Covalent labeling of a high-affinity, guanyl nucleotide sensitive parathyroid hormone receptor in canine renal cortex.
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.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D006150:Guanine Nucleotides / Q000494:pharmacology*; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010281:Parathyroid Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide
D006150:Guanine Nucleotides; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide