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 recep tor has not been previously accomplished in a broken cell or membrane preparation. The guanylyl cyclase-A (GC-A) receptor is known to requir e both atrial natriuretic peptide (ANP) and an adenine nucleotide for maximal cyclase activation. When membranes from NIH 3T3 cells stably o verexpressing GC-A were incubated with ATP, AMPPNP, or ATP gamma S, on ly 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 t hiophosphorylation had sensitized GC-A to ligand and adenine nucleotid e binding. Consistent with this hypothesis, the ATP gamma S effects we re both time- and concentration-dependent. Protein phosphatase stabili ty of thiophosphorylation (ATP gamma S) relative to phosphorylation (A TP) appeared to explain the differential effects of the two nucleotide s since microcystin, beta-glycerol phosphate, or okadaic acid coincide nt with ATP or ATP gamma S effectively sensitized GC-A to ligand stimu lation over prolonged periods of time in either case, GC-A was phospho rylated in the presence of [gamma(32)P]ATP, and the mag nitude of the phosphorylation was increased by the addition of microcystin. Thus, th e phosphorylation of GC-A correlates with the acquisition of ligand se nsitivity. The establishment of an in vitro system to sensitize GC-A d emonstrates that adenine nucleotides have a daul function in the regul ation of GC-A through both phosphorylation of and binding to regulator y sites.