Cyclic AMP-dependent protein kinase A and protein kinase C phosphorylate alpha4beta2 nicotinic receptor subunits at distinct stages of receptor formation and maturation.
Neuronal nicotinic receptor alpha4 subunits associated with nicotinic alpha4beta2 receptors are phosphorylated by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), but the stages of receptor formation during which phosphorylation occurs and the functional consequences of kinase activation are unknown. SH-EP1 cells transfected with DNAs coding for human alpha4 and/or beta2 subunits were incubated with (32)Pi, and PKA or PKC was activated by forskolin or phorbol 12,13-dibutyrate, respectively. Immunoprecipitation and immunoblotting of proteins from cells expressing alpha4beta2 receptors or only alpha4 subunits were used to identify free alpha4 subunits, and alpha4 subunits present in immature alpha4beta2 complexes and mature alpha4beta2 pentamers containing complex carbohydrates. In the absence of kinase activation, phosphorylation of alpha4 subunits associated with mature pentamers was three times higher than subunits associated with immature complexes. PKA and PKC activation increased phosphorylation of free alpha4 subunits on different serine residues; only PKC activation phosphorylated subunits associated with mature alpha4beta2 receptors. Activation of both PKA and PKC increased the density of membrane-associated receptors, but only PKC activation increased peak membrane currents. PKA and PKC activation also phosphorylated beta2 subunits associated with mature alpha4beta2 receptors. Results indicate that activation of PKA and PKC leads to the phosphorylation alpha4beta2 receptors at different stages of receptor formation and maturation and has differential effects on the expression and function of human alpha4beta2 receptors.
Pollock
V V
VV
Department of Psychiatry and Behavioral Medicine, USF College of Medicine, Tampa, FL 33613, USA.
Pastoor
T
T
Katnik
C
C
Cuevas
J
J
Wecker
L
L
D000109:Acetylcholine / Q000494:pharmacology; D000704:Analysis of Variance; D000906:Antibodies / Q000494:pharmacology; D001345:Autoradiography; D019921:Biotinylation; D002461:Cell Line, Transformed; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D025941:Protein Interaction Mapping; D011493:Protein Kinase C / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods
D000906:Antibodies; D004791:Enzyme Inhibitors; D007211:Indoles; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000109:Acetylcholine; C064758:Ro 31-8220