The vitronectin receptor integrin αVβ5 can reside in two distinct adhesion structures: focal adhesions (FAs) and flat clathrin lattices (FCLs). Here we investigated the mechanism that regulates the subcellular distribution of β5 in keratinocytes and show that β5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH and Numb, respectively, than for talin; all proteins that bind to the membrane-proximal NPxY motif of the β5 cytoplasmic domain. Using mass spectrometry, we identified β5 interactors including the Rho GEFs p115Rho-GEF and GEF-H1, and the serine protein kinase MARK2; depletion of which diminishes the clustering of β5 in FCLs. Substitution of two serines (S759/762) in the β5 cytoplasmic domain with phospho-mimetic glutamates causes a shift in the localization of β5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates β5 localization. Finally, we present evidence that β5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.