A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors.
Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000138:chemical synthesis / Q000378:metabolism*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000378:metabolism*
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C056607:atriopeptin analog I; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor