Functionally active catalytic domain is essential for guanylyl cyclase-linked receptor mediated inhibition of human aldosterone synthesis.
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 and Kd 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.
Olson
L J
LJ
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Ho
B Y
BY
Cashdollar
L W
LW
Drewett
J G
JG
D000450:Aldosterone / Q000096:biosynthesis*; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism / Q000821:virology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000096:biosynthesis; D004768:Enterotoxins / Q000378:metabolism / Q000494:pharmacology; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D007700:Kinetics; D017354:Point Mutation; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012680:Sensitivity and Specificity; D013268:Stimulation, Chemical; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007457:Iodine Radioisotopes; D018000:Receptors, Peptide; C034151:heat stable toxin (E coli); D000804:Angiotensin II; D000450:Aldosterone; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP