▪ Abstract This review summarizes key aspects of tetraspanin proteins, with a focus on the functional relevance and structural features of these proteins and how they are organized into a novel type of membrane microdomain. Despite the size of the tetraspanin family and their abundance and wide distribution over many cell types, most have not been studied. However, from studies of prototype tetraspanins, information regarding functions, cell biology, and structural organization has begun to emerge. Genetic evidence points to critical roles for tetraspanins on oocytes during fertilization, in fungi during leaf invasion, in Drosophila embryos during neuromuscular synapse formation, during T and B lymphocyte activation, in brain function, and in retinal degeneration. From structure and mutagenesis studies, we are beginning to understand functional subregions within tetraspanins, as well as the levels of connections among tetraspanins and their many associated proteins. Tetraspanin-enriched microdomains (TEMs) are emerging as entities physically and functionally distinct from lipid rafts. These microdomains now provide a context in which to evaluate tetraspanins in the regulation of growth factor signaling and in the modulation of integrin-mediated post-cell adhesion events. Finally, the enrichment of tetraspanins within secreted vesicles called exosomes, coupled with hints that tetraspanins may regulate vesicle fusion and/or fission, suggests exciting new directions for future research.

Relatively little attention has been given to the large family of abundantly expressed transmembrane proteins known as tetraspanins. Now, the importance of tetraspanins is strongly supported by emerging genetic evidence, coupled with new insights into the biochemistry and functions of tetraspanin protein complexes.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9–β1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.