation of LFA-1 a and 13subunits. The location of repeated domains (I-VII) and the inserted (I) domain are indicated in the Q subunlt, and the conserved region and cysteine.rich repeats are indicated in the 13 subunit. TM, transmembrane domain. (b) Hypothetical model of LFA-1 structure, indicating the putative divalent.cation.binding sites (+) and Ilgand.blnding sites (filled boxes) mapped to the a subunlt. For tile 13subunlt, regions corresponding to the binding sites mapped on B3 are indicated by open boxes. Binding sitescould act cooperatively to form a single 'maxl.pocket' for Ilgand; could act sequentially, with binding to one site inducing or stabilizing binding through a second site; or could act independently to crosdink llgand molecules to each site.

a

subunlt T domain: another blndln9 site

Attention has focused on domains V-VII where llgand-bindlng and metal-bindlng sitesare closely associated.More recently,however, another type of metal-blnding sitehas been located in a part of the cx subunit known as the T domain. In LFA-I, the I domain isa -200 residue sequence Insc,t~dbetween domains IIand III(Fig.la).The I domain Isfound In only seven of 21 integrins: LFA-I, Mac-1 and plS0,95, which allcontain the 132subunit,and a new ~2 integrin found predominantly on macrophages (M. Gallatin, pets. commun.); the collagen and laminln receptorsVLA.1 and VLA-2; and the recently *The 'A' domains cloned IntraepithellalT-cell integrln,%[%;6. The I of the integrins are domains of Integrlnsmake them members of a superusuallytermed family of A-domaln-containlng proteins*, which I domains.The share homology with the prototype molecule yon nomenclature for Willebrand factor;other members of the superfamthis superfamily ily are complement factors B and C2, several nonhas not yet been fibrillarcollagens (types VI, VII, XII and XIV) and standardized. other extracellularmatrix (ECM) components such 380

as cartilage matrix protein and undulin t7. Binding sites for a number of ligands have been mapped to the three A domains of yon Willebrand factor, including platelet glycoprotein lb, heparin and collagens types l and Ill TM. The vast majority of Aftdomain-containing proteins are either ECM proteins or receptors for ECM components, which suggests that this domain may play a general adhesive function in many proteins involved in cell-matrix or matrix-matrix interactions. Comparison of the genomic organization of integrin genes suggests that sequences encoding the I domain have been inserted into an ancestral integrin gene t9.2°. Several initial reports focused interest on the I domain as a site of importance for integrin function: mutation in the Mac-1 I domain affected metal and ligand binding21; and mAbs that block Mac-1 function zz, as well as mAb MEM83, which is able to activate LFA-1, were all found to recognize the I domain z3. These first observations have now been extended by mapping experiments with e~her integrins. The I domain contains epitopes for functionblocking mAbs of LFA-124,2s,p150,9526 and VLA-227; a Mac-1 'activation reporter' epitopeg; and distinctive recognition sites for the LFA-1 ligands ICAM-1 and ICAM-32s. The immunogenicity of this domain suggests that it may be prominently displayed on integrins, but it must be noted that many of these mAbs were selected on the basis of affecting integrin function. A role for the I domain In ligand binding is further supported by the observation that removal of this domain caused a substantial drop In the ability of ICAM.1 to precipitate LFA-1 a subunit flagments ~°. All these observations could be interpreted to suggest that the l domain is involved in ligand binding either by regulating access to a binding site or by directlybinding llgand. Studieswith an isolatedrecombinant form of the l domain have helped to resolvethismatter. Directevl. deace for llgand binding to the I domain came with th~ demonstration that this domain of LFA-I could bind ICAM.1 in a solld-phase adhesion assay and block adhesion to ICAM.I in a T-cell-bindingassay (Ref. 24; P. Sanfllippo and C. Lau, pets. commun.). This has been extended to the Mac-1 1 domain, which binds to fibrinogen and ICAM-1 but only poorly to factorX zB,and the VLA.21 domain, which binds collagen but not lamlnln (D. Tuckwell and M. Humphries, pets.commun.). Thus binding of ligand is a general featureof the integdn I domain. A link between metal binding and llgand binding for the l domain was first demonstrated by Michishita and colleagues who described a Mg~*/MnZ+-binding site in the I domain of Mac-I through the use of a blotting techniqueZL Mutagenesis studies showed the metal-ion-binding site to be dependent on discontinuous residues at three positions throughout the domain (Asp140 to Ser142, Asp242 and Prol9S) and therefore possibly not an EF-hand. Mutations at these residues also abolish the ability of the Mac-11 domain to bind its ligand iC3b u (see Fig. 3). Mutations of the corresponding two ~spartate residues in the VLA-21 domain (Asp151 and Asp2S4) abolish binding to both collagen and certain TRENDS IN CELL BIOLOGYVOL. 4 NOVEMBER 1994

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mAbs that block function z~. Whether these residues are required directly for ligand binding or indirectly through a role for metal ions in ligand binding has still to be determined.

