Solubilization and Molecular Characterization of the Atrial Natriuretic Peptide (ANP) Receptor in Human Platelets: Comparison with ANP Receptors in Rat Tissues*

Abstract

We have previously demonstrated the presence of binding sites for atrial natriuretic peptide (ANP) in human platelets. These sites have pharmacological characteristics similar to those of rat vascular smooth muscle. They are subject to regulation by circulating levels of ANP in plasma, varying inversely with the latter after high sodium intake, in arterial hypertension and congestive heart failure. We have now solubilized these platelet receptors with the nonionic detergent Triton X-100 (0.6%). The preparations were incubated with [125I]ANP in the presence of increasing concentrations of ANP-(99-126), ANP-(101-126), ANP-(103-126), and ANP-(103-123). The order of potency of these peptides to displace [125I]ANP was similar for the solubilized and particulate receptor. Bound [125I]ANP was covalently cross-linked to the receptor with 5 mM disuccinimidyl suberate. Autoradiography of the sodium dodecyl sulfate-polyacrylamide gel showed that [125I]ANP specifically interacts with a 125-kDa membrane component, some of which may be reduced by 2% mercaptoethanol or 10 mmol/L dithiothreitol to a 70-kDa species. A small proportion of a 70-kDa peptide is also found under nonreducing conditions. The concentration of ANP-(99-126) that inhibits binding of [125I]ANP by 50% to both the 125-kDa and the 70-kDa species was 0.1 nM, while that for ANP-(103-123) was 3 nM. The internally ring-deleted analog Des(Gln116,Ser117,Gly118,Leu119,Gly120)ANP -(102-121) or C-ANP displaced with equal potency ANP binding to the high and low mol wt (Mr) bands, as also found in cultured rat vascular smooth muscle cells, but not in the mesemteric arteries these cells are derived from. In the latter, C-ANP displaced only binding from the lower Mr band. These results show that the ANP receptor in human platelets is heterogeneous. There is one nonreducible species with of 125,000 Mr, another of similar Mr containing two disulfide-linked subunits of 70,000 Mr, and, to a lesser extent, a nonreducible 70-kDa species, in agreement with findings in other tissues in experimental animals.