title: Binding of intact parathyroid hormone to chicken renal plasma membranes: evidence for a second binding site with carboxyl-terminal specificity.
abstract: We studied the binding of bovine PTH (bPTH) to chicken renal plasma membranes using an intact hormone radioligand, [125I]bPTH-(1-84). In contrast to previous studies using amino-terminal radioligands, our experiments revealed the presence of two distinct binding sites for intact bPTH. Scatchard analysis of competition curves consistently gave a biphasic curve, and computerized nonlinear regression analysis indicated a high affinity [dissociation constant (Kd) = 1.21 nM] and a low affinity (Kd = 333 nM) site (P less than 0.001). Analysis of binding of [125I] bPTH-(1-34) using identical techniques revealed only one site, similar to the high affinity sites seen with intact hormone tracer. The low affinity site for [125I]bPTH-(1-84) had carboxyl-terminal specificity since analysis of competition curves with unlabeled human PTH-(53-84) gave virtually identical binding parameters to the low affinity site obtained by competition with unlabeled native hormone. Binding to the low affinity site was inhibited in the presence of 10 mM Mg2+ and was reduced after storage of membranes for over 1 month at -70 C. Association of [125I]bPTH-(1-84) to the low affinity site took distinctly longer (approximately 4 h) than to the high affinity site (essentially complete by 2.25 h). Our data suggest that the high affinity site is coupled to adenylate cyclase in these membranes, while the low affinity site is not. The physiological significance of the low affinity carboxyl-terminal PTH binding is not known, and further studies are indicated.
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D010281:Parathyroid Hormone / Q000378:metabolism*; D011309:Preservation, Biological; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D006165:Guanylyl Imidodiphosphate; D000262:Adenylyl Cyclases