Allostery and proteolysis: Two novel modes of regulating integrin function

Attostery and Proteo[ysis: Two Novet Modes of Regulating Integrin Function JEFFREYW. SMITH The Cancer Research Center, The Burnham Institute, La Jolla, California, USA

Abstract Integrins are involved in transmitting signals between the cytoplasm and the extracellular matrix. Importantly, the transfer of information is bi-directionah signals flow inside-out and outside-in. Here, I discuss two potential modes by which integrin function is likely to be regulated. It is hypothesized that the integrin cytoplasmic tails are proteolytic substrates, and that cleavage of the cytoplasmic domain regulates the ligand binding affinity of integrins. It is also hypothesized that the ligand binding site is allosterically regulated by separate divalent ion binding sites that independently control ligand association and dissociation rate. Both hypotheses are suggested by reports in the literature and can be tested experimentally. Key words: Integrin regulation, ligand binding

Introduction Although originally considered to be the physical link between the cell surface and the extracellular matrix, integrins are now known to connect with important intracellular signaling pathways (Hynes, 1992; Yamada and Miyamoto, 1995; Ruoslahti, 1996). Signaling through integrins is bi-directional. Inside-out signals regulate the affinity of integrin for ligand and, consequently, control cell adhesion. Outside-in signals emanate from integrin binding to the extracellular matrix and can ultimately change gene expression. Both outside-in and inside-out signals require the transfer of information between two key domains on the integrin. These are the ligand binding domain and the cytoplasmic tails. Most models of an integrin depict the ligand binding domain as a single site with a static structure. Such models also imply that the cytoplasmic domains are always presented to the cytoplasmic milieu. This brief essay will consider two hypotheses that chalMatrix Biology Vol. 16/1997, pp. 173-178 © 1997 by Gustav Fischer Verlag

lenge these assumptions. First, I will discuss the idea that integrin activation (inside-out signaling) could be regulated at two separate steps in the ligand binding reaction. Evidence suggests that divalent ions control these steps independently. Second, I will discuss the possibility that the integrin cytoplasmic tails are proteolytically cleaved in situ, and that cleavage is a key part of regulating both inside-out and outside-in signaling. Although both hypotheses are still untested, and could be considered speculative, there is ample evidence in the literature to indicate that they warrant further study.

Are Integrin Activation and De-activation Regulated at the Level of Ugand Association or Ligand Dissociation? Although integrin expression levels can vary on the cell surface, this parameter does not seem to be the primary means by which the adhesive capacity of the cell is

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regulated. Rather, most cells are able to turn integrins on and off. This on/off switch is really a modulation of the integrins' affinity for ligand. For the purposes of this discussion, I will refer to an increase in ligand binding affinity as "activation" and a decrease in ligand binding affinity as "de-activation." Hence, activation is an increase in ligand binding affinity, and not an increase in the number of available binding sites. To understand the mechanism of integrin activation, some consideration should be given to the structure and function of the ligand binding site. This domain is often considered a single site, or a single entity. Yet there is ample evidence to indicate that the integrin ligand binding site is more complex, especially because it can be regulated by divalent ions. In fact, the integrin ligand binding site could be considered analogous to the active site of an enzyme which uses divalent metal ions as cofactors. This laboratory has begun to apply real-time kinetic analysis to study ligand binding to integrins (Hu et al., 1995, 1996). It is important to emphasize the advantage of kinetics in this regard, because it allows over-all binding affinity (Kd) to be separated into two parameters, ligand association rate (described by the constant kl) and ligand dissociation rate (described by the constant k_0 (Segal, 1975). The relationship between these constants is shown in equations 1 and 2, where L is ligand and R is receptor (integrin). kl

Equation 1:

L+R ,

Equation 2:

kd= k_l/k 1

k_t

Alloateric Inhlbltory Site

tCa2+ Regulates Dissociation

;IGD Ligand Competent Metal BindingSite

Regulates Association

Mg2+

Cytoplasmic Domains Figure 1. Model of the integrin ligand binding site. Although the integrin is depicted as binding to RGD, the model is likely to apply to non-RGD binding integrins. Two classes of divalent ion binding sites are shown. These are the Ligand Competent Sites, which prefer M g 2~ or Mn2~and which regulate the rate of ligand association. The other class of sites are inhibitory (I). This site is allosteric to the ligand binding site and is specific for Ca2÷.The I site regulates the rate of ligand dissociation.

