Formation of complex AChR aggregates in vitro requires alpha-dystrobrevin

Efficient function at the neuromuscular junction requires high-density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. alpha-Dystrobrevin (alpha DB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of alpha DB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel-like AChR aggregates on primary muscle cell cultures derived from alpha DB knockout (alpha DB-KO) mice in the absence of nerve or agrin. In myotubes lacking alpha DB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of alpha DB are expressed in skeletal muscle: alpha DB1, alpha DB1(-), alpha DB2, alpha DB2(-), and alpha DB3. Expression of alpha DB1 or alpha DB1(-) in alpha DB-KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of alpha DB2, alpha DB2(-), alpha DB3, or an alpha DB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that alpha DB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that alpha DB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in alpha DB-KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo.