Identification of the G protein-activating sequence of the single-transmembrane natriuretic peptide receptor C (NPR-C)

Rat natriuretic peptide clearance receptor (NPR-C) contains four sequences capable of inhibiting adenylyl cyclase. We have undertaken mutational and deletion studies on the intracellular domain of rat NPR-C to determine which of these sequences is functionally relevant. Nine mutant receptors were constructed by deletion of 11 or 28 COOH-terminal residues or by site-directed mutagenesis of basic residues in a 17-amino acid sequence, R(469)RNHQEESNIGKHRELR(485), corresponding to the main active peptide. Substitution of arginine residues ((RR470)-R-469) flanking the NH2 terminus abolished G(i1) and G(i2) and PLC-beta activities and inhibition of adenylyl cyclase. Substitution of one or two basic residues (H-481 and/or R-482 or R-485) in the COOH-terminal motif ((HRELR485)-R-481) greatly decreased or abolished G protein and PLC-beta activities and inhibition of adenylyl cyclase. This implies that sequences NH2-terminal to the motif or COOH-terminal to R-470 could not sustain receptor activity in situ, although they exhibited activity when used as synthetic peptides. Deletion of the 11 COOH-terminal residues (E-486 to A(496)) suggested an autoinhibitory function for this sequence. We conclude that the 17-amino acid sequence (R-469 to R-485) in the middle region of the intracellular domain of NPR-C is both necessary and sufficient for activation of G proteins and effector enzymes.