Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor: Selectivity of a modified PTH(1-15) radioligand for GαS-coupled receptor conformations

Abstract

Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, 125I-[Aib 1,3,Nle8,Gln 10,homoarginine 11,Ala12,Trp14,Tyr15]rPTH(1-15) NH2, 125I-[Aib1,3,M]PTH(1-15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed 125I-PTH(1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of 125I-[Aib1,3,M]PTH(1-15) to the wild-type PTHR was strongly (∼90%) inhibited by guanosine 5′-O-(3-thio)triphosphate (GTPγS), whereas the binding of 125I-PTH(1-34) was only mildly (∼25%) inhibited by GTPγS. Of these two radioligands, only 125I-[Aib1,3,M]PTH(1-15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTPγS. Binding of 125I- [Aib1,3,M]PTH(1-15) to the constitutively active receptor, PTHR-H223R, was only mildly (∼20%) inhibited by GTPγS, as was the binding of 125I-PTH(1-34). In membranes prepared from cells lacking GαS via knockout mutation of Gnas, no binding of 125I-[Aib1,3,M]PTH(1-15) was observed, but binding of 125I-[Aib1,3,M]PTH(1-15) was recovered by virally transducing the cells to heterologously express GαS. 125I-PTH(1-34) bound to the membranes with or without GαS. The overall findings confirm the hypothesis that 125I-[Aib1,3,M]PTH(1-15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to GαS-containing heterotrimeric G proteins, whereas the binding of 125I-PTH(1-34) can occur in the absence of such coupling. Thus, 125I-[Aib1,3,M]PTH(1-15) appears to function as a selective probe of GαS-coupled, active-state PTHR conformations. Copyright © 2006 by The Endocrine Society.