Abstract
The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.
Highlights
The parathyroid hormone (PTH1)-2 receptor, a recently identified PTH receptor subtype, responds fully to PTH but not at all to PTH-related peptide (PTHrP) (1)
The pronounced difference in the ligand selectivity profiles of the PTH-1 and PTH-2 receptors suggested that these two receptors could be used in such an analysis, since the difference in selectivity can most be explained by structural differences in the receptors at sites that are involved in ligand recognition or ligandinduced receptor activation
The difference in the ligand selectivities of the PTH-1 receptor and the PTH-2 receptor can be seen in the cAMP response profiles shown in Fig. 1, panels A and B
Summary
Peptides and Reagents—The preparation and initial characterization of [Trp23,Tyr36]PTHrP-(1–36)amide and [Ile5,Trp23,Tyr36]PTHrP-(1–. Cells were transfected in 24-well plates using plasmid DNA (200 ng/well) that was purified by cesium chloride/ ethidium bromide gradient centrifugation, except for the initial screening of the cassette mutants, in which phenol-extracted miniprep DNA was used. This miniprep DNA was quantified by ethidium bromide staining of agarose gels, and was transfected at a concentration of 100 ng/well. Cell surface receptor numbers were estimated from Scatchard analyses of homologous competition binding studies that were performed with 125I-[Nle8,21,Tyr34]rPTH-(1–34)NH2 (26 fmol/well) and varying amounts (1.2–300 pmol) of the same unlabeled ligand. Dose-response analyses yielded EC50 values (ligand dose resulting in 50% of maximum response (Emax) attained by that ligand), which were calculated from plots of log(E/Emax Ϫ E) versus log[ligand], where E is the cAMP response measured at the corresponding dose of ligand (6)
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