Divalent Cations Differentially Regulate Integrin αIIb Cytoplasmic Tail Binding to β3 and to Calcium- and Integrin-binding Protein

Abstract

We have used recombinant or synthetic ␣ IIb and ␤ 3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. ␣⅐␤ heterodimerization occurred in a 1:1 stoichiometry with a weak K D in the micromolar range. Divalent cations were not required for this association but stabilized the ␣⅐␤ complex by decreasing the dissociation rate. ␣⅐␤ complexation was impaired by the R995A substitution or the KVGFFKR deletion in ␣ IIb but not by the ␤ 3 S752P mutation. Integrins are ␣␤ heterodimeric cell-surface receptors that promote not only adhesion to components present within the extracellular matrix or on the surface of opposite cells but also transfer information into and out of a cell (1). The adhesive functions of integrins can be regulated by intracellular processes referred to as "inside-out signaling." Conversely, ligand binding to the extracellular domain of integrins initiates a cascade of intracellular events termed "outside-in signaling" that generate a large spectrum of cellular responses, such as cell migration, proliferation, differentiation, and gene expression (2). Integrin cytoplasmic tails appear to be key elements in these bidirectional signaling pathways, despite their short size as compared with other signaling receptors and the absence of any demonstrable catalytic activity (3, 4). Integrin ␣ and ␤ cytoplasmic domains are thought to mediate signaling events through modifications of their own structural and spatial organization and/or through interactions with specific cytoplasmic components. Various proteins have been identified that bind, at least in vitro, to the cytoplasmic tail of ␣ and ␤ subunits and are likely to play a role in regulating integrin signaling functions. These include cytoskeletal components such as talin and ␣-actinin, as well as several signaling or regulatory proteins such as integrin-linked kinase p59 ILK , focal adhesion kinase pp125FAK , Grb2, ␤ 3 -endonexin, cytohesin-1, integrin cytoplasmic domain-associated protein ICAP-1, calreticulin and calcium-and integrin-binding protein CIB 1 (reviewed in Refs. 5 and 6).Recently used methods for studying protein-protein interactions, such as the two-hybrid system, have allowed the identification of integrin-specific intracellular ligands (7-12). These methods are based on the use of a unique linear amino acid sequence as a bait and consequently do not take into account the secondary and tertiary structural features of the interacting molecules. However, numerous studies tend to demonstrate that ␣ and ␤ cytoplasmic domains adopt a defined conformation and that the preservation of these structural constraints is crucial to maintain the functional properties of integrin receptors (13)(14)(15)(16)(17)(18)(19)(20).One of the best studied integrins is the platelet fibrinogen receptor, integrin ␣ IIb ␤ 3 , that undergoes conformational changes necessary for receptor function. In order to elucidate further the structural relationship of the cytoplasmic tails of ␣ IIb and ␤ 3 , we...

The MIG-2/Integrin Interaction Strengthens Cell-Matrix Adhesion and Modulates Cell Motility

“…It is widely accepted that interactions of talin-H with the membrane proximal region and consequently separation of the membrane proximal "clasp" are final intracellular steps in integrin activation (7)(8)(9)(11)(12)(13). On the other hand, the membrane distal region appears to regulate integrin activation indirectly (28,51). On the basis of these considerations, we propose that MIG-2 promotes integrin activation through an indirect mechanism.…”

A structural basis for integrin activation by the cytoplasmic tail of the α IIb -subunit

“…5a), or with both. It is noteworthy that studies of complex formation have implicated both the C-and N-terminal regions of the ␣ IIbcytoplasmic tail in interactions with ␤ 3 -cytoplasmic tail (6,12,27,28). Opening of the switch by either protein-protein interaction (Fig.…”

“…A proposed Ca 2ϩ -binding site is also shown in the figure involving R997, E1001, D1003, and D1004, which were identified by comparing the two-dimensional 1 H total correlation spectroscopy spectra of Ca 2ϩ -free and Ca 2ϩ -bound forms (Ca 2ϩ was added up to 5-fold). The binding of Ca 2ϩ to the ␣IIb-tail has been shown by biochemical studies (12,27). (B) Backbone overlay of m-␣ IIb-wt (pink) and m-␣IIb-mut (yellow), showing the structural difference.…”

Oligomerization of the integrin αIIbβ3: Roles of the transmembrane and cytoplasmic domains

“…It has been proposed that a salt-bridge involving a membrane-proximal hinge region, which encompasses a conserved GFFKR motif in ␣IIb and a LLITIHD motif in ␤3, maintains ␣IIb␤3 in an inactive state (5). Although ␣IIb and ␤3 CYTO peptides were initially reported to interact (6,7), more recent studies on related systems failed to show a specific interaction (10). Our data on the full TM-CYTO domain constructs are consistent with this recent article, showing a lack of heteromeric interaction.…”

“…1). These peptides are significantly longer than the CYTO peptides used in previous studies (6,7,16,17), and hence may provide a more realistic model for exploring their interactions. …”