Jean-Pierre Vilardaga - US grants

DESCRIPTION (provided by applicant): The PTH/PTHrP receptor (PTHR), a G protein-coupled receptor (GPCR) of the B family, transmits both parathyroid hormone (PTH) and PTH-related Peptide (PTHrP) signals to initiate and regulate vital biochemical processes in bone and renal physiology. It is unknown how this single receptor discriminates between the two ligand signaling systems: PTH; endocrine and homeostatic, and PTHrP; a paracrine mediator of developmental and diverse organ biology. It is unclear also why in a clinical setting PTH(1-34) stimulates more prolonged increases in serum levels of 1,25-dihydroxy-vitamin-D, calcium, and bone resorption markers than does PTHrP(1-36), when the ligands are administered by continuous infusion so as to mimic conditions of primary hyperparathyroidism and humoral hypercalcemia of malignancy. We now advance a comprehensive model to account for these activities. Our recent studies show that PTH(1-34) differentiates itself from PTHrP(1-36) by inducing prolonged cAMP responses in cultured cells, and in vivo, which are mediated at the receptor level, and not by extended bioavailability of ligands. We discovered that during the time frame of cAMP production, PTHrP(1-36) action, is restricted to the cell surface, whereas PTH(1-34) trafficked to internalized sub-cellular compartments where it forms a stable complex with the PTHR, and continues to stimulate cAMP production. Such marked differences provide a mechanistic basis whereby PTH and PTHrP induce distinctly different responses and suggests that PTHR signaling to cAMP can continue from intracellular domains. Based on these novel findings, we propose the central hypothesis that cAMP production by the PTHR occurs both at the plasma membrane and from intracellular domains, with distinct lifetimes that have different consequences for cell signaling. This concept, supported by our recent findings and preliminary data described here, challenge the classical paradigm that cAMP production triggered by GPCRs originates exclusively at the cell membrane. The proposed experiments seek to determine a) the mechanisms of sustained cAMP responses triggered by the PTHR; and b) the consequences of sustained cAMP levels for cell signaling. These will be experimentally tested in the native environment of living cells by using optical technologies, as well as pharmacological, biochemical and proteomic approaches. These experiments will contribute to a fundamental understanding of the molecular and trafficking mechanisms of activation and signaling of the PTH/PTHrP/PTHR system in the native environment of living cells, which are needed to guide the development of safer and more specific and effective drugs for bone and mineral diseases.

DESCRIPTION (provided by applicant): The PTH/PTHrP receptor (PTHR), a G protein-coupled receptor (GPCR) of the B family, transmits both parathyroid hormone (PTH) and PTH-related Peptide (PTHrP) signals to initiate and regulate vital biochemical processes in bone and renal physiology. It is unknown how this single receptor discriminates between the two ligand signaling systems: PTH; endocrine and homeostatic, and PTHrP; a paracrine mediator of developmental and diverse organ biology. It is unclear also why in a clinical setting PTH(1-34) stimulates more prolonged increases in serum levels of 1,25-dihydroxy-vitamin-D, calcium, and bone resorption markers than does PTHrP(1-36), when the ligands are administered by continuous infusion so as to mimic conditions of primary hyperparathyroidism and humoral hypercalcemia of malignancy. We now advance a comprehensive model to account for these activities. Our recent studies show that PTH(1-34) differentiates itself from PTHrP(1-36) by inducing prolonged cAMP responses in cultured cells, and in vivo, which are mediated at the receptor level, and not by extended bioavailability of ligands. We discovered that during the time frame of cAMP production, PTHrP(1-36) action, is restricted to the cell surface, whereas PTH(1-34) trafficked to internalized sub-cellular compartments where it forms a stable complex with the PTHR, and continues to stimulate cAMP production. Such marked differences provide a mechanistic basis whereby PTH and PTHrP induce distinctly different responses and suggests that PTHR signaling to cAMP can continue from intracellular domains. Based on these novel findings, we propose the central hypothesis that cAMP production by the PTHR occurs both at the plasma membrane and from intracellular domains, with distinct lifetimes that have different consequences for cell signaling. This concept, supported by our recent findings and preliminary data described here, challenge the classical paradigm that cAMP production triggered by GPCRs originates exclusively at the cell membrane. The proposed experiments seek to determine a) the mechanisms of sustained cAMP responses triggered by the PTHR; and b) the consequences of sustained cAMP levels for cell signaling. These will be experimentally tested in the native environment of living cells by using optical technologies, as well as pharmacological, biochemical and proteomic approaches. These experiments will contribute to a fundamental understanding of the molecular and trafficking mechanisms of activation and signaling of the PTH/PTHrP/PTHR system in the native environment of living cells, which are needed to guide the development of safer and more specific and effective drugs for bone and mineral diseases. PUBLIC HEALTH RELEVANCE: The goal of this research plan is to understand how the medically important parathyroid hormone receptor transmits both parathyroid hormone (PTH) and PTH-related peptide (PTHrP) signals to regulate different physiological processes: PTH, endocrine and homeostatic regulates concentrations of calcium, phosphate ions, and vitamin D in blood and extracellular fluids, and PTHrP, a paracrine mediator of developing tissues such as bone. Pharmacological, biochemical and biophysical methods will be employed to advance a comprehensive model at the molecular and cellular levels to account for the functional differences between PTH and PTHrP, which will guide new therapies for osteoporosis.

