Differential lipid binding of vinculin isoforms promotes quasi-equivalent dimerization

Abstract

The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP 2 ) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP 2 binding sites is preserved, the symmetric MV dimer that bridges two PIP 2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP 2 . Unlike vinculin, wild-type MV and the DCM/ HCM-associated R975W mutant bind PIP 2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform's dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling.ardiomyopathies are a major worldwide health problem, with patients often suffering cardiac arrest and premature death. Over the past decade, several inherited and sporadic mutations in genes encoding components of adhesion complexes and of intercalated discs, which are required for the coordinated movement of heart tissue, have been pinpointed as the cause of many cardiomyopathies. Vinculin and its muscle-specific splice variant, metavinculin (MV), are essential and highly conserved cytoskeletal proteins that play critical regulatory roles in cell-cell adherens-type junctions and cell-matrix focal adhesions (1-3). Both isoforms localize to the cell membrane, the I band in the sarcomere, and to intercalated discs (4). MV is coexpressed with vinculin (5) and colocalizes with vinculin in cardiac myocytes (6). MV only differs from vinculin by an insertion of 68 residues between α-helices H1 and H2 of the 5-helix bundle vinculin tail domain Vt (5, 7), whereby H1′ of the insert of MV structurally replaces H1 of vinculin. Several MV mutations have been shown to be associated with dilated cardiomyopathies (DCMs) and hypertrophic cardiomyopathies (HCMs) in human (8-10), where they disrupt intercalated discs in the hearts of...

Lipid binding promotes the open conformation and tumor-suppressive activity of neurofibromin 2

“…Lipid binding to wild type and mutant neurofibromin 2 FERM domains was assayed as described previously 55 . Briefly, lipid vesicles of phosphatidylcholine (PC) and PIP 2 were prepared in chloroform to a final composition of 80% chicken egg PC and 20% porcine brain PIP 2 (Avanti polar lipids).…”

The interaction of talin with the cell membrane is essential for integrin activation and focal adhesion formation

“…Importantly, large pools of GFP-tagged lipid-binding-deficient mutant talin (5M or 8M) accumulated in the cytosol compared with wild-type or mutant (3M) expressing cells. When we previously mutated our vinculin or metavinculin lipid-binding residues (9,36,44), focal adhesions were never completely disassembled; however, the focal adhesions were largely disrupted for cells expressing the lipid-binding-deficient talin mutant. Thus, lipid binding seems necessary for talin localization to the focal adhesion membrane sites and for talin regulation of the scaffolding effects.…”

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using twodimensional polyacrylamide gel electrophoresis and mass spectrometry

“…Aging of the myocardium results in fibrosis and decreased cardiac contractility, with a correlating increase of vinculin [105]. Human mutations in vinculin and vinculin knockout mice have been related to increased incidences of dilated cardiomyopathy and sudden cardiac death [106,107]. Vinculin absence has been shown to both facilitate contractile dysfunction and increase prevalence of ectopic beats in the myocardium, including ventricular tachycardia [106,107].…”

“…Human mutations in vinculin and vinculin knockout mice have been related to increased incidences of dilated cardiomyopathy and sudden cardiac death [106,107]. Vinculin absence has been shown to both facilitate contractile dysfunction and increase prevalence of ectopic beats in the myocardium, including ventricular tachycardia [106,107]. NDRG1 is involved in stress response pathways by inhibiting cell growth and encouraging DNA repair, and is considered an important preventer of carcinogenesis [108,109].…”