title: 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 (125)I-ANP-(1-28) from intact CHOGCAWT cells at 37 degrees C was extremely rapid (K(off) = 0.49 +/- 0.02 min(-1)), whereas in isolated membranes prepared from these cells, the dissociation at 37 degrees C was >10-fold slower (K(off) = 0.035 +/- 0.006 min(-1)). The dissociation of ANP from CHOGCAWT cells showed remarkable temperature dependence. Between 22 and 37 degrees C, K(off) increased approximately 8 times, whereas between 4 and 22 degrees 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 K(off) to 0.064 +/- 0.006 min(-1), and also abolished the temperature dependence of dissociation. Substitution of Ser(506) by Ala and particularly the double substitution of Gly(505) and Ser(506) by Ala within the kinase-like domain markedly reduced ANP-induced increases in cGMP, whereas K(off) 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 temperature per 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.
authors.affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Vieira
authors.forename: M
authors.initials: M
authors.lastname: Gao
authors.forename: L N
authors.initials: LN
authors.lastname: Nikonova
authors.forename: T
authors.initials: T
authors.lastname: Maack
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism*; D001667:Binding, Competitive; D016466:CHO Cells; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D006152:Cyclic GMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D006162:Guanylate Cyclase; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface; D012694:Serine; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D006152:Cyclic GMP; D000409:Alanine; D005998:Glycine