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Transmembrane (TM) helices engage in homomeric and heteromeric interactions that play essential roles in the folding and assembly of TM proteins. However, features that explain their propensity to interact homomerically or heteromerically and determine the strength of these interactions are poorly understood. Integrins provide an ideal model system for addressing these questions because the TM helices of full-length integrins interact heteromerically when integrins are inactive, but isolated TM helices are also able to form homodimers or homooligomers in micelles and bacterial membranes. We sought to determine the features defining specificity for homointeractions versus heterointeractions by conducting a comprehensive comparison of the homomeric and heteromeric interactions of integrin alphaIIbbeta3 TM helices in biological membranes. Using the TOXCAT assay, we found that residues V700, M701, A703, I704, L705, G708, L709, L712, and L713, which are located on the same face of the beta3 helix, mediate homodimer formation. We then characterized the beta3 heterodimer by measuring the ability of beta3 helix mutations to cause ligand binding to alphaIIbbeta3. We found that mutating V696, L697, V700, M701, A703. I704, L705, G708, L712, and L713, but not the small residue-X(3)-small residue motif S699-X(3)-A703, caused constitutive alphaIIbbeta3 activation, as well as persistent focal adhesion kinase phosphorylation dependent on alphaIIbbeta3 activation. Because alphaIIb and beta3 use the same face of their respective TM 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 side chains 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 homooligomer formation. We conclude that the alphaIIbbeta3 TM heterodimer shows the hallmark of finely tuned heterodimeric interaction, while homomeric interaction is less specific., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Summary: Integrins link the cytoskeleton to the extracellular matrix and regulate key signaling events that coordinate cellular processes such as secretion, migration, and proliferation. A single integrin molecule can exist in a resting state that does not bind extracellular ligands or in an active state that can engage ligands and form large signaling complexes. Activation signals are transduced between the cytosolic region and the extracellular region by a binary on/off switch in the integrin''s transmembrane (TM) domain; the integrin''s α and β subunits each have a single TM helix that forms an α/β heterodimer in the resting state, and the TM heterodimer separates to transduce an activation signal across the membrane. In this article, two methods used to generate models of the TM heterodimer, both converging on the same structure, are described. The first model was generated by a Monte Carlo algorithm that selected conformations based on their agreement with published experimental mutagenesis results. The second model was generated by threading the integrin''s sequence onto TM helix dimers parsed from the Protein Data Bank and by selecting conformations based on their agreement with published experimental cysteine crosslinking results. The two models have similar structures; however, they differ markedly from some previously published models. To distinguish conformations that reflect the native integrin, we compared the Monte Carlo model, the threaded model, and four published models with experimental mutagenesis and cysteine crosslinking results. The models presented here had high correlation coefficients when compared with experimental findings, and they are in excellent agreement, both in terms of accuracy and in terms of precision, with a recent NMR structure. These results demonstrate that multiple approaches converged on the same structure of the resting integrin''s TM heterodimer, and this conformation likely reflects the integrin''s native structure. [Copyright &y& Elsevier]

Homomeric and heteromeric interactions between the αllb and β3 transmembrane domains are involved in the regUlation of integrin αIIbβ3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of αllβ3 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 αllbβ3 transmembrane domain. One mutation that enhanced the dimerization of the αllbβ transmembrane domain indeed induced constitutive αllbβ3 activation. However, a series of mutations that disrupted homodimerization also led to αllbβ3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the αllbβ3 transmembrane domain, and that mutations that disrupt the αllbβ3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for αllbβ3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an αllbβ3 transmembrane domain heterodimer and by stabilizing the resulting αllb and β3 transmembrane domain homodimers. [ABSTRACT FROM AUTHOR]