The goal of this proposed research is to determine the structural and cellular basis underlying the mechanism of parathyroid hormone (PTH) receptor (PTHR) signaling. The PTHR is a major G protein-coupled receptor (GPCR) that regulates Ca2+ homeostasis and bone turnover and is the most effective therapeutic target for osteoporosis. The recently recognized capacity of PTH and certain PTH analogs to prolong G-protein activity and cAMP production after PTHR internalization into early endosomes has drastically changed how we think about cellular signaling of the PTHR, and how we study drugs that target this receptor. This new and unexpected behavior of a GPCR is of particular relevance for understanding how the recently developed long-acting PTH analogs, including LA-PTH that is in preclinical development for the treatment of hypoparathyroidism, induce remarkable prolonged signaling (cAMP and calcium) responses in cells and in mice. The structural determinants of the PTHR and cellular mechanisms responsible for these actions are not known and this is an obstacle to further progress for identifying clinically relevant analogs. We therefore propose a research program to overcome this obstacle.
Two specific aims are proposed:
Aim 1 determines the structural determinants of PTHR action through crystallography of full-length PTHR, and PTHR bound to PTH, or to LA-PTH. We will focus initially on the structural basis for the different functional properties of PTH ligands and on ionic interactions likely to be sensitive to pH changes encountered in endosomes because our preliminary findings support a critical role of endosomal pH on sustained PTHR signaling. This structural and molecular information will be further applied to Aim 2, which determines the cellular mechanism regulating endosomal PTHR signaling, focusing on the role of low pH conditions found in the endosome in determining the stability of PTH-PTHR-G protein complexes. Knowledge of structural details and differences of how PTH and LA-PTH bind to the PTHR and trigger signaling in specific subcellular locations (endosomes) provides insights into molecular and cellular processes, which can provide new opportunities for therapy. Selective targeting of PTHR-mediated endosomal Gs signaling might offer more effective and selective treatments than global targeting of cell-surface signaling.

Public Health Relevance

Bone and mineral diseases such as osteoporosis and hypocalcemia are a major causes of death and disability in the United States and worldwide. Our studies show that some long-acting analogs of the parathyroid hormone (PTH) are promising therapies for hypoparathyroidism, a leading cause of hypocalcemia. It is critical to determine the structural and cellular mechanisms that determine the protective action of PTH-analogs. These mechanisms have not been previously investigated and may be exploited for the development of novel therapeutic approaches.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01DK102495-01
Application #
8747187
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Malozowski, Saul N
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Gidon, Alexandre; Al-Bataineh, Mohammad M; Jean-Alphonse, Frederic G et al. (2014) Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase. Nat Chem Biol 10:707-9
Vilardaga, Jean-Pierre; Jean-Alphonse, Frederic G; Gardella, Thomas J (2014) Endosomal generation of cAMP in GPCR signaling. Nat Chem Biol 10:700-6
Calero, Guillermo; Cohen, Aina E; Luft, Joseph R et al. (2014) Identifying, studying and making good use of macromolecular crystals. Acta Crystallogr F Struct Biol Commun 70:993-1008