I~ s u b u n i t : a third Iigand site

There is also an apparent link between ligand binding and metal binding to the I~subunit. Crosslinking of an RGD-containing peptide to the vitronectin receptor (%13a) resulted in 80% of the peptide localizing to the I~ subunit and only 20% bound to the % subunit z9.3°. The RGD-binding site (residues 119-130) is located in a region that is highly conserved among all integrins, suggesting that other 13 subunits might similarly bind ligand. The site overlaps the naturally occurring 'Cam' mutation (Asp119 to Tyr) in the ~ subunit that abolishes both ligand and metal bincl':~g of the GPIIblIIa integrin, affirming the linkage between these two functions ~ (see Fig. 3). This mutation coincides with the +× position of a sequence that Ginsberg and colleagues speculate b.- be an evolved EF-hcnd motif. Interestingly, there is homology between the ligand-binding site of the I~ subunit and residues surrounding the Asp140 to Ser1,12 sequence in the I domain (Fig. 3), but further study of the I~ subunlt and the I domain will be required to establish whether there is any homology between them. However, the relationship between metal-ion and ligand binding is not inevitable as a second llgand-blnding sequence in IBa (defined by a peptide of residues 211-222 that blocks adhesion to fibrlnogen) so far seems not to bind metal ions 3z. Llgand. and nmtal.lon-blndlng sites: t w o sides of t h e same coin?

FIGURE 2 Diagram of domains V and Vl of the LFA-1 a subunit, modelled after known EF.hand proteins 1°. The b,vo helix-loop-helix (or EF.hand) motifs in domain V are shown in two shades of blue as A-B and C-D, and in domain Vl in two shades of green as A'-B' and C'-D'. Each helix is represented in cylinder form. Sequences corresponding to the two binding sites for synthetic peptides that block the LFA-I-ICAM-1 interaction are shown in red and the positions of putative metal ions are indicated by yellow circles. Computer graphics were obtained with the program 'PREPI' (S. A. Islam, ICRF, London, UK).

lntegrins are thought to contain five or six metaf. ion.binding sites: three or four in domains IV-VII, and at least one each in the I domain and the subunit, The coincidence of these sites to llgand-blndlng areas raises EF.handposition 1 the obvious question of how the s s ¢ 9 ~z] Y Z -Y -X Coordinatingidle X metal-ion.binding sites function in the heterodlmer to regulate ConsensusreeidueO integrin activation and ligand binding. Unfortunately, at present 296 the answers are largely specuGPllbllla domain V IT i!~JV iiiNii~ i~iiG! R H ii::~L! 3o~ lative, big 2+ and Ca 2+ are impliLFA-1 domainV 443 !i~iV i~j Q i~lii~lE T !!~! L L L I O A[P L F V O E 'O R O O| 46"] cated directly in the ligand-bindLFA-1 domainVl 505 ii~i I' ~i ii~ i~! !o! L V Ii~! V A YI 516 ing event because bound divalent cations are obligatory for ligand S Vi:~::~ii M Z Diil)~ L W Sl X30 133 . 9 ~ L binding. As discussed, the RGD 134~ L S f::!~Si:!iIVl L D !!i~ L R N 145 motif might supply a coordinatMac-1 I domain ing residue for cation binding by 151 E $ N ii:~ I Y P W D /~ V 162 VLA-21domain EF h~nds. However, several lines 137i~ O S MI:~L O P E ~ Q J4, LFA-1 I domain of evidence suggest that at least some effects of divalent cations may be indirect, potentially af- FIGURE 3 fecting the association of a and 13 Alignment of integrin sequences implicated in ligand b~ding and metal.ion binding. The EF.hand subunits and ther~.~y revealing consensus sequences of domains V and VI of GPllbllla and LFA-1 a subunits are compared with regions a cryptic ligand-binding site. from the 132and 133subunits and from the Mac.l, VLA2 ~,.~dLFA.1 I domain sequences. The EF-hand Analysis of active and inactive consensus motif was adapted from a more detailed review 3s. Integrin residues that correspond to the conformations of GPllbllla EF..hand consensus are shaded, l'esidues required for ligand binding are boxed and residues required for suggests that a ligand-binding divalent-cation binding are circled. The continuation of the LFA.1 domain Vl ligand.binding region, site, situated between the a and determined by peptide blocking studies to be within residues 497-516, is indicated by dotted lines 1°.

