This research proposal is focused on elucidating the interactions of parathyroid hormone (PTH) with its receptor (Rc, the hPTH1-Rc). Formation of the complex between PTH and the hPTH1-Rc leads to a sequence of events, namely hormone binding, Rc activation, and signal transduction, which culminate in expression of hormonal bioactivity. The impetus for this research program comes from a desire to understand: (1) the fundamental nature of molecular recognition between a peptide hormone and its G protein-coupled Rc; (2) the mechanism of action of the hormone (PTH) responsible for minute-to-minute regulation of calcium levels in blood; and (3) the differences in Rc states (conformations) which translate into hormone agonism, antagonism, inverse agonism, etc. The introduction of PTH as a major new agent for treatment of osteoporosis also focuses attention on the mechanism of anabolic action of this hormone. By gaining insight into the nature of the hormone-Rc complex, the discovery of small molecule PTH-mimetics may be facilitated by structure-guided design in the future. During the previous grant award period, by integrating photoaffinity scanning, molecular biology, pharmacology, and structural biology (conformational studies of hormone and Rc, and molecular modeling), we succeeded in generating an advanced experimentally derived model of the PTH-hPTH1-Rc bimolecular complex that provides structural detail and reveals some of the dynamics of hormone-Rc interaction. We are now positioned to take the next major step in mapping the interface of PTH and its Rc, and to extend our studies to identify the shifts in Rc conformation associated with activation. Specifically, we plan to: (1) improve the resolution of the map of the hormone--Rc interface; (2) study the interaction of PTH ligands covalently tethered to the Rc; (3) investigate the ability of dual-reactive analogs to simultaneously make contact with two sites in Rc; (4) perform """"""""reverse"""""""" crosslinking from Rc to PTH; (5) prepare Rc and constitutively active Rc mutants on a large scale for structural studies of antagonist and inverse agonist interaction; (6) use disulfide bridge formation as a probe of Rc states; and (7) integrate all the above efforts in a molecular modeling initiative.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047940-13
Application #
7158606
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Program Officer
Malozowski, Saul N
Project Start
1995-01-01
Project End
2009-12-31
Budget Start
2007-01-01
Budget End
2009-12-31
Support Year
13
Fiscal Year
2007
Total Cost
$374,119
Indirect Cost
Name
Tufts University
Department
Physiology
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Thomas, Beena E; Sharma, Sandhya; Mierke, Dale F et al. (2009) PTH and PTH antagonist induce different conformational changes in the PTHR1 receptor. J Bone Miner Res 24:925-34
Monaghan, Paul; Thomas, Beena E; Woznica, Iwona et al. (2008) Mapping peptide hormone-receptor interactions using a disulfide-trapping approach. Biochemistry 47:5889-95
Wittelsberger, Angela; Mierke, Dale F; Rosenblatt, Michael (2008) Mapping ligand-receptor interfaces: approaching the resolution limit of benzophenone-based photoaffinity scanning. Chem Biol Drug Des 71:380-3
Thomas, Beena E; Woznica, Iwona; Mierke, Dale F et al. (2008) Conformational changes in the parathyroid hormone receptor associated with activation by agonist. Mol Endocrinol 22:1154-62
Monaghan, Paul; Woznica, Iwona; Moza, Beenu et al. (2007) Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor. Protein Expr Purif 54:87-93
Thomas, Beena E; Wittelsberger, Angela; Woznica, Iwona et al. (2007) Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. J Bone Miner Res 22:609-16
Gan, Lu; Alexander, Joseph M; Wittelsberger, Angela et al. (2006) Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells. Protein Expr Purif 47:296-302
Wittelsberger, Angela; Thomas, Beena E; Mierke, Dale F et al. (2006) Methionine acts as a ""magnet"" in photoaffinity crosslinking experiments. FEBS Lett 580:1872-6
Wittelsberger, Angela; Corich, Martina; Thomas, Beena E et al. (2006) The mid-region of parathyroid hormone (1-34) serves as a functional docking domain in receptor activation. Biochemistry 45:2027-34
Scian, Michele; Marin, Massimiliano; Bellanda, Massimo et al. (2006) Backbone dynamics of human parathyroid hormone (1-34): flexibility of the central region under different environmental conditions. Biopolymers 84:147-60

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