Features of the renal parathyroid hormone-parathyroid hormone-related protein receptor derived from structural studies of receptor fragments.

Our earlier results indicated that the binding moiety of the renal PTH receptor is an 85 kD protein that is susceptible to proteolytic cleavage to a 70 kD form that supports high-affinity binding and Gs coupling, and to a 50-55 kD form that contains the ligand binding domain but does not couple to Gs. In the present study we used [125I]hPTHrP-(1-34)amide and a chemical cross-linking technique to discern the structural features of the intact 85 kD PTH/PTHrP receptor that are retained in the proteolyzed forms to "structurally map" the receptor. The results of lectin chromatography and endoglycosidase treatment show that the partially proteolyzed receptor forms retain the complex, N-linked glycans present on the intact receptor. This conclusion is further supported by the finding that wheat germ agglutinin was equally effective at competitively inhibiting specific [125I]hPTHrP-(1-34)A binding to the 70 kD form and the intact 85 kD receptor. Specific binding of [125I]hPTHrP-(1-34)A to the intact 85 kD receptor or to the 70 kD form was completely abolished by treatment with disulfide reducing agents, and both partially proteolyzed receptor forms (70- and 50 kD) were shown to retain the small (less than or equal to 14 kD) labeled fragment that is released from the intact receptor by disulfide reduction. Lectin chromatography and endoglycosidase treatment revealed that the less than or equal to 14 kD receptor component is not glycosylated. The less than or equal to 14 kD fragment does not contain a transmembrane spanning region, as its release from the membrane can be affected without detergent solubilization. Identical partial proteolytic maps of the receptor were obtained whether the receptor was covalently labeled with [125I]hPTHrP-(1-34)amide or [125I]bPTH-(1-34). These results suggest a model of the renal PTH/PTHrP receptor binding moiety as a single-chain protein in which the sites of glycosylation, ligand binding, and the functionally critical disulfide bonds are in extracellular domains near one end of the protein and the sites of proteolysis reside near the other end of the protein. These studies also provide further confirmation that PTH and PTHrP bind to a structurally indistinguishable renal receptor and validate the use of PTHrP as a ligand for studies designed to characterize and purify the PTH receptor.