Identification of a Receptor-Binding Region in Parathyroid Hormone*

Abstract

Analyses of structure/activity relations for parathyroid hormone (PTH) have revealed that the sequence PTH- (1–34) is fully active in all known bioassay systems and that deletion of the first two amino-terminal residues results in the loss of all or virtually all biological activity but not in the loss of the property of binding to renal receptors in vitro. The apparent structural separation of binding and activation regions led to synthesis of the bovine (b) hormone analog [Nle-8,Nle-18,Tyr-34]bPTH-(3–34)amide, which has proved in multiple in vitro assays, based on stimulation of adenylate cyclase, to be a potent inhibitor of PTH action. To define the structural determinants for the binding of PTH to renal receptors, progressively shortened fragments of this analog corresponding to the sequence regions 7–34, 10–34, 15–34, 20–34, and 25–34 were synthesized, and the receptor-binding properties of the fragments were evaluated. In an earlier study, all of the fragments except [Tyr-34]bPTH-(25–34)amide were shown to inhibit PTH-stimulated adenylate cyclase in vitro in renal membranes; the apparent inhibitory constant increased progressively as the fragments were progressively truncated. The purpose of this study was to evaluate these peptides and a still shorter fragment, [Tyr-34]bPTH-(28–34)amide, in a recently developed receptor-binding assay to determine 1) the correlation between inhibitory constants and observed binding constants for these peptides and 2) the minimum chain length required for receptor binding per se. Excellent correlation between apparent binding constants and inhibitory constants was found for analog fragments 3–34, 7–34, 10–34, 15–34, and 20–34. In the receptorbinding assay, which allows assay of peptide doses larger than those in the adenylate cyclase assay, the analog fragment 25–34 bound weakly, but definitely, to renal binding sites. The analog fragment 28–34, even when present in a high concentration, failed to compete for binding sites. The results indicate that a region as short as 10, but not as small as 7, amino acids in length satisfies the structural requirements for occupancy of the PTH renal receptor in vitro.