' LR

A number of reports show that ligand binding to integrins is tightly regulated by divalent ions, like Ca 2÷ and Mg ~÷ (Staatz et al., 1989; Loftus et al., 1990; Dransfield et al., 1992; Mould et al., 1995; Hu et al., 1996). Indeed, the regulatory ion binding sites have opposing effects on ligand binding; some ions promote ligand contact, whereas others inhibit ligand binding. Without breaking the ligand binding reaction down into the separate processes of association and dissociation, it has been difficult to understand how this can occur. Based on our kinetic analysis, a model of the ligand binding domain is proposed in Figure 1. Two classes of ion binding sites are shown. These include the Ligand Competent (LC) sites, which must be filled for ligand to bind and which regulate the rate of ligand association (kl). Another class of ion binding sites are inhibitory (I), and they regulate the rate of ligand dissociation (k_0. What are the implications of this model to activation of integrins? There are essentially two mechanisms by

which the affinity between integrin and ligand can be regulated. Either ligand association (kl) can be increased, or ligand dissociation (k_i) can be reduced. Under equilibrium conditions, either change results in a higher over-all binding affinity and a more stable integrin-ligand complex. At present we do not know which step is changed upon activation, but the resolution of this issue has profound implications for understanding the mechanism. For example, if activation is really an increase in ligand association rate, then it is likely to be regulated by the LC ion binding sites. We and others recently reported on the structure-function relationships of the I-domain like site present in the integrin 13 subunits (Bajt, 1995; Puzon-McLaughlin and Takada, 1996; Tozer et al., 1996; Linet al., 1997). Our data indicate that this site is probably within the class of LC sites. Hence, if activation is controlled by changes in association rate, this region of the 13 subunit is likely to be involved. Indeed, Takada's group did map the epitope for several activating antibodies to this region within the 131 subunit (Kamata et al., 1995).

Regulation of integrin function On the other hand, if activation is a decrease in dissociation rate, then it could largely be regulated by the I CaZ+-binding site. In this case, one might propose that activation is simply a reduction in affinity of the 1 site for Ca 2÷ such that, at physiologic levels of Ca 2÷, the site is no longer occupied. The elimination of ion bound at the I site would decrease the ligand dissociation rate, thereby increasing over-all ligand binding affinity (kd), or essentially "activating" the integrin. There are really no clues to the location of the I Ca 2÷ binding site, but its apparent affinity for Ca 2÷ (high jaM to low mM) suggests that it could be a single aspartic acid residue. It is not unreasonable to hypothesize that even subtle changes in the integrins' cytoplasmic domain that cause activation could change the angle at which a Ca2÷-binding aspartare in the ectodomain is oriented. One might propose that such a small conformation shift in this key aspartate would force it into a salt bridge with another residue within the integrin, masking its ability to bind Ca 2÷. Perhaps this is the mode of inside-out signal transduction. This type of scheme is also analogous to that proposed for the changes that occur in the integrin cytoplasmic tails during activation (Hughes et al., 1996) (see below). The two routes to activation have different implications for cell behavior. For example, consider an adherent cell which encounters an activation stimulus. If activation is a change in association rate of integrin for ligand, the stimulus is unlikely to rapidly effect the integrin, which is already present in a multimeric complex with adhesive proteins and cytoskeletal components. In contrast, if activation is a reduction in dissociation rate, then the integrins which have already made contact with matrix could be greatly influenced by activation. Their binding to adhesive ligand could be greatly stabilized by activation, preventing cell motility. On cells other than platelets, the integrin off-switch, or de-activation, may be as important as the on-switch. Migrating cells typically have a leading edge and a trailing edge. Recent work from Maxfield's group indicates that the c~v[33 integrin recycles from the trailing edge, where it is released from the matrix, to the leading edge, where it again makes contact with matrix (Hendey et al., 1992; Lawson and Maxfield, 1995). Thus, motility is a process that requires several cycles of "activation" and "de-activation" of the integrin. Given that integrin affinity could be regulated via two separate routes (on-rate vs. off-rate), it is important to consider whether activation and de-activation are regulated in different ways. In other words, is it possible that activation is regulated by increasing ligand association rate, but that de-activation is caused by increasing the dissociation rate? One might

