Cyclic AMP-dependent phosphorylation of two size forms of alpha 1 subunits of L-type calcium channels in rat skeletal muscle cells.
Skeletal muscle dihydropyridine-sensitive calcium channels are in vitro substrates for cAMP-dependent protein kinase. In the present work, alpha 1 subunits were isolated from cultured skeletal muscle cells by immunoprecipitation with a specific monoclonal antibody under conditions where proteolysis and dephosphorylation were prevented. Two forms of alpha 1 subunit, 200 and 160 kDa, were identified by back phosphorylation in vitro with cAMP-dependent protein kinase, specific immunoprecipitation, and phosphopeptide mapping. Treatment of cells with forskolin, isoproterenol, calcitonin gene-related peptide, or 8-bromo-cAMP to increase intracellular cAMP reduced 32P incorporation into all phosphopeptides in vitro by 60-80% indicating that increases in cAMP caused endogenous phosphorylation of all sites on both alpha 1(200) and alpha 1(160) to nearly maximal levels. The extents of basal and stimulated phosphorylation in vivo were estimated by back phosphorylation methods to be 35-40% and 83-86%, respectively. In muscle cells metabolically labeled with 32P, 3 mol of phosphate were incorporated into alpha 1 subunits. Forskolin stimulated 32P incorporation into alpha 1 subunits 1.6-fold. Taken together, our results show that skeletal muscle cells contain two forms of the alpha 1 subunit which both are basally phosphorylated on cAMP-dependent phosphorylation sites and are further phosphorylated in response to agents that increase intracellular cAMP.
Lai
Y
Y
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Seagar
M J
MJ
Takahashi
M
M
Catterall
W A
WA
D000818:Animals; D000911:Antibodies, Monoclonal; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D005333:Fetus; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D046911:Macromolecular Substances; D010748:Phosphopeptides; D005576:Colforsin; D000242:Cyclic AMP; D011494:Protein Kinases