DESCRIPTION (provided by applicant): 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.

Project Summary The goal of this project is to determine the mechanisms underlying the molecular and cellular endocrinology of parathyroid hormone receptor (PTHR) signaling as it relates to phosphate and vitamin D balance. PTHR is uniquely expressed on both apical and basolateral membranes of the polarized cells that form renal proximal tubules. The biological consequences of bilateral PTHR expression or its origin are unknown. The premise of the proposal is that characterizing asymmetric PTHR actions will fill a major gap in our understanding of PTHR signaling and function and reconcile conflicting views of PTH action. Pilot studies show that PTHR signals from both basolateral and apical membranes but only basolateral PTHR activation induces transcription of the 1?-vitamin D hydroxylase (CYP27B1). Apical PTHR signaling primarily inhibits phosphate transport. The molecular and cellular mechanisms regulating PTHR actions are incompletely defined. The PTHR interacts at apical membranes with the PDZ scaffolding protein NHERF1, which tethers the receptor and regulates G-protein signaling and function. Absence of NHERF1 or its downregulation causes relocation of PTHR to basolateral membranes with an increased 1,25[OH]2D in mice and humans. Preliminary data show that Scribble, a basolateral PDZ protein, exerts a reciprocal effect, where downregulation causes accumulation of PTHR at apical membranes. We hypothesize that the polarized PTHR arrangement arises from the segregated location of NHERF1 and Scribble. We advance a research program to uncover new aspects of PTHR signaling in polarized kidney cells and test the central hypothesis that polarized PTHR expression is driven by the asymmetric location of the PDZ adaptor proteins, Scribble and NHERF, which in turn underlies the signaling bias of apical and basolateral PTHR actions on vitamin D and phosphate homeostasis. We propose three closely linked aims to evaluate this idea. The first two aims of address the hypothesis that basolateral PTHR activation preferentially stimulates the 1?-vitamin D hydroxylase, whereas apical PTHR signaling primarily blocks Pi transport. Aim 1 will determine the role of Scribble and NHERF in the generation of PTHR polarity and its asymmetric signaling in human kidney proximal tubule epithelial cells. Aim 2 uses live-cell FRET microscopy and other state-of-the art fluorescence techniques to characterize basolateral and apical membrane signaling properties of PTHR and the effect of NHERF1 and Scribble on biased G protein signaling. Aim 3 will delineate the in vivo actions of polarized proximal tubule PTHR by testing the role of Scribble on PTHR-dependent vitamin D and phosphate metabolism using a novel, conditional proximal tubule Scribble knockout mouse model that we generated. The outcomes will frame innovative therapeutic approaches targeting disorders of mineral metabolism.

