Ca sup 2+ -dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells
- Washington Univ. School of Medicine, St. Louis, MO (USA)
Mouse BC3H1 myocytes were incubated with {sup 32}Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the {sup 32}P found in the receptor was bound to the delta 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 approximately the same level in control cells. The divalent cation ionophore, A23187, increased {sup 32}P in site 1 by 40%, but did not affect the {sup 32}P content of site 2. In contrast, isoproterenol increased {sup 32}P in site 2 by more than 60%, while increasing {sup 32}P in site 1 by only 20%. When dephosphorylated receptor was incubated with ({gamma}-{sup 32}P)ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta 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 alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta 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 beta subunit and site 1 of the delta 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 beta subunit and the delta subunit site 1 in cells.
- OSTI ID:
- 5673512
- Journal Information:
- Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 264:22; ISSN JBCHA; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ACETYLCHOLINE
ALKALINE EARTH METAL COMPOUNDS
AMINES
AMINO ACIDS
AMMONIUM COMPOUNDS
AMP
ANIMALS
ATP
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
CALCIUM COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
ESTERS
IMMUNOASSAY
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MAMMALS
MEMBRANE PROTEINS
MICE
MUSCLES
NEUROREGULATORS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PARASYMPATHOMIMETICS
PHOSPHATES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
QUATERNARY COMPOUNDS
RADIOISOTOPES
RECEPTORS
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES