STOICHIOMETRY OF THE ATRIAL NATRIURETIC FACTOR-R1 RECEPTOR COMPLEX INTHE BOVINE ZONA GLOMERULOSA

The atrial natriuretic R1 receptor is a membrane protein that is prese nt as an apparently preassociated noncovalent oligomer in the absence of ligand as suggested by steric exclusion studies and cross-linking e xperiments in physiological and recombinant receptor expression system s. 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-dele ted form obtained by limited proteolysis with trypsin, It was shown by steric exclusion on Superose 6 column that amiloride increased the af finity of ANF for the native and truncated receptor, in contrast with ATP, whose destabilizing effect on ANF binding was abolished by trunca tion of the cytoplasmic domain. Neither amiloride nor ATP exerts its e ffects by altering the aggregation state of the receptor, Comparison o f the measured number of ANF binding sites with immunoassayable recept or protein revealed that the stoichiometry of ANF binding to the R1 re ceptor 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.