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.
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.
|Suofu, Yalikun; Carlisle, Diane L; Vilardaga, Jean-Pierre et al. (2018) Reply to Ahluwalia et al.: Contributions of melatonin receptors are tissue-dependent. Proc Natl Acad Sci U S A 115:E1944|
|Vergara, Sandra; Lukes, Dylan A; Martynowycz, Michael W et al. (2017) MicroED Structure of Au146(p-MBA)57 at Subatomic Resolution Reveals a Twinned FCC Cluster. J Phys Chem Lett 8:5523-5530|
|Jean-Alphonse, Frédéric G; Wehbi, Vanessa L; Chen, Jingming et al. (2017) ?2-adrenergic receptor control of endosomal PTH receptor signaling via G??. Nat Chem Biol 13:259-261|
|de la Cruz, M Jason; Hattne, Johan; Shi, Dan et al. (2017) Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED. Nat Methods 14:399-402|
|Bhattacharyya, Sohinee; Jean-Alphonse, Frédéric G; Raghavan, Venkatesan et al. (2017) Cdc42 activation couples fluid shear stress to apical endocytosis in proximal tubule cells. Physiol Rep 5:|
|Gbahou, Florence; Cecon, Erika; Viault, Guillaume et al. (2017) Design and validation of the first cell-impermeant melatonin receptor agonist. Br J Pharmacol 174:2409-2421|
|Suofu, Yalikun; Li, Wei; Jean-Alphonse, Frédéric G et al. (2017) Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release. Proc Natl Acad Sci U S A 114:E7997-E8006|
|Shi, Dan; Nannenga, Brent L; de la Cruz, M Jason et al. (2016) The collection of MicroED data for macromolecular crystallography. Nat Protoc 11:895-904|
|Gidon, Alexandre; Feinstein, Timothy N; Xiao, Kunhong et al. (2016) Studying the regulation of endosomal cAMP production in GPCR signaling. Methods Cell Biol 132:109-26|
|Barnes, Christopher O; Kovaleva, Elena G; Fu, Xiaofeng et al. (2016) Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography. Arch Biochem Biophys 602:61-68|
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