Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxyl-terminal region of PTH-(1-84)

Abstract

Carboxyl-terminal fragments of PTH (C-PTH) appear to have biological properties different from those mediated by the amino-terminal portions of PTH and PTH-related peptide (PTHrP). To characterize a C-PTH receptor that may be involved in mediating these functions, we performed RRAs and affinity cross-linking studies with several clonal cell lines. Radiolabeled recombinant [Leu8,18,Tyr34]human PTH-(1-84)[mutPTH-(1-84) and [Tyr34] human PTH-(19-84)[mutPTH-(19-84) showed little or no specific binding to stably expressed recombinant PTH/PTHrP receptors. However, high affinity binding was observed using osteoblast-like and rat parathyroid (PT-r3) cells. The apparent Kd values were 20-30 nM for PTH-(1-84), mutPTH-(1-84), and mutPTH-(19-84), respectively; 400-800 nM for PTH-(39-84); and more than 5000 nM for PTH-(53-84). [Nle8,18,Tyr34]bovine PTH-(1-34)amide [PTH-(1-34)], PTH-(44-68), PTHrP-(37-74), and PTHrP-(109-141) showed no displacement of either radioligand. C-PTH receptor number was increased up to 2-fold by pretreating ROS 17/2.8 cells with increasing doses of PTH-(1-34), PTH-(1-84), or 8-bromo-cAMP, whereas no change was observed in response to dexamethasone or PTH-(39-84). Cross-linking studies using radiolabeled mutPTH-(1-84) or mutPTH-(19-84) revealed specific labeling of two proteins in ROS 17/2.8 cells that were approximately 40 and 90 kilodaltons in size (including the radioligand of approximately 10 kilodaltons). The intensity of affinity labeling of both proteins was dose dependently inhibited by increasing concentrations of unlabeled PTH-(1-84) and several carboxyl-terminal PTH-(1-84) fragments, but not by PTH-(1-34). Similar studies with PT-r3 cells revealed only a single protein band of about 90 kilodaltons. These data indicate that the carboxyl-terminal portion of PTH-(1-84) binds specifically to a unique receptor/binding protein distinct from the previously isolated PTH/PTHrP receptor.

Receptors for the Carboxyl-Terminal Region of PTH(1–84) Are Highly Expressed in Osteocytic Cells**This work was supported by the NIH Grant DK-11794.

“…The results of radioreceptor binding assays in OC cells, using the 125 I-[Tyr 34 ]hPTH (19 -84) radioligand, were remarkably similar to those reported previously with ROS17/2.8 cells (6,16), which suggests that the CPTHRs on these different cell types probably are similar or identical. The results of radioreceptor binding assays in OC cells, using the 125 I-[Tyr 34 ]hPTH (19 -84) radioligand, were remarkably similar to those reported previously with ROS17/2.8 cells (6,16), which suggests that the CPTHRs on these different cell types probably are similar or identical.…”

“…Enumeration of CPTHR sites on the OC cells led to estimates of 1,900,000 to 3,400,000 per cell, which was 6-to 10-fold higher than on the osteoblastic (F) cells obtained from the same bones and at least 5-fold higher than on ROS 17/2.8 cells (6). A survey of other available cell lines indicated that, with the exception of a bone marrow stromal cell line (MS-1) (25), little or no binding was detectable on NIH-3T3, HeLa or BHK21 fibroblast cells, as was reported previously for OK and LLC-PK1 kidney cells, YCC cells, and SaOS-2, MG63 and UMR106 -01 osteosarcoma cells (6).…”

“…The [Tyr 34 ]hPTH (19 -84) peptide was radioiodinated with Na[ 125 I] (2000 Ci/mmol) by the chloramine-T method and purified by HPLC, as previously described (6). For binding experiments, cells were plated in 24-well dishes at 100,000 cells/ml and cultured at 33 C for 7-14 days.…”

“…Several such fragments induce cytosolic free calcium transients in human fetal hypertrophic chondrocytes (19). Direct physical evidence of a putative receptor (CPTHR) with binding specificity for C-terminal PTH sequences was obtained by cross-linking of the peptide 125 I-[Tyr 34 ] hPTH (19 -84) (which does not bind to PTH1Rs) to 40-kDa and 90-kDa proteins in ROS 17/2.8 rat osteoblastic cells (6).…”

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“…The C-terminal region binds to another receptor (CPTHR), which responds exclusively to the C-terminal of PTH. [12] It has been shown that the C-terminal fragments may have discrete biological properties acting on the osteocyte to enhance its apoptosis,[13] accounting for the rationale for creating the N-terminal 1–34 PTH fraction.…”