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envision how the machinery to enact these two separate events could be segregated at separate ends of the cell. For example, if activation is the result of phosphorylation of the cytoplasmic tail, then integrin might be co-localized with the appropriate kinase at the leading edge of the cell. Conversely, de-activation might result from the co-localization of phophorylated integrin with the appropriate phosphatase at the trailing edge. Although quite simplistic, this mechanism, or a similar one, might explain the observations of Maxfield's group. Will it be possible to test these hypotheses? It should be possible to identify or construct mutations in the integrin that alter its on-rate and/or off-rate for ligand. Indeed, mutations which increase and decrease over-all ka for ligand have already been identified. These mutations are in the ectodomain and are not expected to disrupt interactions with cytoskeletal components. Hence, the effects of these and similar mutations on association and dissociation rate can be measured and then correlated with their effects on cell motility. It will be interesting to assess how integrins bearing such mutations recycle between the leading and trailing edges of motile cells. Is it possible that the divalent ion binding sites also influence outside-in signaling? Outside-in signaling is regulated both by receptor clustering and by occupation of the iigand binding pocket (Miyamoto et al., 1995). But several lines of evidence show that ion binding and ligand binding can be mutually exclusive (D'Souza et al., 1994; Hu et al., 1996). Therefore, do we need to consider the possibility that ion binding to integrin can also generate intracellular signals? Are integrins only able to sense ligand contact, or are they also sensing bound metal ion? A recent study by Blystone et al. (1995) on the ~v[33 integrin speaks to this issue. In that report, it was demonstrated that the cytoplasmic domain of the 1~3 subunit could be phosphorylated upon ligand binding or by incubating the cells with Mn 2÷ ion, an ion known to bind to ~v[33 with uM affinity. Results from that study also showed that the SH2-binding adapter protein Grb-2 could bind to the c~v]33 integrin when cells were exposed to Mn 2÷. Both observations can be taken as support for the notion that ion binding to the 0~vl~3 integrin may be sufficient to initiate a series of signaling cascades.

Are Integrins Activated By Proteolytic Cleavage of the Cytop|asmic Domains? Most of the study of integrin function assumes that the integrin cytoplasmic tails are intact. We recently

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made an observation that forced us to question this idea. To understand the mechanism of integrin activation, we set out to purify the active and dormant conformers of the platelet integrin aIIbp3 from platelet lysates. Prior studies have outlined the purification protocols for these of IIb-IIIa (Fitzgerald et al., 1985; two “isoforms” Pytela et al., 1987; Kouns et al., 1992). The active form of the integrin can be purified by ligand affinity chromatography. The dormant form is recovered by a series of conventional chromatographic separations. We have recently confirmed that both forms can be purified to homogeneity and that their ligand binding properties are quite distinct. The “active” conformer is capable of binding to fibrinogen with high affinity. The dormant conformer of cxIIbP3 cannot bind fibrinogen, although it does bind to model RGD ligands like Fab-9 (Barbas et al., 1993; Smith et al., 1994). As previously reported, we found that the active and dormant conformers migrate identically on SDS-PAGE. Both are recognized by antibodies against the IIb-IIIa complex, and they have identical N-terminal amino acid sequences. All of these data indicate that the two proteins differ only in conformation. However, we were unable to convert the dormant conformer of cxIIbp3 to the active state using numerous denaturants and salt treatments. This led us to suspect that the distinction between the dormant and active integrin is more than a conformational difference. Indeed, we discovered that polyclonal antibodies raised against a peptide encompassing the entire cytoplasmic domain of the p3 subunit

could bind to the dormant integrin, but they are unable to bind the active integrin. This finding indicates that the structures of the cytoplasmic tails of dormant and active integrin are distinct. Although there are several possible modifications that could explain the loss of immune reactivity, including phosphorylation, we favor the hypothesis that the cytoplasmic domain of the active integrin is proteolytically cleaved (Fig. 2). There are several reports in the literature that can be taken as support for the hypothesis that aIIbb3 could be activated by proteolytic cleavage of the cytoplasmic tail of p3. First, Kouns found that the truncation of large segments from the C-terminal domain of the purified cxIIbp3 was able to activate purified aIIbp3 (Kouns et al., 1992). Hence, it is clear that a constraint found in the C-terminal domain can be removed by cleavage. Second, Du and Ginsberg showed that the vast majority of the p3 present in platelets is cleaved by calpain coincident with platelet aggregation (Du et al., 1995). Although these data were interpreted to have implications for events that follow ligand binding (i.e., clot retraction and outside-in signaling), they are also consistent with the idea that crIIbp3 could be activated by calpain cleavage. Indeed, Fox et al. (1993) demonstrated that, upon treatment of platelets with agonist, u-calpain becomes localized beneath the plasma membrane, in a position to cleave the integrin tails. The most direct evidence in support of the proteolytic activation hypothesis comes from a recent report by Inomata et al. (1996), which showed that E64d, an inhibitor of calpain that

Cleavage of Cytopiasmic Tail \\

C

C

New binding partners

& Cytoskeietai + signaling complex

Figure 2. The proteolytic hypothesis. An integrin is shown in complex cytoplasm. cytoplasm. their own.

I u”,

I N

? Signals

0’ Cytoskeietai + signaling complex

C

with cytoskeletal and signaling components in the Cleavage of the cytoplasmic tails with protease is likely to disrupt these linkages and present a new C-terminus to the The new C-terminus may bind with new partners. Additionally, the released peptides might be signaling molecules on

Regulation of integrin function can cross the plasma membrane, can prevent platelet aggregation. This indicates that inhibition of platelet calpain prevents the activation of c~IIb]33. Collectively, all of these data indicate that one way in which integrins could be "activated" is by proteolytic cleavage in their cytoplasmic tails. The "proteolytic hypothesis" is also consistent with the "integrin hinge hypothesis" put forth by Hughes et al. (1996). The hinge hypothesis suggests that a key salt bridge between the cytoplasmic tail of the integrin 0~ and 13 subunits maintains the integrin in a low affinity, or dormant, state. Changes which disrupt this salt bridge activate the integrin. The removal of one or both cytoplasmic tails by proteolysis would eliminate this structural constraint. Indeed, even the limited cleavage on one cytoplasmic tail may induce sufficient conformational change to break the integrin hinge. A proteolytic cleavage could ensure that activation is irreversible. Interestingly, the activation of (xIIb~33 on platelets can be either reversible or permanent. Activation can be reversed when the agonist is not of sufficient strength, like ADP or platelet activating factor (van Willigen and Akkerman, 1991). On the other hand, strong agonists like Ix-thrombin place the integrin in a permanently activated state (van Willigen and Akkerman, 1992). Clearly, proteolytic cleavage cannot be reversed in the cytoplasm, so any such modifications to the cytoplasmic domains would be expected to be permanent. Thus, activation may proceed in two sequential steps, the first step toward activation being reversible and the second, consisting of proteolysis and following from the first, being permanent. Integrins also make important connections with the cytoskeleton through their cytoplasmic tails. It is not evident how proteolytic modification of cytoplasmic tails would impact on these linkages. The removal of substantial portions of the cytoplasmic tails is likely to eliminate binding to cytoskeletal components. Less "severe" cleavages may preserve the cytoskeletal linkages, but still alter the conformation of the integrin enough to cause a change in ligand binding affinity. The impact of proteolysis of the cytoplasmic domains on outside-in signaling could also be profound. For example, is it possible that the peptides released from the integrin as a result of cleavage are independent signaling molecules? Several reports demonstrate that fusion proteins containing the cytoplasmic domain of integrin 13 subunits can localize in adhesion plaques and also induce cellular de-adhesion (Akiyama et al., 1994; Chen et al., 1994; LaFlamme et al., 1994; Lukashev et al., 1994). Perhaps proteolysis releases such functional cytoplasmic

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domains to operate independent of the rest of the integrin. Alternatively, the new C-termini generated by cleavage may interact with an entirely different set of adapter and signaling proteins or even with the nucleus. Although at present these ideas are purely speculative, the known cleavage of the cytoplasmic domains (Potts et al., 1994; D u e t al., 1995) suggests that such concepts warrant further investigation. Is the proteolytic hypothesis of activation testable? If one is able to identify precise cleavage points within the integrin tails, it may be possible to mutate these sites so that they are no longer recognized by protease. One would hypothesize that integrins harboring such mutations would not be cleaved and could not be activated, or at least not permanently activated. Alternatively, it may be possible to identify cytoplasmic proteases that cleave the integrin tails and determine if inhibitors of these proteases prevent activation. Clearly, this approach has already indicated that integrins are likely to be physiologic substrates for the protease calpain (Inomata et al., 1996), but other proteases should not be excluded at this juncture.

