Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells.
It is generally accepted that G protein-coupled receptors stimulate soluble guanylyl cyclase (sGC)-mediated cGMP production indirectly, by increasing nitric oxide (NO) synthase activity in a calcium- and kinase-dependent manner. Here we show that normal and GH(3) immortalized pituitary cells expressed alpha(1)beta(1)-sGC heterodimer. Activation of adenylyl cyclase by GHRH, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and forskolin increased NO and cGMP levels, and basal and stimulated cGMP production was abolished by inhibition of NO synthase activity. However, activators of adenylyl cyclase were found to enhance this NO-dependent cGMP production even when NO was held constant at basal levels. Receptor-activated cGMP production was mimicked by expression of a constitutive active protein kinase A and was accompanied with phosphorylation of native and recombinant alpha(1)-sGC subunit. Addition of a protein kinase A inhibitor, overexpression of a dominant negative mutant of regulatory protein kinase A subunit, and substitution of Ser(107)-Ser(108) N-terminal residues of alpha(1)-subunit with alanine abolished adenylyl cyclase-dependent cGMP production without affecting basal and NO donor-stimulated cGMP production. These results indicate that phosphorylation of alpha(1)-subunit by protein kinase A enlarges the NO-dependent sGC activity, most likely by stabilizing the NO/alpha(1)beta(1) complex. This is the major pathway by which adenylyl cyclase-coupled receptors stimulate cGMP production.
Kostic
Tatjana S
TS
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Andric
Silvana A
SA
Stojilkovic
Stanko S
SS
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006152:Cyclic GMP / Q000096:biosynthesis*; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D006162:Guanylate Cyclase; D009479:Neuropeptides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D021122:Protein Subunits; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000187:drug effects / Q000378:metabolism*; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000187:drug effects / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
C495403:Adcyap1 protein, rat; D004791:Enzyme Inhibitors; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D005576:Colforsin; D009569:Nitric Oxide; D014660:Vasoactive Intestinal Peptide; D012694:Serine; D013007:Growth Hormone-Releasing Hormone; D019001:Nitric Oxide Synthase; D017868:Cyclic AMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; C048250:somatotropin releasing hormone receptor; D006152:Cyclic GMP; D000409:Alanine