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.
Highlights
Clearance receptors of atrial natriuretic peptide (ANP), the most abundant class of the natriuretic peptide receptors, have a single transmembrane domain, a short cytoplasmic tail of 37 amino acids, and an extracellular binding domain that has a significant homology to the extracellular domain of guanylyl cyclase (GC) receptors (1, 4 – 6)
We postulated that the rapid dissociation of ANP from surface Guanylyl cyclase subtype A (GCA) receptors at physiologilike; CHO, Chinese hamster ovary; GCAWT, wild-type GCA receptor; GCACYTϪ, GCA receptor with the cytoplasmic domain deleted; GCATKϪ, GCA receptor with the tyrosine kinase-like (TK) domain deleted; GCAGCϪ, GCA receptor with the GC domain deleted; CHOGCAWT, CHO cells transfected with GCAWT; CHOGCACYTϪ, CHO cells transfected with GCACYTϪ; CHOGCATKϪ, CHO cells transfected with GCATKϪ; BSA, bovine serum albumin; ATP␥S, adenosine 5Ј-O-(3-thiotriphosphate)
Removal of the kinase-like and catalytic guanylyl cyclase domains decreased receptor-ligand dissociation to the slow rates of 0.064 Ϯ 0.006 and 0.043 Ϯ 0.004 minϪ1 for CHOGCATKϪ and CHOGCAGCϪ cells, respectively. These results show that the cytoplasmic domain of GCA is involved in the mediation of the fast receptor-ligand dissociation at physiological temperatures and that major deletions within the cytoplasmic domain markedly reduce the ability of GCA receptors to physiologically modulate receptor-ligand dissociation
Summary
Clearance receptors of ANP, the most abundant class of the natriuretic peptide receptors, have a single transmembrane domain, a short cytoplasmic tail of 37 amino acids, and an extracellular binding domain that has a significant homology to the extracellular domain of GC receptors (1, 4 – 6). Removal of the kinase-like and catalytic guanylyl cyclase domains decreased receptor-ligand dissociation to the slow rates of 0.064 Ϯ 0.006 and 0.043 Ϯ 0.004 minϪ1 for CHOGCATKϪ and CHOGCAGCϪ cells, respectively.
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