GA-Binding Protein Is Dispensable for Neuromuscular Synapse Formation and Synapse-Specific Gene Expression

During neuromuscular synapse formation, postsynaptic proteins, including acetylcholine receptors (AChRs) and the muscle-specific receptor tyrosine kinase, MuSK, become localized to the synaptic membrane of skeletal muscle fibers. Similarly, the mRNAs encoding many postsynaptic components become concentrated in the synaptic region of myofibers, because their corresponding genes are transcribed selectively in subsynaptic muscle nuclei (4, 28). The transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression (32). GABP is a dimer of GABPα, which contains an Ets domain that binds DNA, and GABPβ, which contributes a nuclear localization sequence and the transcriptional activation domain to GABP (18, 31). Dimerization of GABPα and GABPβ is mediated by interactions between four amino-terminal ankyrin repeats in GABPβ and the Ets domain, plus a short α-helix adjacent to the Ets domain, in GABPα (1, 39). While there is one known gabpα gene, two gabpβ genes, gabpβ1 and gabpβ2, have been found in mammals, and gabpβ1 gives rise to at least four distinct splice isoforms (7, 10, 18, 41). Certain GABPβ isoforms mediate formation of a heterotetramer, composed of two GABPα/β dimers, which binds to paired Ets sites, whereas other GABPβ isoforms are recruited to a single Ets site as part of a GABPα/β dimer (7). Synapse-specific transcription of the AChR δ gene is dependent upon a single Ets site in its promoter region (16). GABP is the major Ets protein in myotube nuclear extracts that binds this Ets site (9, 33). The AChR ɛ promoter also contains an Ets site that binds GABP, and mutations in this Ets site are responsible for certain congenital myasthenic syndromes in humans (22, 23, 35). While the importance of Ets sites in synapse-specific gene expression is widely accepted, the role of GABP in synapse formation is less clear. Transfection of a dominant-negative form of GABPβ, lacking sequences required for transcriptional activation, inhibits AChR cluster formation and induction of an AChR ɛ reporter construct by ectopic agrin expression in adult skeletal muscle (2). Transfection of a dominant-negative form of GABPβ also attenuates the activation of a MuSK reporter construct by agrin in vitro (17). Furthermore, transfection of a mutant form of GABPα that cannot be phosphorylated at threonine 280 interferes with the induction of AChR ɛ gene expression by neuregulin 1 (Nrg-1) in cultured muscle cells (38). Forced expression of the DNA-binding domain of Ets2, another Ets domain-containing transcription factor, in skeletal muscle leads to defects in the organization and size of primary gutters and secondary folds at neuromuscular synapses and reduces the expression levels of “synaptic” genes (6). These results indicate that interfering with the function of multiple members of the Ets family of transcription factors by transfection of dominant-negative constructs affects the expression of genes that are preferentially transcribed in subsynaptic nuclei at the neuromuscular synapse. These studies, however, do not address whether GABP is required for neuromuscular synapse formation or synapse-specific gene expression. Deletion of GABPα in mice results in embryonic lethality prior to embryonic day 7.5 (E7.5) of development (26), preventing an analysis of a potential role for GABP in synapse formation, which begins at E13. Here we conditionally inactivated gabpα specifically in skeletal muscle and analyzed synaptic development. We find that GABPα is dispensable for synapse-specific gene expression and clustering of synaptic AChRs during synapse formation. Furthermore, postnatal synaptic maturation is normal in conditional gabpα mutant mice. These findings suggest that additional proteins bind the Ets site in AChR genes and stimulate their expression in synaptic nuclei.