Acknowledgments This work was supported by grants from the National Cancer Institute (CA56483) and the National Institute for Arthritis and Musculoskeletal and Skin Disease (AR42750). Jeffrey W. Smith is an Established Investigator from the American Heart Association and Genentech. Special thanks to Olivier Morand at Roche, who suggested during a phone conversation that the cleaved integrin tail may be a signaling molecule.

References Akiyama, S.K., Yamada, S.S., Yamada, K.M. and LaFlamme, S.E.: Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J. Biol. Chem. 269: 15961-15964, 1994. Bajt, M.L., Goodman,T. and McGuire, S.L.: 132 (CD18) mutations abolish ligand recognition by I domain integrins LFA-1 (~L~2, CD11a/CD18) and MAC-1 (~M[32, CD11b/CD18). J. Biol. Chem. 270: 94-98, 1995. Barbas, C.E, Languino, L.R. and Smith, J.W.: High-affinity self-reactive human antibodies by design and selection: targeting the integrin ligand binding site. Proc. Natl. Acad. Sci. U S A 90: 10003-10007, 1993. Blystone, S.D., Lindberg, EP., LaFlamme, S.E. and Brown, E.J." Integrin 133 cytoplasmic tail is necessary and sufficient for regulation of ~x5131phagocytosis by avb3 and integrin-associated protein. J. Cell Biol. 130: 745-754, 1995. Chen, Y.E, O'Toole, T.E., Shipley, T., Forsyth, J., LaFlamme, S.E., Yamada, K.M., Shattil, S.J. and Ginsberg, M.H.: Insideout signal transduction inhibited by isolated integrin cytoplasmic domains. J. Biol. Chem. 269: 18307-18310, 1994.

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D'Souza, S.E., Haas, T.A., Piotrowicz, R.S., Byers-Ward, V., McGrath, D.E., Soule, H.R., Ciernieswki, C., Plow, E.E and Smith, J.W." Ligand and cation binding are dual functions of a discrete segment of the integrin [53 subunit: cation displacement from this site is implicated in ligand binding. Cell 79: 659-667, 1994. Dransfield, I., Cabanas, C., Craig, A. and Hogg, N.: Divalent cation regulation of the function of the leukocyte integrin LFA-1. J. Cell Biol. 116: 219-226, 1992. Du, X., Saido, T.C., Tsubuki, S., Indig, EE., Williams, M.J. and Ginsberg, M.H.: Calpain cleavage of the cytoplasmic domain of the integrin [33 subunit. J. Biol. Chem. 270: 26146-26151, 1995. Fitzgerald, L.A., Leung, B. and Phillips, D.R.: A method for purifying the platelet membrane glycoprotein IIb-IIIa complex. Anal. Biochem. 151: 169-177, 1985. Fox, J.E., Taylor, R.G., Taffarel, M., Boyles, J.K. and Goll, D.E.: Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIla. J. Cell Biol. 120: 1501-1507, 1993. Hendey, B., Klee, C.B. and Maxfield, ER.: Inhibition of neutrophil chemokinesis on vitronectin by inhibitors of calcium. Science 258: 296-299, 1992. Hu, D.D., Barbas, C.E and Smith, J.W.: An allosteric Ca 2" binding site on the [33-integrins that regulates the dissociation rate for RGD ligands. J. Biol. Chem. 271: 21745-21751, 1996. Hu, D.D., Hoyer, J.R. and Smith, J.W.: Ca 2" suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin 0~v133.J. Biol. Cbem. 270: 9917-9925, 1995. Hughes, EE., Diaz-Gonzalez, E, Leong, L., Wu, C., McDonald, J.A., Shattil, S.J. and Ginsberg, M.H.: Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J. Biol. Chem. 271: 6571-6574, 1996. Hynes, R.O.: Integrins: versatility, modulation and signaling in cell adhesion. Cell 69: 11-25, 1992. Inomata, M., Hayashi, M., Ohno-Iwashita, Y., Tsubuki, S,, Saido, T.C. and Kawashima, S.: Involvement of calpain in integrin-mediated signal transduction. Arch. Biochem. Biophys. 328: 129-34, 1996. Kamata, T., Puzon, W. and Takada, Y." Identification of putative ligand-binding sites of the integfin (x4131 (VLA-4, CD49d/CD29). Biochem. J. 305: 945-951, 1995. Kouns, W.C., Hadvary, P. and Steiner, B.: Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-llla. J. Biol. Chem. 267: 18844-18851, 1992. LaFlamme, S.E., Thomas, L.A., Yamada, S.S. and Yamada, K.M.: Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J.Cell Biol. 126: 1287-1298, 1994. Lawson, M.A. and Maxfield, ER.: Ca 2÷- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. Nature 377: 75-79, 1995. Lin, E.C.K., Ramikov, B.I., Tsai, P.M., Gonzalez, R., McDonald, S., Pelletier, A.J. and Smith, J.W.: Evidence that the inte-

