Human natriuretic peptide receptor-A guanylyl cyclase is self-associated prior to hormone binding.
The human natriuretic peptide receptor-A (NPR-A) guanylyl cyclase is specifically activated to synthesize cGMP by binding of atrial natriuretic peptide (ANP) to the receptor's extracellular domain. In this report, NPR-A monoclonal and polyclonal antibodies were used to assess the aggregation status of wild-type NPR-A and a truncation mutant lacking most of the NPR-A cytoplasmic domain. On intact human embryonic kidney 293 cells, in the absence of ANP, recombinant human NPR-A is self-aggregated through disulfide bonds in an M(r) > 500,000, possibly tetrameric, complex. Under nonreducing conditions, truncated NPR-A was a monomer, indicating that the cytoplasmic domain is necessary for NPR-A self-association. In the presence of the homobifunctional cross-linker dithiobis(succinimidyl propionate), or disuccimidyl suberate, truncated NPR-A could be cross-linked as a dimer and trimer only in the presence of ANP. Wild-type NPR-A was cross-linked with disuccinimidyl suberate to an M(r) > 500,000 species in the absence of ANP, and with ANP, a smaller, M(r) approximately 400,000 receptor trimer cross-linking product was observed, together with the larger, possibly tetrameric complex. When whole cell stimulation of cGMP production by ANP was tested on the low level of endogenous 293 cell NPR-A, maximal stimulation was observed regardless of truncated NPR-A overexpression. The absence of a dominant negative effect by the truncated NPR-A, together with the cross-linking data, demonstrates that preassociated NPR-A is the functionally relevant form of this receptor.
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
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D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D003432:Cross-Linking Reagents; D006152:Cyclic GMP / Q000096:biosynthesis; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D011233:Precipitin Tests; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism*; D015398:Signal Transduction; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP; C019358:disuccinimidyl suberate