(Taken directly from the application): Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) play critical roles in the regulation of blood mineral ion levels and organ development, respectively. They mediate these actions through the PTH/PTHrP receptor (PTH1R), a class 2 G protein-coupled receptor. A solid understanding of the molecular mechanisms by which these molecules interact and function, both in normal and disease states, is crucial to human health. Recent studies suggest that the (15-34) domain of PTH (and PTHrP ) """"""""docks"""""""" to the N-terminal domain of the PTH1R and this high affinity interaction enables the PTH(1-14) domain to engage the heptahelical region of the receptor and thus induce receptor activation. But there are many unknowns. For example, the specific contacts that occur between the ligand and the receptor; the three-dimensional topologies of the ligand and receptor in the bound and free states, and the conformational changes that occur upon activation. These problems will be experimentally addressed in the proposed studies. We will apply """"""""reductionist"""""""" approaches that expand on our prior work. We will use PTH(1-14) and PTH(15-34) analogs for structure-activity relationship analyses aimed at defining the ligand determinants of receptor activation and binding affinity, respectively. New types of amino acids and conformational constraints will be introduced into these ligands, some via our new collaboration with Dr. Greg Verdine of the Harvard University Chemistry Department, and. the resulting peptides will be used to functionally explore the PTH/PTH1R interaction mechanism. Thus, second-site suppression analyses will be performed with the ligands and mutant PTH1Rs altered by mutagenesis. As a complementary approach, photo-crosslinking studies will be performed in which photoderivatized PTH or PTHrP ligands are used to map sites of physical interaction with the receptor. We will also use intra-molecular, second-site suppression analyses, as well as divalent metal ion-chelation strategies to investigate the topology and dynamics of the heptahelical transmembrane domain region of the PTH1R. Our new collaborations with Dr. Mierke at Brown University will afford structural NMR analyses of the new ligand analogs and computer modeling of the PTHPTH1R complex. The overall studies will provide important new insights into the molecular mechanisms by which the PTH/PTHrP/PTH1R system mediates its crucial biological functions.
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