Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor: EFFECT OF RECEPTOR-G-PROTEIN INTERACTION ON THE LIGAND BINDING MECHANISM AND RECEPTOR CONFORMATION

Abstract

Ligand binding to the PTH1 receptor is described by a "two-site" model, in which the C-terminal portion of the ligand interacts with the N-terminal domain of the receptor (N interaction), and the N-terminal region of the ligand binds the juxtamembrane domain of the receptor (J interaction). Previous studies have not considered the dynamic nature of receptor conformation in ligand binding and receptor activation. In this study the ligand binding mechanism was compared for the G-protein-coupled (RG) and uncoupled (R) PTH1 receptor conformations. The two-site model was confirmed by demonstration of spatially distinct binding sites for PTH(3-34) and PTH(1-14): PTH(1-14), which binds predominantly to the J domain, only partially inhibited binding of 125I-PTH(3-34); and PTH(3-34), shown to bind predominantly to the N domain, only partially inhibited PTH(1-14)-stimulated cAMP accumulation. To assess the effect of R-G coupling, ligand binding to R was measured by displacement of 125I-PTH(3-34) with 30 microM guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) present, and binding to RG was measured by displacement of 125I-[MAP]PTHrP(1-36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1-34) affinity for RG; residue 5 of MAP-PTHrP(1-36) was a determinant of R/RG binding selectivity, and PTH(1-14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3-34) binding was GTPgammaS-insensitive. Finally, several observations suggest the receptor conformation is more "closed" at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1-14) and PTH(3-34). At RG PTH(1-14) better occluded binding of 125I-PTH(3-34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states.