Radioiodinated rat parathyroid hormone-(1-34) binds to its receptor on rat osteosarcoma cells in a manner consistent with two classes of binding sites

Abstract

Binding of ¹²⁵I-labeled rat (r) PTH-(1-34) to ROS 17/2.8 osteoblastic bone cells and to membranes from these cells was examined. Competitive binding inhibition experiments were performed using unlabeled rPTH-(1-34) with particular emphasis on concentrations of peptide below 1 nM. In intact cells, binding of labeled rPTH-(l-34) was highly specific, and inhibition of binding by unlabeled ligand suggested the presence of two classes of binding sites, one with high affinity and low capacity (K_D = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (K_D = 2 nM, approximately 80% of the sites). Membranes prepared from ROS cells also exhibited a pattern of binding from competitive inhibition curves consistent with two distinct binding sites (K_D = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10⁻¹¹-10⁻⁹ M rPTH-(l-34) with an ED₅₀ intermediate between the two K_D values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the K_D of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.