The long-term goal of this project is to elucidate the cellular mechanisms by which the 50-kDa ezrin-bind protein (EBP50) regulates parathyroid hormone receptor (PTHR)-mediated signaling and function in bone. Mice with targeted deletion of EBP50 exhibit a bone phenotype, as do patients with EBP50 mutations. Although it is thought that the bone disorder arises as a secondary consequence of renal dysfunction, our preliminary data identify direct effects of EBP50 on bone. This suggested a novel mechanism by which mutations interfere with EBP50 function and, by extension, that EBP50 is dynamically regulated by PTH in open and closed conformations. The unifying idea of the present proposal is that novel structural determinants in EBP50 and their posttranslational modification dictate EBP50 function on PTHR activity in bone.
Three specific aims are developed to test this idea.
In Aim 1 we will characterize EBP50 conformations and dimerization to test the hypothesis that the described mutations lock EBP50 in a closed configuration that interferes with PTHR function. These experiments will use molecular biological maneuvers to examine static interactions, molecular modeling to predict the effect of amino acid mutation on binding affinity, and biophysical measurements of fluorescence resonance energy transfer microscopy to acquire dynamic interactions in living cells and in real time, and surface plasmon resonance to quantify protein-protein interactions.
Aim 2 will define post- translational modifications of EBP50 that determine its function. This will be accomplished by testing the hypothesis that PTH-induced phosphorylation of EBP50 induces the closed configuration. We will apply mass spectrometry to identify site-specific EBP50 phosphorylation, and molecular biological tools with phospho-mimics and phospho-resistant EBP50 derivatives to determine their structural conformation and their actions on bone cells.
Aim 3 will delineate the direct effects of EBP50 on bone to test the hypothesis that EBP50 regulates bone development and turnover. Several approaches will be applied including allograft transplantation to determine if the bone phenotype of EBP50-null mice can be rescued by transplanting marrow stem cells from wild-type mice. Other experiments will involve transfecting bone cell models with mutant EBP50 or EBP50 harboring phospho-mimics or phospho- resistant forms of EBP50 to determine how these influence PTHR action. These studies will quantitatively examine the relations between EBP50 structure and function and characterize a novel mechanism to explain the regulation and origin of EBP50 effects on bone. The findings will generate new information that is relevant to understanding bone turnover. The outcomes will help define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.

Public Health Relevance

The proposed studies will test how the adapter protein EBP50 regulates parathyroid hormone action on bone. Patients with EBP50 mutations and mice lacking EBP50 have decreased bone mineral (osteomalacia). Our preliminary studies show that this results from direct effects of EBP50 in bone and not indirectly from loss of phosphate in the urine, as thought. The actions and mechanism by which EBP50 affects bone is not understood. The planned experiments will fill this gap. The outcome of our studies is highly relevant to understanding bone biology and the factors that cause osteomalacia, osteopenia, osteoporosis, and other related bone disease. The results will help define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK069998-09
Application #
8589589
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Malozowski, Saul N
Project Start
2005-01-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
9
Fiscal Year
2014
Total Cost
$296,561
Indirect Cost
$100,811
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Wang, Bin; Yang, Yanmei; Liu, Li et al. (2013) NHERF1 regulation of PTH-dependent bimodal Pi transport in osteoblasts. Bone 52:268-77
Alonso, Veronica; Friedman, Peter A (2013) Minireview: ubiquitination-regulated G protein-coupled receptor signaling and trafficking. Mol Endocrinol 27:558-72
Song, Gyun Jee; Barrick, Stacey; Leslie, Kristen L et al. (2012) The scaffolding protein EBP50 promotes vascular smooth muscle cell proliferation and neointima formation by regulating Skp2 and p21(cip1). Arterioscler Thromb Vasc Biol 32:33-41
Mamonova, Tatyana; Kurnikova, Maria; Friedman, Peter A (2012) Structural basis for NHERF1 PDZ domain binding. Biochemistry 51:3110-20
Ardura, Juan A; Wang, Bin; Watkins, Simon C et al. (2011) Dynamic Na+-H+ exchanger regulatory factor-1 association and dissociation regulate parathyroid hormone receptor trafficking at membrane microdomains. J Biol Chem 286:35020-9
Vilardaga, Jean-Pierre; Romero, Guillermo; Friedman, Peter A et al. (2011) Molecular basis of parathyroid hormone receptor signaling and trafficking: a family B GPCR paradigm. Cell Mol Life Sci 68:1-13
Romero, Guillermo; von Zastrow, Mark; Friedman, Peter A (2011) Role of PDZ proteins in regulating trafficking, signaling, and function of GPCRs: means, motif, and opportunity. Adv Pharmacol 62:279-314
Ardura, Juan A; Friedman, Peter A (2011) Regulation of G protein-coupled receptor function by Na+/H+ exchange regulatory factors. Pharmacol Rev 63:882-900
Liu, Li; Schlesinger, Paul H; Slack, Nicole M et al. (2011) High capacity Na+/H+ exchange activity in mineralizing osteoblasts. J Cell Physiol 226:1702-12

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