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 (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.
Zhou
Huiping
H
Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Murthy
Karnam S
KS
D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D019556:COS Cells; D004789:Enzyme Activation / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D017353:Gene Deletion; D006162:Guanylate Cyclase / Q000235:genetics / Q000502:physiology*; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics / Q000502:physiology; D054799:Phospholipase C beta; D011869:Radioligand Assay; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C