Although vinculin is present at all sites of F-actin attachment to plasma membranes and is required for linkage of myofibrils to sarcolemma, it is unclear how it promotes attachment of actin to membranes. Because biochemical evidence for a direct interaction of vinculin with F-actin is controversial, current models of actin-membrane linkages depict only an indirect role for vinculin, as a tether for alpha-actinin. We demonstrate here that an intramolecular association between the 95K head and 30K tail domains of vinculin masks an F-actin binding site present in the carboxy-terminal tail domain. Cosedimentation and crosslinking assays, and direct visualization by transmission electron microscopy, reveal an interaction between F-actin and a bacterially expressed fusion protein containing amino acids 811-1066 of vinculin, and between F-actin and a proteolytic fragment of vinculin containing amino acids 858-1066. Vinculin itself neither cosediments with nor crosslinks F-actin. The amino-terminal 95K head fragment of vinculin, but not intact vinculin, inhibits both cosedimentation and crosslinking. We propose that assembly of vinculin into an adherens junction involves disruption of the head-tail interaction, revealing a site that mediates microfilament attachment.

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Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent structural model explains autoinhibition solely in terms of the interaction between vinculin tail (V t ) and residues 1-258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (V h residues 710 -836) and V t . Charge-to-alanine mutations in V t revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their K d values for head binding of 100-and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact V t yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in K d values between V t binding to V-(1-258), as opposed to full-length V h -(1-851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to V h . Whereas V t fails to compete with the talin rod domain for binding to V-(1-258), competition occurs readily with full-length V h , and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-V t interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-V t interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-V t and D4-V t interfaces provide the high affinity (ϳ10 ؊9 ) autoinhibition observed in full-length vinculin. Vinculin (V)1 is a 116-kDa cytoskeletal protein associated with focal adhesions and adherens junctions. Several functions, mostly related to cell adhesion and motility processes, have been ascribed to vinculin. Disruption of vinculin expression in mice results in an embryonic lethal phenotype, with severe cardiac and brain abnormalities (1). Moreover, heterozygotes show increased susceptibility to stress-induced cardiomyopathies (2), consistent with the paralysis and defects in muscle architecture associated with the knockout of vinculin in Caenorhabditis elegans (3). Interestingly, vinculin-null cell lines show defects in mechanical stiffness (4), Rac-mediated lamellipodial protrusion (5), cell shape, and spreading on fibronectin (1, 6, 7), as well as misregulation of apoptotic cues (8).Whereas mechanistic insights into the molecular basis of these phenotypes are limited with a few notable exceptions (7,8), the aforementioned effects are generally consistent with the idea that vinculin functions as a mechanical linker between the plasma membrane and actin cytoskeleton through cross-linking several adhesion proteins. This model derives from in vitro studies that identified interactions between vinculin and numerous focal adhesions proteins including talin (9), ␣-actinin (10, 11), paxillin (12), VASP (13), vinexin/ponsin family members (14, 15), and F-actin (16, 17). However, the binding o...

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