Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells.
Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells.
复制标题
DOI：
10.1016/s0021-9258(18)51559-3
复制
发表时间：
1989-08
期刊：
影响因子：
--
通讯作者：
McHardy M. Smith;John P. Merlie;John C. Lawrence
中科院分区：
文献类型：
--
作者：
McHardy M. Smith;John P. Merlie;John C. Lawrence
Mouse BC3H1 myocytes were incubated with32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the32P found in the receptor was bound to the δ subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to ∼ the same level in control cells. The divalent cation ionophore, A23187, increased32P in site 1 by 40%, but did not affect the32P content of site 2. In contrast, isoproterenol increased32P in site 2 by more than 60%, while increasing32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [γ-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the δ subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The α subunit was phosphorylated more slowly, but phosphorylation of both α and δ subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the β subunit and site 1 of the δ subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the β subunit and the δ subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the α subunit and the δ subunit site 2 in response to elevations in cAMP.
