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

Avery D Posey,Peter Pytel,Konstantina Gardikiotes,Alexis R Demonbreun,Mark Rainey,Manju George,Hamid Band,Elizabeth M Mcnally

doi:10.1074/jbc.m110.157222

Abstract

The mammalian ferlins are calcium-sensing, C2 domain-containing 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.

Highlights

The ferlin family is defined by its homology to FER1, a Caenorhabditis elegans gene required for the fusion of intracellular organelles to the plasma membrane and normal reproduction in worms [1, 2]
We found that Fer1L5 is expressed in myoblasts undergoing fusion to myotubes and that Fer1L5 can bind both EHD1 and EHD2, two EHD family members that are expressed in myoblasts. siRNAmediated reduction of EHD1 and/or EHD2 expression leads to impaired myoblast fusion
A seventh C2 domain can be identified between C2D and C2E in dysferlin and myoferlin by the SMART algorithm and other protein prediction programs [29, 32]; this domain is highly divergent from the standard C2 domain profile

Summary

Introduction

The ferlin family is defined by its homology to FER1, a Caenorhabditis elegans gene required for the fusion of intracellular organelles to the plasma membrane and normal reproduction in worms [1, 2]. 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.

Results

Conclusion