||The upregulation of synapse-specific genes such as acetylcholine receptors (AChR) plays a crucial role in the formation of the neuromuscular junction (NMJ). However, the precise mechanisms through which gene transcription is regulated during NMJ formation remain largely enigmatic. Neuregulin (NRG) has been shown to modulate the transcription of AChR subunits at the NMJ. I have recently demonstrated that the expression of G protein β1 subunit (Gβ1) is also up-regulated upon NRG treatment. This raises the possibility that G protein signaling may participate in the formation of the NMJ. In the current study, we found that loss of neural activity following sciatic nerve cut, nerve crush or application of tetrodotoxin enhanced the expression of Gβ1 and Gβ2 in muscle. In addition, both Gβ1 and Gβ2 subunits proteins were prominently expressed in embryonic and postnatal stages and then were decreased in adult stage of rat skeletal muscles. Expression of Gβ subunits was also enhanced in cultured myotubes following NRG treatment. To further explicate the mechanisms through which Gβ subunits may be involved in the formation of NMJ, the effect of Gβγ subunits overexpression on NRG-activated downstream signaling was examined. While transient transfection with Gβγ subunits suppressed the NRG-induced activation of Erk1/2, overexpression of Gβγ subunits enhanced basal JNK activation. We found that NRG treatment of C2C12 cells resulted in a rapid translocation of Gαz protein to the muscle membrane. The specific translocation suggests that Gαz subunits may act as potential candidates to act in concert with Gβγ subunits to regulate NRG-mediated MAP kinase activation at NMJ. These findings propose that G proteins may affect gene transcription at the NMJ by interfering with the downstream signaling of NRG. On the other hand, the C2C12 myoblast stably expressing constitutive active mutant of Gαz (GαzQL-EE) suppressed the expression of myogenin and myosin heavy chain (MHC) and failed to differentiate into myotube after 4-days of incubation in the differentiation medium. Our observations provide evidence for the potential involvement of G proteins in the regulation of NRG-mediated gene transcription and myogenesis.