Factors Influencing the Homomeric and Heteromeric Association of Platelet Integrin Transmembrane Domains.

Integrin are α/β heterodimers that mediate an array of cell-cell and cell-matrix interactions including platelet adhesion and aggregation. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations that are regulated by transmembrane (TM) domain interactions: when integrins are inactive, the TM domains of their α and β subunits interact; the domains separate when integrins assume their active conformation. Platelets express five α subunits (α2, αIIb, αv, α5, and α6) and two β subunits (β1 and β3) that combine to form five adhesions receptors. Previously, we observed that the αIIb and β3 TM domains undergo both heteromeric and homomeric interactions and there is overlap of the interfaces that mediates these interactions. Less is known about the other platelet integrins. To study their interactions, we used the TOXCAT assay. In TOXCAT, a chimeric protein consisting of an N-terminal ToxR’ DNA binding domain, a C-terminal maltose-binding protein domain, and an interposed TM domain is expressed in the E. coli inner membrane. TM domain-mediated dimerization of the chimeric protein drives the transcriptional activation of a chloramphenicol acetyl transferase (CAT) reporter gene. To enable TOXCAT to measure heteromeric as well as homomeric interactions, we introduced an R68K mutation into the ToxR’ DNA-binding region, thereby preventing CAT synthesis without affecting protein expression. Thus, when both wild-type and disabled ToxR’ are concurrently expressed from the same plasmid, disabled ToxR’ exerts a dominant-negative effect on CAT synthesis. Using this assay, we found that the interaction of platelet integrin TM domains correlated with the presence of a small residue (sr)-xxx-small residue motif (sr-x3-sr) where x = any residue: α2, αIIb and β1, each of which contains a Gx3G motif, had the strongest tendency to undergo specific homomeric association, whereas α2+β1 and αIIb+β3 had the strongest tendency to form heterodimers. In the TM domains of αv, α5 and β3, one or more of the glycines in sr-x3-sr is replaced by Ser or Ala; as a result, homomeric interactions involving these subunits are substantially weaker. Moreover, mutating each of the small residues in sr-x3-sr to Leu precluded the formation of TM domain oligomers, emphasizing the importance of the sr-x3-sr motif. The dominant-negative TOXCAT assay was also used to screen for inactivating αIIbβ3 and αvβ3 mutations. By introducing random mutations into the β3 TM domain and selecting mutants based on a reduction in CAT synthesis, we identified mutations that enhanced heteromeric αIIbβ3 and αvβ3 association. It is noteworthy that mutations that enhanced the interaction of β3 with αIib were present along the face of the β3 TM helix containing the sr-x3-sr motif. By contrast, mutations enhancing αvβ3 association were distributed throughout the β3 TM helix and didn’t cluster around the sr-x3-sr motif. In summary, we have demonstrated that the TM domains of platelet integrin subunits, in addition to αIIb and β3, undergo specific heteromeric and homomeric interactions, suggesting that TM domain interactions may regulate the function of the integrins containing these subunits. Further, our results indicate that sr-x3-sr motifs play an essential role in the oligomerization of these subunits, suggesting that these motifs play a central role in regulating integrin function.