Covalent labeling of a high-affinity, guanyl nucleotide sensitive parathyroid hormone receptor in canine renal cortex

Abstract

Putative parathyroid hormone (PTH) receptors in canine renal membranes were affinity labeled with 125I-bPTH(1-34) using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 4-azidobenzoate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a major 85,000 molecular weight (Mr) PTH binding component, the labeling of which was inhibited by nanomolar concentrations of unlabeled PTH and by micromolar concentrations of 5'-guanylyl imidodiphosphate [Gpp-(NH)p]. Labeling was not influenced by the unrelated peptides insulin and arginine vasopressin. Minor PTH binding components of Mr 55,000 and 130,000 were also seen, and labeling of these was likewise sensitive to unlabeled PTH and to Gpp(NH)p. Omission of protease inhibitors during the isolation of plasma membranes resulted in the loss of the Mr 85,000 PTH binding species and the appearance of an Mr 70,000 form. Several minor PTH binding components also were observed. Equilibrium binding studies showed that such membranes had an affinity for PTH indistinguishable from that in membranes isolated with protease inhibitors and displaying a major Mr 85,000 PTH binding species. We conclude that the major form of the adenylate cyclase coupled PTH receptor in canine renal membranes is an Mr 85,000 protein. An endogenous enzyme, probably a lysosomal cathepsin, can cleave this form to produce an Mr 70,000 receptor that retains full functional activity with respect to high-affinity, guanyl nucleotide sensitive PTH binding. The ability to covalently label the PTH receptor in high yield represents a major step toward the structural characterization of this important detector molecule.

Identification and characterization of parathyroid hormone receptors on dog kidney, human kidney, chick bone and human dermal fibroblast. A comparative study of functional and structural properties

“…The detection of two species of PTH receptor in this as well as other studies (Demay et al, 1985;McKee & Murray, 1985) agrees with the physiological observation that there may be at least two different types of PTH receptors coupled to different second messengers. Photoaffinity labelling of PTH receptors on dog kidney membrane has been performed with several photoaffinity labels (Coltrera et al, 1981;Draper et al, 1982;Goldring et al, 1984;Nissenson et al, 1987). Our results obtained with the heterobifunctional, photoreactive reagent HSAb were in general agreement with previous reports.…”

“…Using a classical radioligand binding assay, parathyroid hormone (PTH) receptors have been identified on chicken (Nissenson & Arnaud, 1979;McKee & Murray, 1985), rat (Chansel et al, 1977), dog (Segre et al, 1979;Teitelbaum et al, 1982), bovine (Zull et al, 1977) and rabbit (Kremer et al, 1982) bone and kidney tissues. However, structural identification using photoaffinity labelling techniques has been performed mainly on dog kidney membrane (Coltrera et al, 1981;Draper et al, 1982;Goldring et al, 1984;Nissenson et al, 1987). Despite the above information on animal models, the functional and structural characteristics of human PTH receptors have not yet been defined.…”

Functional Expression and Signaling Properties of Cloned Human Parathyroid Hormone Receptor in Xenopus Oocytes

“…Parathyroid hormone (PTH) 1 is the primary regulator of calcium and phosphate homeostasis in higher animals (1). This 84-amino acid peptide and truncated forms such as the 1-34 or 1-36 fragments initiate the biological actions of PTH through a specific receptor (PTHR) on the plasma membrane in target tissues, primarily kidney and bone (2)(3)(4)(5). Recently, the cDNAs of PTHR from rat, mouse, and human cells have been cloned (6 -10), and both transient and stable transfection of PTHR has been described (7,11,12).…”

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

“…All probes tested label a major protein with a n apparent molecular weight of 75-85 kDa (Pun et al, 1988;Goldring et al, 1984;Nissenson et al, 1987;Wright et al, 1987;Nissenson et al, 1988;Karpf et al, 1991). In some studies, minor labeling of another protein has been observed (Pun et al, 1988;Nissenson et al, 1987;Wright et al, 1987;Nissenson et al, 1988;Karpf et al, 19911, which probably represents proteolytic products of the 75-85 kDa protein (Nissenson, et al, 1987). The similar dose response curves for PTHmediated cAMP and [Ca" 'I, increase, together with binding and photoaffinity studies, led us to suggest that the same PTH receptor is coupled to two signal transduction systems (Yamaguchi et al, 1987a).…”