Dual role for adenine nucleotides in the regulation of the atrial natriuretic peptide receptor, guanylyl cyclase-A.
The ability to both sensitize and desensitize a guanylyl cyclase receptor has not been previously accomplished in a broken cell or membrane preparation. The guanylyl cyclase-A (GC-A) receptor is known to require both atrial natriuretic peptide (ANP) and an adenine nucleotide for maximal cyclase activation. When membranes from NIH 3T3 cells stably overexpressing GC-A were incubated with ATP, AMPPNP, or ATPgammaS, only ATPgammaS dramatically potentiated ANP-dependent cyclase activity. When the membranes were incubated with ATPgammaS and then washed, GC-A now became sensitive to ANP/AMPPNP stimulation, suggestive that thiophosphorylation had sensitized GC-A to ligand and adenine nucleotide binding. Consistent with this hypo- thesis, the ATPgammaS effects were both time- and concentration-dependent. Protein phosphatase stability of thiophosphorylation (ATPgammaS) relative to phosphorylation (ATP) appeared to explain the differential effects of the two nucleotides since microcystin, beta-glycerol phosphate, or okadaic acid coincident with ATP or ATPgammaS effectively sensitized GC-A to ligand stimulation over prolonged periods of time in either case. GC-A was phosphorylated in the presence of [gamma32P]ATP, and the magnitude of the phosphorylation was increased by the addition of microcystin. Thus, the phosphorylation of GC-A correlates with the acquisition of ligand sensitivity. The establishment of an in vitro system to sensitize GC-A demonstrates that adenine nucleotides have a daul function in the regulation of GC-A through both phosphorylation of and binding to regulatory sites.
Foster
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Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, USA.
Garbers
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D016475:3T3 Cells; D000227:Adenine Nucleotides / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D052998:Microcystins; D010456:Peptides, Cyclic / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*
D000227:Adenine Nucleotides; D008024:Ligands; D052998:Microcystins; D010456:Peptides, Cyclic; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); C078588:microcystin; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A