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

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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 cAMP. Using photoaffinity labeling methods, these cells were examined for the presence of PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analog 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, MW 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from intact human cells derived from giant cell tumors of bone, intact human fibroblasts, and canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and were in terms of molecular size (as determined by the migration position on NaDodSO4-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 wiht PTH was related to a decrease in the number of availability of PTH receptors. The PTH-induced cAMP response and photoaffinity labeling of the PTH binding component were not altered by preincubating cells with a PTH inhibitor which binds to PTH receptors but fails to increase cAMP levels. Simple occupancy of the hormone receptor apparently is not sufficient to produce desensitization to PTH. Biological events that take place after the binding of PTH to the receptor apparently are involved in the subsequent loss of hormone responsiveness.

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