A Conserved Transmembrane Domain Interface Regulates Integrin Function
Authors: Berger Bryan W , Span Lisa M. , Kulp Daniel W , Billings Paul C. , DeGrado William F. , Bennett Joel S. 
Published: April 2020

Integrins are a superfamily of transmembrane (TM) α/β heterodimers that mediate fundamental cellular adhesive functions. Platelet integrins, for example, mediate stable platelet adhesion to collagen and fibronectin and the formation of stable platelet aggregates. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. An essential feature of this equilibrium is interaction of the integrin α and β subunit TM domains. Thus, when integrins are inactive, the α and β TM domains are in proximity, but they separate when integrins assume an active conformation. Moreover, inducing TM domain separation alone is sufficient to cause integrin activation. Previously, we reported that the TM domains of the platelet integrin αIIbβ3 interact both heteromerically and homomerically and that the strength of their heteromeric interaction is necessarily weak to allow regulated TM domain separation. To address whether these observations can be extended to the other members of the integrin superfamily, we focused initially on αvβ3, α2β1 and α5β1, integrins present in platelets, using a dominant-negative ToxR-based assay. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it dimerizes in the inner membrane of E. coli. By co-expressing wild-type ToxR with either wild-type ToxR or an R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure the homomeric and heteromeric interaction of each integrin TM domain. Using alanine and leucine scanning mutagenesis, we found that like αIIb, homo-oligomerization of other integrin α subunit TM domains is preferred over hetero-oligomerization, and that the relative strength of homo-oligomerization correlates with the presence of a canonical small residue-xxx-small residue motif followed one turn of the TM helix by a leucine (G, A, S-xxx-G-xxx-L). This motif also mediates the hetero-oligomerization of these TM domains with either β3 or β1. By contrast, a different motif (V-xxx-I-xxx-G) mediates the heteromeric interaction of both β3 and β1 with their complementary α subunits. Mutations that disrupt either the αIIb or β3 interaction motif induce constitutive αIIbβ3 activation. To determine if this is also the case for β1-containing integrins, we introduced disruptive interfacial mutations into the full-length integrins and expressed the mutants in either the β1-deficient Jurkat A1 cells or in HEK293 suspension cells. We found that the β1 mutations V716A, I720A and G724L caused a substantial increase in the static adhesion of A1 cells to laminin, fibronectin, the α4β1-specific peptide H1, as well as type I, II and type IV collagen, whereas mutation of the canonical G-xxx-G motif did not. On the other hand, an increase in binding to type I collagen and fibronectin was observed for mutations of the interfacial α2 residues S1009, G1013, and L1017 and the interfacial α5 residues A964, G968, and L972, respectively. Thus, our studies indicate that β1 and β3 integrins employ a novel, specific, and conserved reciprocating ‘large-small’ TM packing interface that interacts less strongly than the canonical small-residue-xxx-small residue motif. It is also noteworthy that this interface is present in all integrins except β4 and is overrepresented in databases of TM helix-helix interaction as well. Accordingly, it is likely that this type of interface evolved to mediate TM domain interactions that are capable of regulation.