Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney.

R A Nissenson,D Diep,G J Strewler

doi:10.1016/s0021-9258(18)37641-5

R A Nissenson, D Diep + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)37641-5

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Abstract

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.

Highlights

From the Endocrine Unit, Veterans AdministrationMedical Center and Departments of Medicineand Physiology, University of California, Sun Francisco, California94121
We compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr3‘]PLP-(136)amide, with those of bovine parathyroid hormone-(1-34) on receptors and adenylate cyclase in proteins are immunologically distinguishable from PTH, yet reproduce the major biochemical and physiologic effects of PTH in model in uitro systems, including activation of adenylate cyclase in bone and kidney (1-8), stimulation of resorption of fetal rat limb bones (4, 9-12), and inhibition of bone cells and in renal membranes
A competitive PTH receptor antago- cDNA sequence, PLP derived from lung carcinoma cells (14)

Summary

EXPERIMENTAL PROCEDURES

Materials-Synthetic bPTH-(1-34) (5000 units/mg) was purchased from Bachem, Inc. (Torrance, CA). Radioligand Binding Assays-Bovine PTH-(1-34) was iodinated by an electrolytic procedure to a specific activity of [100-200] pCi/pg, as described previously (20). 100 pCi/pg.'261-labeled PTH and PLP peptides were purified by chromatography over Sephadex LH-20 followedby reverse-phase high performance liquid chromatography over a C-18 column, as described previously for '261-bPTH-(1-34) (22). Binding of '251-bPTH-(1-34) and '*'I-labeled peptides to canine renal plasma membranes was assessed as described previously for '251-bPTH-(1-34) (23). 1. Stimulation of adenylatecyclase activity by synwith 1.0 ml of ice-cold phosphate-buffered saline and were thetic PLP peptides and by bPTH-(1-34). Adenylate cyclase activity in UMR-106 cells was assessed as 36)amide. Photoaffinity Cr0ss-linki~-'~~I-bPTH-(1-34a)nd '251-PLP-(134)amide were covalently attached to binding sites in canine renal about 6-fold less potent thanbPTH-(1-34) (E& value of 0.5 membranes using the heterobifunctional cross-linker HSAB, as de- nM). This using gels polymerized in a 5-20% gradient of acrylamide

RESULTS

DISCUSSION

Methods