| The 117 kDa adhesion plaque protein, vinculin, is implicated in regulation of cell adhesion. Vinculin is thought to affect cell adhesion by altering mechanical linkages between the membrane and the cytoskeleton. Although isolated vinculin domains bind several cytoskeletal proteins, including talin and F-actin, these interactions are masked in full-length vinculin by an autoinhibitory head-tail (Vh-Vt) interaction. Prior to this work, the structural basis for head-tail interaction and its physiological relevance was poorly understood. The work presented herein describes my contribution to a 3.3A resolution model of vinculin, a structure-function analysis defining the full requirements for autoinhibition, and the role of autoinhibition in driving the dynamics of focal adhesion complex. In addition to a large hydrophobic interface between D1 (V1-258) and Vt, previously implicated in head-tail interaction, I found a requirement for a small, polar interface between Vt and the D4 (V710-836). Although the D4:Vt interface buried only 530A2, it strengthened Vh-Vt binding by one-hundred fold, and completely suppressed activation of vinculin by talin. Loss of the D4:Vt interface led to constitutive vinculin:talin association in cells and a hypertrophy of focal adhesions. Importantly, this localization phenotype correlated with decreased turnover of activated vinculin. FRAP (fluorescence recovery after photobleach) measurements revealed a two to three fold reduction in the dissociation rate of activated vinculin from focal adhesions. This FRAP phenotype was suppressed by disruption of the talin-binding site in vinculin. Furthermore, activation of vinculin specifically inhibited talin dynamics, whereas paxillin and alpha-actinin remain unaffected. These findings establish a physiological function of head-tail interaction in regulation of ligand-binding and focal adhesion residency of vinculin. Furthermore, they suggest a mechanism by which activation of vinculin may promote cell adhesion by stabilizing the association of talin with focal adhesions. |
