Cytoplasmic Domain of Natriuretic Peptide Receptor-C Inhibits Adenylyl Cyclase

doi:10.1074/jbc.271.32.19324

Cytoplasmic Domain of Natriuretic Peptide Receptor-C Inhibits Adenylyl Cyclase

INVOLVEMENT OF A PERTUSSIS TOXIN-SENSITIVE G PROTEIN*

From the ^‡ Department of Physiology, University of Montreal, Montreal, Quebec H3C-3J7, Canada and
the ^¶ Cardiovascular Research Department, Genentech, South San Francisco, California 94080

^§ Recipient of the Medical Research Council Scientist Award from the Medical Research Council of Canada. To whom correspondence should be addressed:
Dept. of Physiology, Faculty of Medicine, University of Montreal, CP 6128, Succursale centreville, Montreal, Quebec, Canada, H3C-3J7.
Tel.: 514-343-2111.

Abstract

Natriuretic peptide receptor C (NPR-C) is a disulfide-linked homodimer with an approximately 440-amino acid extracellular domain and a 37-amino acid cytoplasmic domain; it functions in the internalization and degradation of bound ligand. The use of NPR-C-specific natriuretic peptide analogs has implicated this receptor in mediating the inhibition of adenylyl cyclase or activation of phospholipase C. In the present studies we have investigated the role of the cytoplasmic domain of NPR-C in signaling the inhibition of adenylyl cyclase. Polyclonal rabbit antisera were raised against a 37-amino acid synthetic peptide (R37A) corresponding to the cytoplasmic domain of NPR-C. Incubation of anti-R37A with rat heart particulate fractions blocked atrial natriuretic peptide-dependent inhibition of adenylyl cyclase. The cytoplasmic domain peptides R37A and TMC (10 residues of transmembrane domain appended on R37A) were equipotent in inhibiting adenylyl cyclase (K_i ~1 nM) in a GTP-dependent manner, whereas K37E (a scrambled peptide control for R37A) did not inhibit adenylyl cyclase activity. Prior incubation of membranes with pertussis toxin blocked R37A or TMC inhibition of cAMP production. Detergent solubilization of the rat heart particulate fraction destroyed natriuretic peptide inhibition of adenylyl cyclase, but TMC was able to inhibit cAMP production in a dose-dependent manner. Our results provide evidence that the 37-amino acid cytoplasmic domain of NPR-C is sufficient for signaling inhibition of adenylyl cyclase through a pertussis toxin-sensitive G protein.

Footnotes

↵* This study was supported in part by Grant MT-11024 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ANP

rat atrial natriuretic peptide (28 amino acids)

BNP

brain natriuretic peptide

CNP

C-type natriuretic peptide

cANP(4-23)

[des-Gln¹⁸-Ser¹⁹-Gly²⁰-Leu²¹-Gly²²]rat ANP(4-23)

NPR

natriuretic peptide receptor

NPR-A

natriuretic peptide receptor A

NPR-B

natriuretic peptide receptor B

NPR-C

natriuretic peptide receptor C

G protein

heterotrimeric guanine nucleotide-binding regulatory protein

PT

pertussis toxin

GTPγS

guanosine 5′-3-O-(thio)triphosphate.
↵² M. B. Anand-Srivastava, P. D. Sehl, and D. G. Lowe, unpublished observations.
- Received February 26, 1996.
- Revision received April 26, 1996.