Homomeric and heteromeric interactions between the ␣IIb and ␤3 transmembrane domains are involved in the regulation of integrin ␣IIb␤3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of ␣IIb␤3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the ␣IIb transmembrane domain. One mutation that enhanced the dimerization of the ␣IIb transmembrane domain indeed induced constitutive ␣IIb␤3 activation. However, a series of mutations that disrupted homodimerization also led to ␣IIb␤3 activation. These results suggest that the homo-and heterodimerization motifs overlap in the ␣IIb transmembrane domain, and that mutations that disrupt the ␣IIb͞␤3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for ␣IIb␤3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an ␣IIb͞␤3 transmembrane domain heterodimer and by stabilizing the resulting ␣IIb and ␤3 transmembrane domain homodimers.␣IIb␤3 ͉ integrin regulation ͉ transmembrane domains I ntegrins reside on cell surfaces in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor integrins in cell membranes (1). At the cellular level, integrin activation is regulated by cellular agonists, but how this occurs is uncertain. In the case of the platelet integrin ␣IIb␤3, membrane-proximal segments of the ␣IIb and ␤3 cytoplasmic (CYT) domains are thought to directly interact to constrain the integrin in an inactive state (2). Agonist-stimulated talin binding to the ␤3 CYT domain may relieve this constraint, inducing ␣IIb␤3 activation (3).The ␣IIb and ␤3 transmembrane domains are also in proximity when the integrin is inactive and separate upon integrin activation (4). Moreover, these domains readily undergo homomeric interactions in micelles (5), and both homomeric and heteromeric interactions have been detected in biological membranes (6, 7). Thus, in the platelet membrane where the concentration of these domains is high, the ␣IIb and ␤3 helices might be expected to form homooligomers in the activated state, crosslinking individual molecules and stabilizing focal adhesions (8). Indeed, we tested this possibility previously by placing Asn, a residue known to strengthen homomeric transmembrane (TM) interactions (9, 10), at successive positions across a 10-residue segment of the ␤3 TM domain and found that mutations along one face of the helix led to constitutive ␣IIb␤3 activation and integrin clustering (11).However, there are two distinct mechanisms by which TM domain mutations might activate integrins. Besides increasing the tendency of a highly expressed integrin to form homooligomers, TM domain ...

The integrin αIIbβ3 is a transmembrane (TM) heterodimeric adhesion receptor that exists in equilibrium between resting and active ligand binding conformations. In resting αIIbβ3, the TM and cytoplasmic domains of αIIb and β3 form a heterodimer that constrains αIIbβ3 in its resting conformation. To study the structure and dynamics of the cytoplasmic domain heterodimer, we prepared a disulfide-stabilized complex consisting of portions of the TM domains and the full cytoplasmic domains. NMR and hydrogendeuterium exchange of this complex in micelles showed that the αIIb cytoplasmic domain is largely disordered, but it interacts with and influences the conformation of the β3 cytoplasmic domain. The β3 cytoplasmic domain consists of a stable proximal helix contiguous with the TM helix and two distal amphiphilic helices. To confirm the NMR structure in a membrane-like environment, we studied the β3 cytoplasmic domain tethered to phospholipid bilayers. Hydrogen-deuterium exchange mass spectrometry, as well as circular dichroism spectroscopy, demonstrated that the β3 cytoplasmic domain becomes more ordered and helical under these conditions, consistent with our NMR results. Further, these experiments suggest that the two distal helices associate with lipid bilayers but undergo fluctuations that would allow rapid binding of cytoplasmic proteins regulating integrin activation, such as talin and kindlin-3. Thus, these results provide a framework for understanding the kinetics and thermodynamics of protein interactions involving integrin cytoplasmic domains and suggest that such interactions act in a concerted fashion to influence integrin stalk separation and exposure of extracellular ligand binding sites.platelets | fibrinogen receptors | receptor regulation

Background: Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N-and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed.

We present a molecular modeling protocol that selects modeled protein structures based on experimental mutagenesis results. The computed effect of a point mutation should be consistent with its experimental effect for correct models; mutations that do not affect protein stability and function should not affect the computed energy of a correct model while destabilizing mutations should have unfavorable computed energies. On the other hand, an incorrect model will likely display computed energies that are inconsistent with experimental results. We added terms to our energy function which penalize models that are inconsistent with experimental results. This creates a selective advantage for models that are consistent with experimental results in the Monte Carlo simulated annealing protocol we use to search conformational space. We calibrated our protocol to predict the structure of transmembrane helix dimers using glycophorin A as a model system. Inclusion of mutational data in this protocol compensates for the limitations of our force field and the limitations of our conformational search. We demonstrate an application of this structure prediction protocol by modeling the transmembrane region of the BNIP3 apoptosis factor.

Transmembrane helices engage in homomeric and heteromeric interactions that play essential roles in folding and assembly of transmembrane proteins. However, features that explain their propensity to interact homomerically or heteromerically and determine the strength of these interactions are poorly understood. Integrins are an ideal model system to address these questions because the transmembrane helices of full-length integrins interact heteromerically when integrins are inactive, but the isolated transmembrane helices are also able to form homo-dimers or homooligomers in micelles and bacterial membranes. We sought to determine the features defining specificity for homo versus hetero interactions by conducting a comprehensive comparison of the homomeric and heteromeric interactions of the integrin αIIbβ3 transmembrane helices in biological membranes. Using the TOXCAT assay, we found that residues V700, M701, A703, I704, L705, G708, L709, L712, and L713, located on the same face of the β3 helix, mediate homodimer formation. We then characterized the β3 heterodimer by measuring the ability of β3 helix mutations to cause ligand binding to αIIbβ3. We found that mutating V696, L697, V700, M701, A703. I704, L705, G708, L712, and L713, but not the small-X 3 -small motif, S699-X 3 -A703, caused constitutive αIIbβ3 activation, as well as persistent αIIbβ3 activation-dependent FAK phosphorylation. Because αIIb and β3 use the same face of their respective transmembrane helices for homomeric and heteromeric interactions, the interacting surface on each has an intrinsic "stickiness" predisposing towards helix-helix interactions in membranes. The residues responsible for heterodimer formation comprise a network of interdigitated sidechains with considerable geometric complementarity; mutations along this interface invariably destabilize heterodimer formation. By contrast, residues responsible for homomeric interactions are dispersed over a wider surface. While most mutations of these residues are destabilizing, some stabilized homo-oligomer formation. We conclude that the αIIbβ3 transmembrane heterodimer shows the hallmark of finely-tuned heterodimeric interaction, while the homomeric interaction is less specific.