Integrin structures and conformational signaling

Integrins are cell adhesion molecules that play critical roles in development, wound healing, hemostasis, immunity and cancer. Advances in the past two years have shed light on the structural basis for integrin regulation and signaling, especially on how global conformational changes between bent and extended conformations relate to the inter-domain and intra-domain shape shifting that regulates affinity for ligand. The downward movements of the C-terminal helices of the alpha I and beta I domains and the swing-out of the hybrid domain play pivotal roles in integrin conformational signaling. Experiments have also shown that integrins transmit bidirectional signals across the plasma membrane by coupling extracellular conformational change with an unclasping and separation of the alpha and beta transmembrane and cytoplasmic domains.     

Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.     

Trans-dominant inhibition of integrin function.

Occupancy of integrin adhesion receptors can alter the functions of other integrins and cause partition of the ligand-occupied integrin into focal adhesions. Ligand binding also changes the conformation of integrin extracellular domains. To explore the relationship between ligand-induced conformational change and integrin signaling, we examined the effect of ligands specific for integrin alpha IIb beta 3 on the functions of target integrins alpha 5 beta 1 and alpha 2 beta 1. We report that binding of integrin-specific ligands to a suppressive integrin can inhibit the function of other target integrins (trans-dominant inhibition). Trans-dominant inhibition is due to a blockade of integrin signaling. Furthermore, this inhibition involves both a conformational change in the extracellular domain and the presence of the beta cytoplasmic tail in the suppressive integrin. Similarly, ligand-induced recruitment of alpha IIb beta 3 to focal adhesions also involves a conformational rearrangement of its extracellular domain. These findings imply that the ligand-induced conformational changes can propagate from an integrin's extracellular to its intracellular face. Trans-dominant inhibition by integrin ligands may coordinate integrin signaling and can lead to unexpected biological effects of integrin-specific inhibitors.     

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.     

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.     

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.     

Integrin bi‐directional signaling across the plasma membrane

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi‐directional signals across the plasma membrane. Inside‐out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside‐in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi‐directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside‐out and outside‐in signaling with specific focus on the mechanism how integrins transmit bi‐directional signals through transmembrane/cytoplasmic domains. J. Cell. Physiol. 228: 306–312, 2013. © 2012 Wiley Periodicals, Inc.     

Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.     

Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal

We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.     

Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state

The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha(L)beta(2)) or Mac-1 (alpha(M)beta(2)) with mutations in the cytoplasmic domain. Complete truncation of the beta(2) cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha(L)beta(2) and alpha(M)beta(2), demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha(L) and beta(2) cytoplasmic domains with acidic and basic peptides that form an alpha-helical coiled coil caused inactivation of alpha(L)beta(2). Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha(L) and beta(2) cytoplasmic domains with two basic peptides that do not form an alpha-helical coiled coil activated alpha(L)beta(2). Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha and beta subunits, constrains an integrin in the inactive conformation.     

A role for caveolin and the urokinase receptor in integrin-mediated adhesion and signaling

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with beta1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with beta1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes beta1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, beta1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with beta1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal beta1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.     

Selective recruitment of src family kinase Hck by leukocyte integrin alphaMbeta2 but not alphaLbeta2 or alphaXbeta2

Integrins are type I heterodimeric (alpha/beta) cell adhesion molecules. They trigger cell-signaling by recruiting cytosolic molecules to their cytoplasmic tails. Integrin alpha cytoplasmic tail contributes towards integrin function specificity, an important feature of integrins having different alpha subunits but sharing the same beta subunit. Herein, we show that the src family kinase Hck co-capped selectively with leukocyte integrin alpha(M)beta(2) but not alpha(L)beta(2) or alpha(X)beta(2). This was disrupted when the alpha(M) cytoplasmic tail was substituted with that of alpha(L) or alpha(X). Co-capping was recovered by alpha(L) or alpha(X) cytoplasmic tail truncation or forced separation of the alpha and beta cytoplasmic tails via salt-bridge disruption.     

Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions (review)

Integrins are heterodimeric membrane-spanning adhesion receptors that are essential for a wide range of biological functions. Control of integrin conformational states is required for bidirectional signalling across the membrane. Key components of this control mechanism are the transmembrane and cytoplasmic domains of the alpha and beta subunits. These domains are believed to interact, holding the integrin in the inactive state, while inside-out integrin activation is accompanied by domain separation. Although there are strong indications for domain interactions, the majority of evidence is insufficient to precisely define the interaction interface. The current best model of the complex, derived from computational calculations with experimental restraints, suggests that integrin activation by the cytoplasmic protein talin is accomplished by steric disruption of the alpha/beta interface. Better atomic-level resolution structures of the alpha/beta transmembrane/cytoplasmic domain complex are still required for the resting state integrin to corroborate this. Integrin activation is also controlled by competitive interactions involving the cytoplasmic domains, particularly the beta-tails. The concept of the beta integrin tail as a focal adhesion interaction 'hub' for interactions and regulation is discussed. Current efforts to define the structure and affinity of the various complexes formed by integrin tails, and how these interactions are controlled, e.g. by phosphorylation and localization, are described.     

