Stoichiometry of the atrial natriuretic factor-R1 receptor complex in the bovine zona glomerulosa.
The atrial natriuretic R1 receptor is a membrane protein that is present as an apparently preassociated noncovalent oligomer in the absence of ligand as suggested by steric exclusion studies and cross-linking experiments in physiological and recombinant receptor expression systems. The association state of this receptor oligomer was studied in the presence of amiloride and ATP, two known modulators of the R1 receptor functions with both the intact receptor and a cytoplasmic domain-deleted form obtained by limited proteolysis with trypsin. It was shown by steric exclusion on Superose 6 column that amiloride increased the affinity of ANF for the native and truncated receptor, in contrast with ATP, whose destabilizing effect on ANF binding was abolished by truncation of the cytoplasmic domain. Neither amiloride nor ATP exerts its effects by altering the aggregation state of the receptor. Comparison of the measured number of ANF binding sites with immunoassayable receptor protein revealed that the stoichiometry of ANF binding to the R1 receptor was 1:2. This was confirmed by using an ANF analog that bears a photoactivatable group at both of its ends, showing that ANF, as for the growth hormone/receptor complex, interacts with both the receptor subunits and specifically cross-links a dimeric form of the receptor. The potential pharmacological consequences of this 1:2 stoichiometric ratio of the ANF-receptor complex are discussed.
Faculty of Pharmacy, University of Montreal, Canada.
J J
JJ
Rondeau
N
N
McNicoll
J
J
Gagnon
N
N
Bouchard
H
H
Ong
A
A
De Léan
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents; D003593:Cytoplasm; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011940:Receptor Aggregation / Q000187:drug effects; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D015384:Zona Glomerulosa / Q000378:metabolism*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010455:Peptides; D000584:Amiloride; D009320:Atrial Natriuretic Factor; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A