Proximity-Dependent Biotinylation (BioID) of Integrin Interaction Partners.

Integrins are heterodimeric adhesion receptors that maintain cell-extracellular matrix (ECM) interactions in diverse tissue microenvironments. They mediate cell adhesion and signaling through the assembly of large cytoplasmic multiprotein complexes that focally connect with the cytoskeleton. Integrin adhesion complexes (IAC) are specialized by the type of integrin-ECM contact and are sensitive to mechanical forces. Thus, they encrypt context-dependent information about the microenvironment in their composition. Signals mediated through IACs modulate many aspects of cell behavior, which allows cells to adapt to their surroundings. To gain insights into their function, IACs have been isolated from cultured cells and explored by proteomics. IACs are insoluble by nature and held together by transient/weak interactions, which makes it challenging to isolate intact IACs. Usually all IACs coupled to a specified ECM, which may employ different integrins, are isolated. Here we describe an alternative method based on proximity-dependent biotin identification (BioID), where specific integrin interaction partners are labeled in live cells and isolated without the need to isolate intact IACs.

The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation.

Functioning as signal receivers and transmitters, the integrin α/ß cytoplasmic tails (CT) are pivotal in integrin activation and signaling. 18 α integrin subunits share a conserved membrane-proximal region but have a highly diverse membrane-distal (MD) region at their CTs. Recent studies demonstrated that the presence of α CTMD region is essential for talin-induced integrin inside-out activation. However, it remains unknown whether the non-conserved α CTMD regions differently regulate the inside-out activation of integrin. Using αIIbß3, αLß2, and α5ß1 as model integrins and by replacing their α CTMD regions with those of α subunits that pair with ß3, ß2, and ß1 subunits, we analyzed the function of CTMD regions of 17 α subunits in talin-mediated integrin activation. We found that the α CTMD regions play two roles on integrin, which are activation-supportive and activation-regulatory. The regulatory but not the supportive function depends on the sequence identity of α CTMD region. A membrane-proximal tyrosine residue present in the CTMD regions of a subset of α integrins was identified to negatively regulate integrin inside-out activation. Our study provides a useful resource for investigating the function of α integrin CTMD regions.

Integrins uncouple Src-induced morphological and oncogenic transformation.

Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.