Functionally Active Catalytic Domain Is Essential for Guanylyl Cyclase-Linked Receptor Mediated Inhibition of Human Aldosterone Synthesis
- Departments of 1Pharmacology and Toxicology (L.J.O., B.Y.H., J.G.D.) and 2Microbiology (L.W.C.), Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and 3Department of Pharmacology and Toxicology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202 (B.Y.H., J.G.D.)
Abstract
An aspartate-to-alanine point mutation in the catalytic domain (D853A) of guanylyl cyclase-C (GC-C), the heat-stable enterotoxin (STa) receptor, rendered the enzyme catalytically inactive. Mn2+/Triton X-100-stimulated guanylyl cyclase activity was detected in membranes from COS7 cells overexpressing GC-C but not GC-CD853A. STa treatment of paired cells resulted in cGMP production in those transiently expressing GC-C but not GC-CD853A. GC-C and GC-CD853A showed similar Bmax andKd values for [125I]STa binding in these cells, indicating that the lack of catalytic activity in the latter was not due to differing expression levels or reduced binding affinity. The involvement of the catalytic domain in aldosteronogenesis was studied in human adrenocortical H295R cells. COS7 and H295R cells infected with vaccinia virus-expressing GC-C and GC-CD853A (VVGC-CD853A) had [125I]STa-binding characteristics akin to those in transfected cells. Immunoblot confirmed that both GC-C and GC-CD853A formed similar higher order oligomers in infected cells. Virus-mediated expression of GC-C in H295R cells revealed concentration-dependent STa-stimulated cGMP formation that was undetectable in VVGC-CD853A-infected cells. STa decreased angiotensin II-stimulated human aldosterone generation in a concentration-dependent manner in vaccinia virus-expressing GC-C-infected cells but not in those infected with VVGC-CD853A. These results demonstrate that a catalytically active guanylyl cyclase is required for the inhibition of aldosteronogenesis.
Footnotes
-
Send reprint requests to: James G. Drewett, Ph.D., Department of Pharmacology and Toxicology, University of North Dakota School of Medicine and Health Sciences, 501 North Columbia Road, Grand Forks, ND 58202-9037. E-mail:drewett{at}badlands.nodak.edu
-
↵1 Current affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226-4801.
-
↵2 Current affiliation: Department of Pharmacology and Toxicology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037.
-
This work was supported by National Institutes of Health Grant R29-HL54717 (J.G.D.), American Heart Association, Wisconsin Affiliate, Grant-in-Aid 94-GB-38 (J.G.D.), a National Heart Foundation Research Starter Grant and the 1995 Ruth Salta Junior Investigator Award (both to J.G.D. from the American Health Assistance Foundation), and institutional support from the Medical College of Wisconsin and the University of North Dakota.
- Abbreviations:
- Ang
- angiotensin
- ANOVA
- analysis of variance
- ANP
- type A atrial natriuretic peptide
- BSA
- bovine serum albumin
- HEK
- human embryonic kidney
- GC
- guanylyl cyclase
- MOI
- multiplicity of infection
- PBS
- phosphate-buffered saline
- sANP
- natriuretic peptide receptor/GC-A selective agonist
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
- STa
- heat-stable enterotoxin
- TBS
- Tris-buffered saline
- VVWT
- wild-type vaccinia virus
- VVGC-C
- recombinant vaccinia virus expressing GC-C
- VVGC-C D853A
- recombinant vaccinia virus expressing GC-C D853A
-
- Received June 25, 1998.
- Accepted July 31, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
