Cytoplasmic domain of natriuretic peptide receptor C constitutes G(i) activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhib ited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) prot ein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase ac tivity. The peptides used were KKYRITIER-RNH (peptide 1), RRNHQEESNIGK (pep tide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITYKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y h ave partial G(i) activator sequences with truncated carboxyl or amino termi nus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inh ibited adenylyl cyclase activity in a concentration-dependent manner with a pparent K-i between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyc lase activity with a higher K-i of about 10 nM, and peptides X, Y, and Z we re unable to inhibit adenylyl cyclase activity. The maximal inhibitions obs erved were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleot ides and was completely attenuated by PT treatment. In addition, the stimul atory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These resu lts suggest that the small peptide fragments of the cytoplasmic domain of t he NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibi t adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptid es having complete structural specificity of G(i) activator sequences at bo th amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus , whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.