Inverse agonism and the PTH/PTHrP receptor

The PTH/PTHrP receptor (PPR) is a family B G protein-coupled receptor that plays a vital role in calcium homeostasis and skeletal development. We are using photoaffinity cross-linking methods to explore how inverse agonists interact with constitutively active mutant PPRs. The analog [parabenzoyl-phenylalanine (Bpa)(2)]PTHrP(I - 36) is a selective inverse agonist for PPR-H223R (altered in transmembrane domain (TMD) 2), while [D-Bpa(12)]PTHrP(5-36) is an inverse agonist for both PPR-H223R and PPR-T410P (altered in TMD 6). In each ligand, the photolabile Bpa group is the key determinant of inverse agonism. The [Bpa(2)]PTHrP(I - 36) analog cross-linked to two sites in PPR-H223R: a primary site near the extracellular end of TMD 6 and a secondary site within TMD 4, extracellular loop 2 or TMD 5. Mutational studies revealed that the intensity of cross-linking to TMD 6, relative to TMD 4/5, increased as receptor signaling activity increased. Our preliminary mapping studies with [D-Bpa(12)]PTHrP(5-36) indicate that this analog cross-links to a different site, possibly near the boundary of TMD I and the N-terminal domain. Thus, there appear to be two different mechanisms by which inverse agonism can be achieved in constitutively active PPRs. The studies also reveal conformational differences between active and inactive state PPRs, at least in the vicinity of residue 2 of the ligand. (C) 2003 Elsevier Science B.V. All rights reserved.