Isolated integrin β3 subunit cytoplasmic domains require membrane anchorage and the NPXY motif to recruit to adhesion complexes but do not discriminate between β1- and β3-positive complexes

Integrin adhesion receptors consist of non-covalently linked alpha and beta subunits each of which contains a large extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Engaged integrins recruit to focal structures globally termed adhesion complexes. The cytoplasmic domain of the P subunit is essential for this clustering. beta 1 and beta 3 integrins can recruit at distinct cellular locations (i.e. fibrillar adhesions vs focal adhesions, respectively) but it is not clear whether individual beta subunit cytoplasmic and transmembrane domains are by themselves sufficient to drive orthotopic targeting to the cognate adhesion complex. To address this question, we expressed full-length beta 3 transmembrane anchored cytoplasmic domains and truncated beta 3 cytoplasmic domains as GFP-fusion constructs and monitored their localization in endothelial cells. Membrane-anchored full-length beta 3 cytoplasmic domain and a beta 3 mutant lacking the NXXY motif recruited to adhesion complexes, while beta 3 mutants lacking the NPXY and NXXY motifs or the transmembrane domain did not. Replacing the natural beta subunit transmembrane domain with an unrelated (i.e. HLA-A2 alpha chain) transmembrane domain significantly reduced recruitment to adhesion complexes. Transmembrane anchored beta 3 and cytoplasmic domain constructs, however, recruited without discrimination to beta 1- and beta 3-rich adhesions complexes. These findings demonstrate that membrane anchorage and the NPXY (but not the NXXY) motif are necessary for beta 3 cytoplasmic domain recruitment to adhesion complexes and that the natural transmembrane domain actively contributes to this recruitment. The beta 3 transmembrane and cytoplasmic domains alone are insufficient for orthotopic recruitment to cognate adhesion complexes.