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

Abstract

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 (R(469)R(470)) flanking the NH(2) 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 (H(481)RELR(485)) greatly decreased or abolished G protein and PLC-beta activities and inhibition of adenylyl cyclase. This implies that sequences NH(2)-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.