Quantitative cell membrane-based radioligand binding assays for parathyroid hormone receptors.
Most current assays of PTH receptor ligand binding employ whole cells as the vehicle for receptor. Whole cell binding does not easily permit the estimation of physically meaningful binding parameters, the detection of multiple receptor states, or the evaluation of the effects of receptor modulators such as guanine nucleotides. We have developed quantitative methods for the measurement of equilibrium ligand binding parameters at cloned parathyroid hormone (PTH) receptors in cell membrane preparations. Centrifugation is used to separate bound and free [125I]-labeled peptide radioligands, and nonfat dried milk is used as a blocking agent to reduce nonspecific binding. This method is useful for measurement of agonist and antagonist radioligand binding at the PTH-1 receptor and binding of [125I]PTH(1-34) at the PTH-2 receptor. Less than 25% of [125I]PTH(1-34) or [125I]PTHrP(1-36) is degraded during the assay incubation. We demonstrated the utility of the assay using measurements of ligand binding properties at the PTH-1 receptor. (1) Homologous displacement experiments provided estimates of Kd and Bmax for the radioligands. (2) Displacement of radiolabeled antagonist binding ([125I]PTH(3-34)) by an unlabeled agonist (RS-66271) revealed multiple affinity states of agonist-receptor interaction. (3) Comparison of RS-66271 displacement in the presence and absence of GTP-gammaS demonstrated that the highest affinity state is guanine nucleotide-sensitive, suggesting that this state requires stabilization by G-protein. This assay thus allows more mechanistic interpretation of binding data than PTH binding assays in current use. A more convenient rapid-filtration method was also developed for measurement of radioligand binding at PTH-1 and PTH-2 receptors.
Hoare
S R
SR
Unit on Cell Biology, Laboratory of Genetics, National Institute of Mental Health, Bethesda, MD 20892, USA.
Usdin
T B
TB
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D044183:Receptor, Parathyroid Hormone, Type 2; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D019379:Teriparatide / Q000031:analogs & derivatives / Q000378:metabolism
D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D044183:Receptor, Parathyroid Hormone, Type 2; D018016:Receptors, Parathyroid Hormone; D019379:Teriparatide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C104917:RS 66271