Molecular and Cellular Physiology of the Dissociation of Atrial Natriuretic Peptide from Guanylyl Cyclase A Receptors

Abstract

Guanylyl cyclase subtype A (GCA) is the main receptor that mediates the effects of atrial natriuretic peptide (ANP) in the regulation of plasma volume and blood pressure. The dynamics of the dissociation of ANP from GCA were investigated in cultured Chinese hamster ovary (CHO) cells stably transfected with wild-type (WT) or mutant GCA receptors. The rate of dissociation of specifically bound 125I-ANP-(1-28) from intact CHOGCAWT cells at 37 °C was extremely rapid (Koff = 0.49 ± 0.02 min -1), whereas in isolated membranes prepared from these cells, the dissociation at 37 °C was >10-fold slower (Koff = 0.035 ± 0.006 min-1). The dissociation of ANP from CHOGCAWT cells showed remarkable temperature dependence. Between 22 and 37 °C, K off increased ∼8 times, whereas between 4 and 22 °C, it increased only 1.5 times. Total deletion of the cytoplasmic domain or of the catalytic guanylyl cyclase sequence within this domain abolished ANP-induced increases in cGMP, dramatically slowed receptor-ligand dissociation by at least 10-fold, and abolished the temperature dependence of the dissociation of ANP. Deletion of the kinase-like domain led to maximal constitutive activation of guanylyl cyclase, markedly decreased Koff to 0.064 ± 0.006 min-1, and also abolished the temperature dependence of dissociation. Substitution of Ser506 by Ala and particularly the double substitution of Gly505 and Ser506 by Ala within the kinase-like domain markedly reduced ANP-induced increases in cGMP, whereas Koff decreased modestly (albeit significantly) to 0.36 ± 0.03 and 0.24 ± 0.02 min-1, respectively. As a whole, the results demonstrate for the first time that temperatureper se or ATP alone cannot account for rapid GCA receptor-ligand dissociation under physiological conditions and suggest that ligand dissociation is modulated in part by the interaction of still unidentified cytosolic factors with the cytoplasmic domain of GCA.