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

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298 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- 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- 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. phospholipase C- ; adenylyl cyclase; G protein-coupled receptors; natriuretic peptide clearance receptor