Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.

Abstract

Abstract. Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

“…In agreement with previous work (Gillespie et al, 1996), we find that coexpression of MuSK and rapsyn significantly increased phosphorylation of the AChR β and δ subunits (Fig 1A,B). The MuSK/rapsyn-induced increase in phosphorylation was greatest for the β subunit, whereas basal phosphorylation was highest for the δ subunit; these findings in heterologous cells mirror previous observations in muscle cells (Meier et al, 1995. Moreover, most phosphorylated AChR was found in the pellet fraction, consistent with previous findings in both heterologous and muscle cells (Meier et al, 1995, Borges andFerns, 2001).…”

“…Moreover, both β and δ subunit phosphorylation are dependent on rapsyn, with phosphorylation levels being significantly decreased in rapsyn null myotubes . Basal levels of phosphorylation are higher for δ than β, however, and agrin-induced phosphorylation occurs with higher stoichiometry on β (Meier et al, 1995). In addition, several findings suggest that β and δ subunit phosphorylation may serve different functions.…”

“…In muscle cells, agrin-or nerve-induced clustering of the receptor involves tyrosine phosphorylation of both the AChR β and δ subunits (Wallace et al, 1991, Meier et al, 1995, Ferns et al, 1996. Following MuSK activation, phosphorylation of each subunit occurs with a similar time-course and is inhibited by the same pharmacological agents , Mohamed et al, 2001.…”

Roles of Rapsyn and Agrin in Interaction of Postsynaptic Proteins with Acetylcholine Receptors

“…MuSK is activated by agrin, and mice lacking MuSK are similar to agrin-deficient animals in that they lack AChR clusters . Agrin also induces tyrosine phosphorylation of the ␤ subunit of the AChR (Wallace et al, 1991;Meier et al, 1995;Ferns et al, 1996), a modification whose significance for AChR clustering is unclear at present (Meyer and Wallace, 1998). This phosphorylation appears not to occur through the direct action of MuSK (Fuhrer et al, 1997) but rather through one or several downstream kinases, possibly of the src family Huganir, 1993, 1994;Fuhrer and Hall, 1996).…”

Neural Agrin Induces Ectopic Postsynaptic Specializations in Innervated Muscle Fibers

“…Similarly, phosphorylation of the AChR ␤-subunit seems to be required for agrin-induced AChR aggregation (Wallace et al, 1991;Meier et al, 1995Meier et al, , 1996.…”