'1

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TRENDS IN CELL BIOLOGY VOL. 4 NOVEMBER 1994

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subunits, is revealed following integrin activation, through the widening of a 'narrow tunnel' leading to the site 33. Subtle mutations in the divalent-cationbinding regions (domains IV-VII) of VLA-4 frequently lead to failure of the a and 13subunits to associate appropriately34. Furthermore, binding of ICAM-1 to the isolated domain V-VI region of LFA1 a subunit is independent of divalent cations ~°. Therefore, divalent cations may exert at least some of their effects over a long range, rather than being directly involved in ligand binding. A similar situation is seen in other proteins such as annexin V, in which bound Ca z÷affects the protein's tertiary structure but makes little difference at the CaZ+-binding site 3s,36. Another intriguing possibility is that there may be domain-specific roles for the metal ions. For example, Mg 2÷(and Mn z÷) but not Ca z÷binds well to the Mac-1 1domain 2~, and Ca z÷but not Mg z+ appears to support LFA-1 clustering, suggesting that it may act as a linker between adjacent integrins 7.

Speculation on Ilgand.blndlng mechanisms How do domains V-VI and the ! domain of the c~ subunit and a consewed area in the 13subunit operate together to bind ligand (Fig. lb)? The sites may collectively form a 'maxi' pocket with several points of contact between integrin and ligand. Alternatively different sites may act sequentially, each with a distinct :ole in the regulation of receptor-llgand pairing. For example, initial binding of ilgand to one site, say to an exposed ! domain, might induce an alteration In lntegrin structure that reveals another site In domain ~-Vl. leading to more stable binding. Support for a sequential model comes from the observation that stable binding of LFA.I to ICAM,I must be preceded by a distinct Interaction wtth ICAM-1 as part of the LFA.1 activation process 37, However, such a hypothetical role for the [ domain cannot be generally applied because other lntegrlns such as GPllbllla do not possess an I domain yet undergo a similar 'two-stage' process of llgand binding 3~, It is also possible that a single integrtn binds more than one ligand; for example, LFA.1 may use the different binding sites to bind more than one ICAM.1 molecule, This might also mean that orie ICAM-1 molecule could bind to two LFA.1 molecules causing receptor dlmerizatlon, as described for growth hormone and its receptor 39. Discriminating between these possibilities will be the next challenge. References 1 HYNES,R.O. (1992) Cell 69, 11-25 2 DIAMOND,M. S.and SPRINGER,T, A. (1994)Curr. 8/o/.4, 506-517 3 AGER,A, (1994) Trends Cell Biol. 4, 326-333 4 WILLIAMS,M. J., HUGHES,P. E., O'TOOLE,T. E. and GINSBERG,M. H. (1994) Trends CellBioL 4, 109-112 5 SMITH,J.W, and CHERESH,D. A. (1991)1. Biol. Chem,266, 11429-11432 6 DRANSFIELD,I. and HOGG,N, (1989) EMBOI, 8, 3759-3765 7 VAN KOOYK,Y., WEDER,P., HEIjE,K. and FIGDOR'C. G. (1994) J. Cel/Biol. 124, 1061-1070 8 DIAMOND,M. S. and SPRINGER,T. A. (1993)1. CellBiol. 120, 545-556 9 TUFTY,R.M. and KRETSINGER'R.H. (1975)Sc/ence187,167-169 382

