We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.

Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/ RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins.Increased affinity ("activation") of cellular integrins is central to physiological events such as cell migration, assembly of the extracellular matrix, the immune response, and hemostasis (1). Each integrin comprises a type I transmembrane ␣ and ␤ subunit, each of which has a large extracellular domain, a single transmembrane domain, and a cytoplasmic domain (tail). Talin binds to most integrin ␤ cytoplasmic domains and the binding of talin to the integrin ␤ tail initiates integrin activation (2-4). A small, PTB-like domain of talin mediates activation via a twosite interaction with integrin ␤ tails (5), and this PTB domain is functionally masked in the intact talin molecule (6). A central question in integrin biology is how the talin-integrin interaction is regulated to control integrin activation; recent work has implicated Ras GTPases as critical signaling modules in this process (7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound active form and the GDP-bound inactive form. Guanine nucleotide exchange factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas GTPase-activating proteins (GAPs) 3 enhance the hydrolysis of Ras-bound GTP to GDP (for review, see Ref. 8). The Ras subfamily members Rap1A and Rap1B stimulate integrin activation (9, 10). For example, expression of constitutively active Rap1 activates integrin ␣M␤2 in macrophage, and inhibition of Rap1 abrogated integrin activation induced by inflammatory agonists (11-13). Murine T-cells expressing constitutively active Rap1 manifest enhanced integrin dependent cell adhesion (14). In platelets, Rap1 is rapidly activated by platelet agonists (15, 16). A knock-out of Rap1B (17) Recently we used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway...

MDC-15 (ADAM-15, metargidin), a membrane-anchored metalloprotease/disintegrin/cysteine-rich protein, is expressed on the surface of a wide range of cells and has an RGD tripeptide in its disintegrin-like domain. MDC-15 is potentially involved in cell-cell interactions through its interaction with integrins. We expressed a recombinant MDC-15 disintegrin-like domain as a fusion protein with glutathione S-transferase (designated D-15) in bacteria and examined its binding function to integrins using mammalian cells expressing different recombinant integrins. We found that D-15 specifically interacts with ␣v␤3 but not with the other integrins tested (␣2␤1, ␣3␤1, ␣4␤1, ␣5␤1, ␣6␤1, ␣6␤4, ␣v␤1, ␣IIb␤3, and ␣L␤2). Mutation of the tripeptide RGD to SGA totally blocked binding of D-15 to ␣v␤3, suggesting that D-15-␣v␤3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is recognized by both ␣IIb␤3 and ␣v␤3, suggesting that the receptor binding specificity is mediated by the sequence flanking the RGD tripeptide, as in snake venom disintegrins. These results indicate that the disintegrin-like domain of MDC-15 functions as an adhesion molecule and may be involved n ␣v␤3-mediated cell-cell interactions.Metalloprotease/disintegrin/cysteine-rich proteins (MDCs, also called ADAMs) 1 are membrane-anchored proteins with several domains including a metalloprotease domain, a disintegrin-like domain, a cysteine-rich sequence, an epidermal growth factor-like sequence, a transmembrane domain, and a short cytoplasmic domain (1). The biological functions of MDCs are not clear; however, we do know that fertilins (MDC-1 and -2) (2) are involved in sperm-egg binding and fusion (3), meltrins (MDC-12) (4) are involved in myoblast fusion during muscle development, and KUZ (a Drosophila MDC protein) (5) assists in neurogenesis. The MDC cytoplasmic domain has a proline-rich potential SH3 binding motif, suggesting that MDCcounter receptor interaction may induce signal transduction.Integrins are a family of cell adhesion receptors that bind to a variety of ligands, including extracellular matrix proteins and other cell surface molecules (6 -10). MDCs are potential ligands for integrins, since most snake venom disintegrins interact with integrins ␣IIb␤3 and ␣v␤3 (reviewed in Ref. 11 and references therein). However, little is known about the receptor specificity of MDCs, except that mouse egg integrin ␣6␤1 has been proposed as a receptor for fertilin (2). Evans et al. (12) recently expressed recombinant fertilin fragments in bacteria as fusion proteins with maltose-binding protein (12). The recombinant fertilin-␤ fragment has been shown to bind to the egg membrane to which sperm bind and to block sperm from binding to the egg. These results suggest that the disintegrinlike domains of MDCs may be properly folded in bacteria, that glycosylation of the disintegrin-like domain may not be required for interaction with receptors, and that a strategy using recombinant MDC proteins is a viable alternative to those using puri...

ADAMs (a disintegrin and metalloproteases) mediate several important processes (e.g. tumor necrosis factor-alpha release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin alpha(9)beta(1) specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and alpha(9)beta(1) supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for alpha(9)beta(1)/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/alpha(6)beta(1) interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that alpha(9)beta(1) may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and alpha(9)beta(1), this interaction may be of potential biological and pathological significance.

Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin ␣v␤3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while ␣v␤1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the ␤1 and ␤3 subunits. When the sequence CTSEQNC (residues 187-193) of ␤1 is replaced with the corresponding CYD-MKTTC sequence of ␤3, the ligand specificity of ␣v␤1 is altered. The mutant (␣v␤1-3-1), like ␣v␤3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The ␣v␤1-3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal ␤3-1-3 mutation blocks binding of ␣v␤3 to these multiple ligands and to LM609, a function-blocking anti-␣v␤3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of ␤, in which the sequence is located in the ligand binding site.Integrins are a family of ␣/␤ heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions (1-5). Integrin-ligand interactions are critically involved in the pathogenesis of many diseases in human and animal models. Although integrin-ligand interaction is a therapeutic target, we poorly understand at the molecular level how integrins recognize multiple ligands. Evidence suggests that the I or A domain, a set of inserted sequences consisting of about 200 amino acid residues, of several integrin ␣ subunits (␣M, ␣L, ␣1, ␣2) is important in ligand binding and receptor activation (reviewed in Ref. 6 and references therein). The presence of an I domain-like structure within the ␤ subunit has been suggested based on the similarity in hydropathy profiles between the I domain and part of the ␤ subunit (7). Interestingly, this region of ␤ has been reported to be critical for ligand binding and its regulation (reviewed in Ref. 8) (Fig. 1). The Asp-119 (␤3) (9) and Asp-130 (␤1) (10, 11) and the corresponding residues in ␤2 and ␤6 are critical for ligand binding (12, 13). A synthetic peptide of ␤3 (MDLSYSMKDDLWSI, residues 118 -131) has been shown to produce a ternary complex with cations and ligand (14). Also, the sequence DDLW (residues 126 -129 of ␤3) was shown to be critical for interaction with the RGD sequence using a phage display system (15). A synthetic peptide of ␤3, DAPEGGFDAIMQATV (residues 217-231 of ␤3), has been shown to bind to immobilized fibrinogen (Fg), 1 von Willebrand's factor (vWf), and fibronectin (Fn) (16,17). A synthetic peptide of ␤3, SVSRNRDAPEG (residues 211-221 of ␤3), has been reported to block binding of Fg to ␣IIb␤3 (18, 19). We identified a small region of ␤1 (residues 207-218, a regulatory epitope) that is recognized by both activating and inhibiting anti-␤1 antibodies (...

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)‐1 and fibronectin (CS‐1). The alpha 4 beta 1‐ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine‐scanning mutagenesis of the previously localized putative ligand binding sites (residues 108–268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM‐1 and CS‐1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta‐turn structure (residues 181–190) of the third N‐terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin‐alpha 5 beta 1 interaction. These findings suggest that the predicted beta‐turn structure could be ubiquitously involved in ligand binding of non‐I domain integrins.

Integrin ␣ IIb ␤ 3 , a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with ␣ IIb ␤ 3 has been controversial. Ligand-mimetic anti-␣ IIb ␤ 3 antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in ␣ IIb and ␤ 3 using human-to-mouse chimeras, which we expect to maintain functional integrity of ␣ IIb ␤ 3 . Here we report that these antibodies recognize several discontinuous binding sites in both the ␣ IIb and ␤ 3 subunits; these binding sites are located in residues 156 -162 and 229 -230 of ␣ IIb and residues 179 -183 of ␤ 3 . In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2-3 but not repeats 4 -7, of ␣ IIb ). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous binding sites in both subunits, which may constitute a ligand-binding pocket.Integrin ␣ IIb ␤ 3 is a platelet fibrinogen receptor that is critically involved in platelet aggregation (1). Thus ␣ IIb ␤ 3 -fibrinogen interaction is a therapeutic target for thrombosis and hemostasis. However, how ligands interact with the integrin ␣ IIb and ␤ 3 subunits has been the subject of much discussion.The ␣ IIb subunit has seven repeated sequences of 60 -70 residues each in its N-terminal portion. Two different regions of the ␣ IIb subunit have been implicated in ligand binding. The second metal binding site of ␣ IIb (residues 294 -314 in N-terminal repeat 5 of ␣ IIb ) has been identified as a ligand binding site by chemically cross-linking the ␥-peptide (HHLGGAKQ-AGDV 400 -411 ) of the fibrinogen ␥ chain C-terminal domain (2). Both the peptide derived from this ␣ IIb sequence and its antibodies have been shown to block fibrinogen binding to ␣ IIb ␤ 3 (3). Consistently, recombinant bacterial proteins that consist of repeats 4 -7 of ␣ IIb (residues 171-464) have been shown to bind to ligands in a cation-dependent manner (4). On the other hand, alanine-scanning mutagenesis (5) 1 suggests that the predicted loops in repeats 2 and 3 are critical for ligand binding. Also, function-blocking anti-␣ IIb ␤ 3 monoclonal antibodies (mAbs) 2 are mapped in repeats 2 and 3 (5). 1 It has not been established which regions of ␣ IIb actually interact with ligands.The presence of an I-domain-like structure within the ␤ subunit has been suggested based on the similarity in hydropathy profiles between the I-domain of the ␣ M subunit and part of the ␤ subunit (6). The N-terminal region of the ␤ 3 subunit has components that are critical for ligand...

The molecular mechanisms that regulate integrin–ligand binding are unknown; however, bivalent cations are essential for integrin activity. According to recent models of integrin tertiary structure, sites involved in ligand recognition are located on the upper face of the seven-bladed β-propeller formed by the N-terminal repeats of the α subunit and on the von Willebrand factor A-domain-like region of the β subunit. The epitopes of function-altering monoclonal antibodies (mAbs) cluster in these regions of the α and β subunits; hence these mAbs can be used as probes to detect changes in the exposure or shape of the ligand-binding sites. Bivalent cations were found to alter the apparent affinity of binding of the inhibitory anti-α5 mAbs JBS5 and 16, the inhibitory anti-β1 mAb 13, and the stimulatory anti-β1 mAb 12G10 to α5β1. Analysis of the binding of these mAbs to α5β1 over a range of Mn2+, Mg2+ or Ca2+ concentrations demonstrated that there was a concordance between the ability of cations to elicit conformational changes and the ligand-binding potential of α5β1. Competitive ELISA experiments provided evidence that the domains of the α5 and β1 subunits recognized by mAbs JBS5/16 and 13/12G10 are spatially close, and that the distance between these two domains is increased when α5β1 is occupied by bivalent cations. Taken together, our findings suggest that bivalent cations induce a conformational relaxation in the integrin that results in exposure of ligand-binding sites, and that these sites lie near an interface between the α subunit β-propeller and the β subunit putative A-domain.