Abstract
The integrin αIIbβ3 resides on the platelet surface in an equilibrium between inactive and active conformations that can be shifted in either direction by altering the distance between the stalks that anchor αIIbβ3 in the platelet membrane. Accordingly, the αIIb and β3 transmembrane (TM) domains, located near the ends of the stalks, are in proximity when αIIbβ3 is inactive and separate upon αIIbβ3 activation. Peptides corresponding to these domains undergo both homomeric and heteromeric interactions in biological membranes. Thus, it is possible that the shift between inactive and active αIIbβ3 conformations is accompanied by a shift from heteromeric to homomeric αIIb and β3 TM domain interactions. Indeed, we reported that introducing Asn, a residue known to strengthen homomeric TM helix interactions, into the β3 TM domain shifts αIIbβ3 to an active state. As a further test of this model of αIIbβ3 regulation, we studied the effects of mutations of the αIIb TM domain. First, we placed Asn at 10 consecutive positions in the αIIb TM domain, extending from residues V969 to L978, and co-expressed each mutant with WT β3 in CHO cells. Only one of the mutants, G972N, was constitutively active, binding ~ 8-fold more fibrinogen than WT αIIbβ3. Moreover, G972N was expressed in non-uniform patches on the CHO cell surface, consistent with the formation of αIIbβ3 clusters. G972 is the first residue of a GxxxG motif that is essential for dimerization of the αIIb TM domain. Using the TOXCAT assay to assess TM domain dimerization, we observed that G972N results in a 55% decrease in TOXCAT activity. This implies that the effect of G972N on the αIIbβ3 activation state was not a result of increased homo-dimerization of αIIb, but it is more likely that the mutation disrupted its heteromeric interaction with β3. To test this suggestion, we introduced mutations known to disrupt αIIb homo-dimerization (G972L, G976A, and G976L) into αIIbβ3 and measured their effect on αIIbβ3 function. Like G972N, each mutation induced constitutive αIIbβ3 activation and clustering. Lastly, we measured the effect of L980A, a mutation in the αIIb TM domain that unlike G972N, results in a 2.5-fold increase in TOXCAT activity. CHO cells expressing L980A constitutively bound ~ 6.5-fold more fibrinogen than did cells expressing WT αIIbβ3. Taken together, our results suggest a mechanism for αIIbβ3 regulation that involves both the heteromeric and homomeric association of the αIIb and β3 TM domains. Any process that destabilizes the heteromeric association of the αIIb and β3 TM domains would be expected to allow dissociation of these domains with concomitant αIIbβ3 activation. Hence, mutations that disrupt the heteromeric αIIb/β3 TM domain interface “push” αIIbβ3 toward activation. Conversely, intermolecular interactions that either require separation of the αIIb and β3 TM domains or are more favorable when they dissociate, such as homo-oligomerization of the αIIb and β3 TM domains, will “pull” the equilibrium toward the activated state.
Structural basis of transmembrane domain interactions in integrin signaling
