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 ATP gamma S, only ATP gamma S dramatically potentiated ANP-dependent cyclase activity. When the membranes were incubated with ATP gamma S 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 hypothesis, the ATP gamma S effects were both time- and concentration-dependent. Protein phosphatase stability of thiophosphorylation (ATP gamma S) 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 ATP gamma S effectively sensitized GC-A to ligand stimulation over prolonged periods of time in either case, GC-A was phosphorylated in the presence of [gamma(32)P]ATP, and the mag nitude 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.