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10 STANLEY,P., BATES,P. A., HARVEY,I., BENNETT,R. I. and HOGG,N. (1994) EMBO J. 13,1790-1798 11 COOK,W. J., EALICK,S. E., BABU,Y. S., COX,J.A. and VIJAY.KUMAR,S. (1991)J. Biol. Chem. 266, 652-656 12 WAS,N. K., WAS, M. N. and QUIOCHO,F.A. (1987) Nature 327, 635-638 13 GULINO,D., BOUDIGNON,C., ZHANG,L., CONCORD,E., RABIET,M-J.and MARGUERIE,G. (1992)J. Biol. Chem. 267, 1001-1007 14 D'SOUZA,S. E., GINSBERG,M. H., BURKE,T. A. and PLOW,E. F. (1990)J. Biol. Chem. 265, 3440-3446 15 D'SOUZA,S. E., GINSBERG,M. H. and PLOW,E. F. (1991) Trends Biochem. Sci. 16, 246-250 16 SHAW,S. K., CEPEK,K. L., MURPHY,E.A., RUSSELL,G. J., BRENNER,M. B. and PARKER,C. M. (1994)J. Biol. Chem. 269, 6016-6025 17 COLOMBATI"I,A., BONALDO,P. and DOLIANA,R. (1993) Motrix 13, 297-306 18 SADLER'J. E. (1991)J. Biol. Chem. 266, 22777-22780 19 CORBI,A. L., GARCIA-AGUILAR,J. and SPRINGER,T. A. (1990) J. Biol. Chem. 265, 2782-2788 20 FLEMING,J. C., PAHL,H. L., GONZALEZ,D. A., SMITH,T. F. and TENEN,D. G. (1993)J. Immunol. 150, 480-490 21 MICHISHITA,M., VIDEM,V. and ARNAOUT,M. A. (1993) Cell 72, 857-867 22 DIAMOND,M. S., GARCIA-AGUILAR'J., BICKFORD,J. K., CORBI,A. L. and SPRINGER,T. A. (1993)[ CellBiol. 120, 1031-1043 23 LANDIS,R. C., BENNETI",R. I. and HOGG,N. (1993)]. Cell Biol. 120, 519-527 24 RANDI,A. M. and HOGG,N. (1994)[ Biol. Chem. 269, 12395-12398 2S LANDIS,R.C., McDOWALL,A., HOLNESS,C. L, L., LITTLER,A. I., SIMMONS,D. L. and HOGG,N. (1994)[ CellBIol, 126, 529-537 26 BILSLAND,C. A, G., DIAMOND,M, S. and SPRINGER,T. A. (1994)J. ImmunoL 152, 4582-4589 27 KAMATA,T,, PUZON,W, and TAKADA,Y, (1994)[ Biol, Chem, 269, 9(59-9663 28 ZHOU,L., LEE,D, H. S., PLESCIA,I., LAU,C. Y. and ALTIERI,D. C, (1994) l, Biol, Chem. 269, 17075-17079 29 SMITH,J.W, and CHERESH,D. A. (1990)1. Biol. Chem. 265, 2168-2172 30 D'SOUZA,S. E., GINSBERG,M. H., BURKF.,T. A,, LAMoS, C.T. and PLOW,E. F. (1988) Science242, 91-9~ 31 LOFTUS,J, C,, Oq'OOLF.,T, E,, PLOW,E, F., GLASS,A,, FRELINGER'A, L,, Ul and GINSBERG,M. H, (1990) Science249, 915-918 32 CHARO,I. F,, NANNIZZI,L., PHILLIPS,D. R,, HSU,M. A. and SCARBOROUGH,R, M, (1991)I. Biol. Chem. 266, 1415-1421 33 CALVETE,J,J,, MANN, K,, SCHAFER'W,, FERNANDEZIAFUENTE,R, and GUISAN,I, M, (1994) 8iochem, ], 298, 1-7 34 MASUMOTO,A, and HEMLER,M, E,(1993)l. Cell BioL 123, 245-253 35 DA SILVA,A, C, R. and REINACH,F. C, (1991) Trends 8iochem. Sci. 16, 53-57 36 LEWIT.BENTLEY,A,, MORERA,S., HUBER,R. and BODO,G. (1992) Eur. I. Biochem. 210, 73-77 37 CABAIr4AS,C, and HOGG,N, (1993) Proc.Natl Acod. Sci. USA 90, 5838-5842 38 DU, X,, PLOW,E, F., FRELINGER'A. L, O'TOOLE,T. E., LOFTUS,]. C and GINSBERG,M. H. (1991) Cell65, 409-416 39 CUNNINGHAM,B. C., ULTSCH,M., DEVOS,A. M., MULKERRIN,M. G., CLAUSER,K, R.and WELLS,I. A. (1991) Science254, 821-825 TRENDSIN CELLBIOLOGYVOL. ~ NOVEMBER1994