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-(1-34) 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 (KD = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (KD = 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 (KD = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10(-11)-10(-9) M rPTH-(1-34) with an ED50 intermediate between the two KD values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the KD of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.

Protein kinase C-activating domains of parathyroid hormone-related protein

“…PTH-(44-68), and PTH-(53-84), do not stimulate membrane PKC activity in ROS 17/2 cells. (ao) It should also be noted that this ability of hPTHrP- (28)(29)(30)(31)(32)(33)(34) to stimulate PKC definitively eliminated a role for adenylyl cyclase in the activation of the PKC-stimulating phospholipid breakdown because PTHrP, like PTH, needs its first two N-terminal amino acids to activate adenylyl cyclase. arate experiments, the PTHrP-( 107-1 11) fragment stimulated membrane-associated PKC activity but strongly and consistently only at picomolar concentrations.…”

“…This peptide inhibits PKC activity because its amino acid sequence is that of their autoinhibitory pseudosubstrate prototopes, PKC- (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). (24,15) The identity of the protein kinase was also confirmed by selectively depleting ("downregulating") the cellular PKC pools by incubating the cells for 20 h in 300 nM TPA (12-0-tetradecanoyl phorbol-13-acetate, a selective PKC activator) and then determining whether this pretreatment had eliminated the responses to PTHrP and a second exposure to…”

Parathyroid hormone stimulates phosphoinositide metabolism and intracellular calcium mobilization in osteoblast-like clone MC3T3-E1 cells

“…Thus, the structure-function relationship of PTH peptide has been reinvestigated (3,22). More recently, Seitz et al reported two classes of binding sites for rPTH on rat osteosarcoma ROS 17/2.8 cells (25). One is with high affinity and low capacity (KD=pM order), and the other is low affinity and high capacity (KD =nM order) site.…”

Type-1 Parathyroid Hormone (PTH)/PTH-Related Peptide (PTHrP) Receptors Activate Phospholipase C in Response to Carboxyl-Truncated Analogs of PTH(1–34)1

“…Second, previous work was conducted using ROS 17/2 osteosarcoma cells that expressed endogenous, rather than transfected, rat PTH1R genes (11,15). Ͼ250,000/cell) than do established osteosarcoma cells, such as ROS 17/2.8 or UMR 106 -01 (20,000 -50,000/cell) (26,31). On the other hand, it is quite possible [perhaps even likely (27)(28)(29)(30)] that ROS 17/2 cells and spleen cells express alternate species of receptors for PTH that could have inhibited the response(s) to hPTH in these cells or that might have supported a PLC-independent activation of PKC by hPTH(1-34) but not by hPTH .…”