Affiliation:
1. Endocrine Unit (T.D., A.L., M.J.M., M.B., H.J., J.T.P., T.J.D.), Massachusetts 02114
2. Department of Pediatrics (H.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Abstract
AbstractMechanisms 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-[Aib1,3,Nle8,Gln 10,homoarginine11,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.
Subject
Endocrinology,Molecular Biology,General Medicine