Identification of the G protein-activating domain of the natriuretic peptide clearance receptor (NPR-C).
We have shown recently that the 37-amino acid intracellular domain of the single-transmembrane, natriuretic peptide clearance receptor, NPR-C, which is devoid of kinase and guanylyl cyclase activities, activates selectively Gi1 and Gi2 in gastric and tenia coli smooth muscle. In this study, we have used synthetic peptide fragments of the N-terminal, C-terminal, and middle regions of the cytoplasmic domain of NPR-C to identify the G protein-activating sequence. A 17-amino acid peptide of the middle region (Arg469-Arg485), denoted Peptide 4, which possesses two N-terminal arginine residues and a C-terminal B-B-X-X-B motif (where B and X are basic and non-basic residues, respectively) bound selectively to Gi1 and Gi2, activated phospholipase C-beta3 via the betagamma subunits, inhibited adenylyl cyclase, and induced smooth muscle contraction, in similar fashion to the selective NPR-C ligand, cANP4-23. A similar sequence (Peptide 3), but with a partial C-terminal motif, had minimal activity. Sequences which possessed either the N-terminal basic residues (Peptide 1) or the C-terminal B-B-X-X-B motif (Peptide 2) were inactive. Peptide 2, however, inhibited G protein activation and cellular responses mediated by the stimulatory Peptide 4 and by cANP4-23, suggesting that the B-B-X-X-B motif mediated binding but not activation of G protein, thus causing Peptide 2 to act as a competitive inhibitor of G protein activation.
Murthy
K S
KS
Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711, USA.
Makhlouf
G M
GM
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D006168:Guinea Pigs; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D054799:Phospholipase C beta; D011485:Protein Binding; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010446:Peptide Fragments; D005576:Colforsin; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C