Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion

Abstract

The mammalian ferlins are calcium-sensing, C2 domaincontaining proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.

GRAF1 promotes ferlin-dependent myoblast fusion

“…The GRAF2 (PS-GAP) antibody was a generous gift from Dr. Wen-Cheng Xiong (Georgia Regents University, GA) (Ren et al, 2001). Derivation of the polyclonal Fer1L5 and GRAF1 antibodies was previously described (Doherty et al, 2011; Posey et al, 2011b). A hamster monoclonal GRAF1 antibody was designed in house using the identical peptide immunogen by standard methodology.…”

“…Dysferlin-deficiency is causal for Limb girdle muscular dystrophy 2B, and studies in muscle fibers lacking dysferlin revealed defects in membrane resealing following mechanical or laser-induced membrane rupture (Bansal et al, 2003; Han and Campbell, 2007). The finding that dysferlin-null muscle retained accumulation of vesicles near membrane damage sites indicates that dysferlin likely mediates the final step of fusion necessary for plasma membrane repair (Posey et al, 2011b). Myoferlin and Fer1L5 which are transiently expressed during myogenesis and are known to facilitate myoblast fusion during the development of nascent muscle fibers likely function in a similar fashion.…”

“…Myoferlin and Fer1L5 which are transiently expressed during myogenesis and are known to facilitate myoblast fusion during the development of nascent muscle fibers likely function in a similar fashion. Ferlins are only transiently expressed on muscle plasma membranes and their active recruitment to the sarcolemma is tightly regulated by endocytic recycling mediated by the Eps15 homology domain (EHD)-containing proteins 1 and 2 (Cai et al, 2009a; Doherty et al, 2008; Posey et al, 2011a, 2011b). While several muscular dystrophies are associated with abnormal plasma membrane localization of dysferlin, indicating the significance of this regulatory process (Cai et al, 2009b; Evesson et al, 2010; Matsuda et al, 2001; Wallace and McNally, 2009), the precise mechanisms that govern ferlin recruitment during myoblast fusion or to sites of injury are still poorly understood.…”

Skeletal Muscle Differentiation and Fusion Are Regulated by the BAR-containing Rho-GTPase-activating Protein (Rho-GAP), GRAF1

“…Indeed, the GRAF1 BAR domain is capable of inducing tubulation of spherical lipids and has been implicated in driving clathrin-independent endocytosis in fibroblasts and HeLa cells (29,30). Interestingly, other mediators of endocytic membrane recycling, including myoferlin, MG53, and Eps15 homology domain-containing proteins 1 and 2, have recently been shown to promote myoblast fusion (31)(32)(33). The presence of unilamellar vesicles observed at sites of myoblast membrane fusion has been proposed to be important for the recruitment of essential fusogenic phospholipids and the subsequent internalization of excess plasma membrane from fusing cells (34).…”

Proteomic Analysis of Plasma Membrane Proteins in an In Vitro Blood-Brain Barrier Model

“…EHD2 binds to lipid membranes and deforms them into tubules. The protein regulates trafficking from the PM by controlling Rac1 activity [Benjamin et al, 2011] and is important for internalization of the glucose-transporter 4 (GLUT-4) [Park et al, 2004] Lastly, EHD2 is required for the translocation of a newly identified ferlin-like protein (Fer1L5) to the PM [Posey et al, 2011].…”