A MUTATION OF THE ATRIAL-NATRIURETIC-PEPTIDE (GUANYLYL CYCLASE-A) RECEPTOR RESULTS IN A CONSTITUTIVELY HYPERACTIVE ENZYME

Mutation of an invariant glutamate residue found within the catalytic domain of guanylyl cyclases resulted in a dramatic 14-fold increase in the activity of the guanylyl cyclase-A receptor, Even in the presence of Mn2+/Triton X-100, a treatment previously thought to yield hormone -independent and maximum cyclase activity, the mutant enzyme remained 7-fold more active; to our knowledge, this is the first example of a p rotein modification or of an added agent that significantly increases cyclase activity in the presence of Mn2+/Triton X-100, Intracellular c oncentrations of cGMP in cells expressing the mutant (E974A) cyclase w ere only marginally elevated by the addition of atrial natriuretic pep tide, and in broken cell preparations, the mutant enzyme also was rela tively insensitive to ligand/regulatory nucleotide, The marked increas e in cyclase activity was not due to a relief of protein kinase domain inhibition, since the point mutation caused 7- to 13-fold elevations in guanylyl cyclase-a activity when the protein kinase homology domain was deleted, The E974A mutation also altered the kinetics from positi ve cooperative to linear with respect to MnGTP, suggesting disruption of subunit-subunit interactions, Thus, a single point mutation within the catalytic domain of a guanylyl cyclase results in a constitutively hyperactive enzyme that is independent of protein kinase domain regul ation.