alpha 2beta 1 integrin is not recognized by rhodocytin but is the specific, high affinity target of rhodocetin, an RGD-independent disintegrin and potent inhibitor of cell adhesion to collagen

We have recombinantly expressed a soluble form of human alpha(2)beta(1) integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble alpha(2)beta(1) integrin binds to its collagen ligands the same way as the wild-type alpha(2)beta(1) integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of alpha(2)beta(1) integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble alpha(2)beta(1) integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble alpha(2)beta(1) integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble alpha(2)beta(1) integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to alpha(2)beta(1) integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with alpha(2)beta(1) integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to alpha(2)beta(1) integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking alpha(2)beta(1) integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, alpha(2)beta(1) integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, alpha(2)beta(1) integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit alpha(2)beta(1) integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.     

Unraveling ICAP-1 function: Toward a new direction?

Cell adhesion to either the extracellular matrix (ECM) or to neighboring cells is of critical importance during both physiological and pathological situations. Integrins are a large family of cell adhesion receptors composed of two non-covalently linked alpha and beta subunits. They have a well-identified dual function of mediating both firm adhesion and signaling. The short cytoplasmic domain of integrin can interact with cytoplasmic proteins that are either shared by several different integrins or specific for one type of integrin. Integrin cytoplasmic domain-associated protein-1 (ICAP-1) is a small cytoplasmic protein that specifically interacts with the beta1 integrin subunit. In this review we will discuss recent findings on ICAP-1, not only at the structural and functional level, but also its possible interconnection in other signaling pathways such as those that control cell proliferation.     

C-terminal opening mimics 'inside-out' activation of integrin alpha5beta1

Integrins are adhesion molecules that convey signals both to and from the cytoplasm across the plasma membrane. In resting cells, integrins in a low affinity state can be activated by 'inside-out signaling', in which signals affecting integrin heterodimer cytoplasmic domains cause a conformational change in the integrin ligand-binding headpiece connected to the membrane by two long, approximately 16 nm stalks. Here we demonstrate a mechanism for conveying a conformational change over the long distance from the plasma membrane to the headpiece. We prepared soluble, alpha5beta1 integrin heterodimer extracellular fragments in which interactions between alpha- and beta-subunit cytoplasmic domains were replaced with an artificial clasp. Release of this C-terminal clasp by specific protease cleavage resulted in an approximately 14 nm separation of the stalks coupled to increased binding to fibronectin. This activation did not require any associated molecules or clustering and was observed with physiological concentrations of divalent cations. These findings suggest that the overall mechanism for integrin inside-out activation involves the spatial separation of the cytoplasmic and/or transmembrane domains.     

Urokinase-type plasminogen activator receptor (CD87) is a ligand for integrins and mediates cell-cell interaction

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.     

CD98hc (SLC3A2) interaction with the integrin beta subunit cytoplasmic domain mediates adhesive signaling

In mammals, beta1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin beta subunit cytoplasmic domains (tails) via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc interaction with the Drosophila integrin betaPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of beta3 (Thr(755)-Thr(802)) in CD98hc binding. We then identified point mutations in the beta3 C terminus (T755K and T758M) that abolish CD98hc association and a double mutation in the corresponding residues in the betaPS tail (K839T,M842T), which resulted in gain of CD98hc interaction. Furthermore, the loss of function beta3(T755K) mutation or the gain of function beta3/betaPS(K839T,M842T) led to a loss or gain of integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction and in doing so have shown that CD98c interaction with the integrin beta tail is required for its ability to mediate integrin signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins to modulate integrin functions and into the evolution of integrin signaling.     

A coiled-coil structure of the alphaIIbbeta3 integrin transmembrane and cytoplasmic domains in its resting state

One of the hallmark features of the integrin receptors is the ability to transmit signals bidirectionally through the cell membrane. The transmembrane integrin domains are pivotal to the signaling events. An understanding of the signaling mechanism requires structural information. Here, we report a structural model of the transmembrane and part of the cytosolic domains of the alphaIIbbeta3 integrin in its resting state. The model was obtained computationally by a restrained conformational search of helix-helix interactions. It agrees with one published NMR structure of the cytoplasmic complex and can put many experimental findings on structural grounds. According to our model, integrins form an intricately designed coiled-coil structure in the resting state. The conserved Glycophorin A (GpA)-like sequence motif of the alpha, but not the beta, subunit, is in the interface of this model. Based on our calculations and other data, a signaling mechanism that involves a transient GpA-like structure is proposed.     

Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.