grin 133 and [55 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain. J. Biol. Chem. 272: 14236-14243, 1997. Loftus, J.C., O'Toole, T.E., Plow, E.E, Glass, A., Frelinger, A.L. and Ginsberg, M.H.: A [53 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 249: 915-918, 1990. Lukashev, M.E., Sheppard, D. and Pytela, R.: Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed 61 integrin cytoplasmic domain. J. Biol. Chem. 269: 18311-18314, 1994. Miyamoto, S., Akiyama, S.K. and Yamada, K.M.: Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267: 883-885, 1995. Mould, A.P., Akiyama, S.K. and Humphries, M.J.: Regulation of integrin a5bl-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn 2., Mg -'~, and Ca 2÷.J. Biol. Chem. 270: 26270-26277, 1995. Potts, A.J., Croall, D.E. and Hemler, M.E.: Proteolytic cleavage of the integrin 134 subunit. Exp. Cell Res. 212: 2-9, 1994. Puzon-McLaughlin, W. and Takada, Y.: Critical residues for ligand binding in an I domain-like structure of the integrin []1 subunit. J. Biol. Chem. 271: 20438-20443, 1996. Pytela, R., Pierschbacher, M.D., Argraves, S., Suziki, S. and Ruoslahti, E.: Arginine-Glycine-Aspartic acid adhesion receptors. Meth. Enzymol. 144: 475-489, 1987. Ruoslahti, E.: Integrin signaling and matrix assembly. Tumor Biol. 17: 117-124, 1996. Segal, I.: Enzyme Kinetics. John Wiley and Sons, New York, 1975. Smith, J.W., Hu, D., Satterthwait, A., Pinz-Sweeney, S. and Barbas, C.E: Building synthetic antibodies as adhesive ligands for integrins. J. Biol. Chem. 269: 32788-32795, 1994. Staatz, W.D., Rajpara, S.M., Wayner, E.A., Carter, W.G. and Santoro, S.A.: The membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg++-dependent adhesion of platelets to collagen. J.Cell Biol. 108: 1917-1924, 1989. Tozer, E.C., Liddington, R.C., Sutcliffe, M.J., Smeeton, A.H. and Loftus, J.C.: Ligand binding to integrin c~IIb[]3 is dependent on a MIDAS-like domain in the [33 subunit. J. Biol. Chem. 271: 21978-21984, 1996. van Willigen, G. and Akkerman, J.W.: Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets. Biochem. J. 273: 115-120, 1991. van Willigen, G. and Akkerman, J.W.: Regulation of glycoprotein IIB/IIIA exposure on platelets stimulated with alphathrombin. Blood 79: 82-90, 1992. Yamada, K.M. and Miyamoto, S.: Integrin transmembrane signaling and cytoskeletal control. Curr. Opin. Cell Biol. 7: 681-689, 1995. Dr. Jeffrey W. Smith, The Cancer Research Center, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92130 Received July 2, 1997