Photoaffinity labeling of parathyroid hormone receptors: comparison of receptors across species and target tissues and after desensitization to hormone

Abstract

Cells derived from human giant cell tumors of bone and fibroblasts derived from human neonatal foreskin respond to parathyroid hormone (PTH) by increasing the intracellular and extracellular levels of adenosine cyclic 3',5'-phosphate (cAMP). Using photoaffinity labeling methods, we examined these cells for the presence of a PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analogue of PTH was reacted with these intact cells. After photolysis, the cells were extracted, and the proteins were denatured, reduced, and separated by electrophoresis on sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels followed by autoradiography. A single membrane component, Mr 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from (a) intact human cells derived from giant cell tumors of bone, (b) intact human fibroblasts, and (c) canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and found to be identical in terms of molecular size (as determined by the migration position on NaDod-SO4-polyacrylamide gels) across species (dog and human) and target tissue (bone, skin, and kidney). Preincubation of cells cultured from giant cell tumors of bone with PTH resulted in loss of the PTH-induced cAMP response (desensitization). Preincubation with PTH was accompanied by a marked decrease in photoaffinity labeling of the PTH binding component and suggests that the loss of hormone response in cells preincubated with PTH was related to a decrease in the number or availability of PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoprecipitation of the parathyroid hormone receptor.

“…An additional observation made by using both the photoaffinity and chemical affinity labeling approaches is the presence of several membrane components that are specifically labeled. Previous reports have described the receptor-PTH complex as a component of60-70 kDa (2)(3)(4). In these studies, using photoaffinity labeling techniques, we observed the appearance of a doublet of apparent molecular mass 68-70 kDa and 95-and 28-to 30-kDa bands that may represent additional components or forms of the receptor.…”

“…4 mass of 70 kDa (2-4). Furthermore, a membrane component of identical apparent molecular mass (as determined by NaDodSO4 gel electrophoresis) was found in several PTHresponsive tissues from dogs and humans, indicating that the physicochemical properties of the PTH receptor have been highly conserved during evolution (4), as has the sequence of PTH (1). This initial characterization of the receptor has also served to guide further efforts directed at harvesting and isolating the PTH receptor.…”

“…A receptor, or a subunit of the receptor, that specifically binds PTH has been identified as a 60-to 70-kDa (70 kDa is the size of receptor-hormone complex) membrane component in canine renal membranes by using photoaffinity techniques based on a biologically active photolabile radiolabeled PTH analog (2,3). Subsequent studies demonstrated that a similar, if not identical, component was the principal plasma membrane constituent binding PTH in both human tumor-derived bone cells and skin fibroblasts (4).…”

Stephen M. Krane: A Scholar and a Gentleman

“…Studies with Steven Goldring established the patterns of parathyroid responses of osteoblasts, synovial and dermal fibroblasts and stromal cells from giant cell tumors of bone (Goldring et al, 1977;Goldring et al, 1978a;Goldring et al, 1978b;Goldring et al, 1979a;Goldring et al, 1979b;Goldring and Krane, 1980;Crisp et al, 1984;Goldring et al, 1984a;Goldring et al, 1984b;Goldring et al, 1986b;Goldring et al, 1990). Studies with Steven Goldring established the patterns of parathyroid responses of osteoblasts, synovial and dermal fibroblasts and stromal cells from giant cell tumors of bone (Goldring et al, 1977;Goldring et al, 1978a;Goldring et al, 1978b;Goldring et al, 1979a;Goldring et al, 1979b;Goldring and Krane, 1980;Crisp et al, 1984;Goldring et al, 1984a;Goldring et al, 1984b;Goldring et al, 1986b;Goldring et al, 1990).…”

Dissociation between parathyroid hormone‐stimulated cAMP and calcium increase in UMR‐106‐01 cells

“…Several approaches to answer the question of PTH receptor subtypes include PTH binding assays (Rosenhlatt et al, 1977;Segre et al, 1979;Keutmann et al, 1985;Demay et al, 1985;Pliam et al, 1982;Rizzoli et al, 1983;Rao and Murray, 1985;McKee and Murray, 1985;Pun et al, 19881, photo-affinity labeling (Pun et al, 1988;Goldring et al, 1984;Nissenson et al, D 1992WILEY-LTSS, INC 1987Wright et al, 19871, and determination of dose responses for hormone-stimulated cAMP accumulation and [Ca2+l, (Yamaguchi et al, 1987a1. In general, all of the above methods conclude that PTH-responsive cells express a single type of PTH receptor.…”