Project Summary The goal of this project is to determine structural mechanisms by which for the parathyroid hormone (PTH) receptor (PTHR) signals in response to its functionally distinct ligands: PTH, PTH-related peptide (PTHrP) and the long-acting PTH analog (LA-PTH). The PTHR is a major G protein-coupled receptor (GPCR) that regulates Ca2+ homeostasis in blood and bone turnover, and is the most effective therapeutic target for osteoporosis. It also is one of the first GPCR found to sustain cAMP production after internalization of the PTH?receptor complex in endosomes. The recently recognized feature that the calcemic action of PTHR in mice and primates is sustained by LA-PTH, which also prolongs endosomal cAMP production, is changing our thinking about how PTHR mediates its physiological actions. Implicit in these findings is that efficient treatment of hypocalcemia might be more approachable with selective targeting of PTHR-mediated endosomal cAMP signaling. The mechanism that differentiating the signaling selectivity of PTH and its analogs are not known and this is an obstacle to further move toward new directions to develop PTH-based therapies that have improved efficacies for treating bone and mineral diseases. We therefore propose a research program to overcome this obstacle. The goal of this project is thus to determine the structural basis by which PTHR function and activate G proteins in response to PTH, PTHrP and LA-PTH. Two specific aims are proposed to discover structural basis of PTHR signaling. The first aim addresses the hypothesis that PTH and LA-PTH promote long endosomal cAMP production by stabilizing unique structural arrangements and phosphorylation pattern in the PTHR that are different from those stabilized by PTHrP, which induces a short cAMP response from the plasma membrane. To this end, we will use quantitative mass spectrometry (MS) based-proteomics technologies, such as Hydrogen-Deuterium exchange coupled to MS (HDXMS) to determine structural changes and structural determinants for PTHR activation upon binding to the distinct ligands, and identify the binding interface in PTHR?G protein complexes. The second aim will complement our understanding of the functional selectivity of PTHR by X-ray crystallography of PTHR and PTHR bound to PTH, PTHrP and LA-PTH. We have obtained X-ray diffractable crystals (4 Å) of the full length PTHR bound to LA-PTH and in complex with the RNA polymerase II. Here we are using the cavity formed in the RNA polymerase II crystal as a ?crystal sponge? that creates a favorable crystallization environment for PTHR. We will initially optimize the crystallization conditions to resolve crystal structure of the PTHR bound to LA-PTH in complex with RNA polymerase II at higher resolution (2-3 Å). We will then focus of resolving PTH and PTHrP-bound states of PTHR. These studies will provide new insights into how PTHR activate G proteins and the structural mechanism differentiating the action of PTH, PTHrP and LA-PTH.

Parathyroid glands (PTGs) control mineral, hormonal, and skeletal homeostasis by adjusting parathyroid hormone (PTH) secretion in response to changes in serum [Ca2+]. It is well documented that activation of homomeric extracellular calcium-sensing receptor (CaSR) by raising serum [Ca2+] suppresses PTH secretion. However, the mechanisms promoting PTH secretion at hypocalcemic and various hyperparathyroidism (HPT) states due to CaSR-deficiency have not been explored. Our pilot data raise a novel hypothesis of a novel autocrine mechanism by which GABA and GABAB1R regulate G protein signaling of the CaSR to promote PTH secretion. Multi-disciplinary approaches to be performed by two highly complementary teams at University of Pittsburgh and University of California San Francisco will be employed to test this hypothesis through 3 specific aims. Aim 1 will first demonstrate the physiopathological relevance of the functional interaction between the CaSR and GABAB1R in PTGs by studying parathyroid cell (PTC)-specific GABAB1R and/or CaSR knockout mice in the contexts of hypocalcemia and different forms of HPT challenges (i.e., CaSR-deficiency or chronical kidney disease) in vivo and human PTGs excised from patients with primary and secondary HPT. Aim 2 will define the biological actions of Gad1/2 in regulating PTG functions by studying the effects of PTC-specific Gad1 and Gad2 double knockout in conditions of Ca2+ deficiency and various HPT states in mice and assessing Gad1/2 and GABA expression in human PTGs excised from patients with primary and secondary HPT. Aim 3 will delineate molecular mechanisms by which the CaSR/GABAB1R heteromers alter efficacy of G-protein activation of the CaSR and its consequence for PTH secretion and GABA production in cultured parathyroid-derived PTH-C1 cells. Optical (FRET, TIRF, BiFC) and biochemical techniques will be used to test the theory that PTH release and GABA synthesis are controlled through mechanisms involving the allosteric action of GABAB1R on CaSR signaling via receptor heteromerization that inhibits Ca2+-mediated Gq/11 and Gi signal transduction and promote PTH secretion. Successful completion of this project will help to develop new regimens to manage PTH hypo- or hyper-secretion and related endocrine and skeletal diseases and prevent unwanted side-effects of GABAB1R agonists and antagonists prescribed to patients with neurological disorders.