Specificity for Homooligomer versus Heterooligomer Formation in Integrin Transmembrane Helices

Abstract

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.

Tests of Integrin Transmembrane Domain Homo-oligomerization during Integrin Ligand Binding and Signaling

“…1A) and applied a similar method to determine whether the ␣IIb or ␤3 TM domains form homo-oligomers during integrin signaling. As suggested by the TOXCAT assay (24,25) and predicted by computer modeling (26 -28), the ␣IIb TM helix forms homodimers, and the ␤3 TM helix forms homotrimers, using surfaces that are similar to those in the heterodimeric interface. If this homomeric interface is actually formed in the mammalian cell membrane, we expected that some singlecysteine mutations of these residues would form homodimeric disulfide bonds in the presence of an oxidation catalyst such as Cu-phenanthroline.…”

“…Instead, they observed that the ␣ and ␤ fragments tend to form homodimers and homotrimers, respectively (23). Later, ␣IIb and ␤3 TM helices were confirmed to form homo-oligomers in bacterial membranes using the TOXCAT assay (24,25). The ␣ and ␤ homomeric interactions have been widely studied by computational modeling (26 -28).…”

“…The ␣ and ␤ homomeric interactions have been widely studied by computational modeling (26 -28). These studies showed that the homo-oligomerization interface and the heterodimerization motifs largely overlap, but it seems that homomeric interaction is less specific than the heterodimeric interaction (25). In 2003, asparagine mutagenesis in the TM region of ␤3 subunit (with most experiments on the mutation ␤3G708N) suggested that TM homo-oligomerization contributes to integrin activation and clustering (29).…”

“…More specifically, it is unclear whether integrin TM homo-oligomerization plays any role in integrin clustering and signaling, even though it has been proposed that it provides structural basis for this process (29,30). Importantly, although ␣ and ␤ TM homooligomerization was found in micelles and bacterial cell membranes (14,23,25,29,31), it has never been observed in mammalian cell membrane using full-length integrins. On the contrary, the TM heterodimeric but not homomeric association of integrin fragments was detected using mini-integrin affinity capture method in mammalian cell membranes (32).…”

The role of protein–protein interactions in Toll-like receptor function

“…The functional significance of TMDs and their mechanisms of assembly have previously been demonstrated for many membrane proteins including integrins, receptor tyrosine kinases and G-protein coupled receptors [78][79][80]. Several biochemical studies have emphasized the functional relevance of TMD interactions and their regulation [81][82][83][84] in the TLR family.…”