R01DK132902

Mechanistic Basis of Calcium Sensing Receptor Signaling - Abstract:

Mechanistic Basis of Calcium Sensing Receptor Signaling

The calcium sensing receptor (CASR) is the master regulator of calcium metabolism in humans and represents an outstanding drug target for the treatment of parathyroid disorders that develop in patients with chronic kidney diseases (CKDs). For patients with renal dysfunction that develop hyperparathyroidism, calcimimetic drugs that act as positive allosteric modulators (PAMs) of the CASR are the favored therapeutics. PAMs, such as cinacalcet, evocalcet, and etelcalcetide, are approved treatments for CKD; however, their clinical use is limited due to their adverse side effects.

By elucidating the dynamic structural mechanisms of receptor activation, its modulation by small-molecule modulators, and the specificity of G protein activation, we seek to understand in detail the CASR signaling mechanism and enable the rational design of improved therapeutics modulating receptor function.

CASR is a Family C member of G protein-coupled receptors (GPCRs), which also include the metabotropic glutamate receptors (mGluRs) and the metabotropic gamma-aminobutyric acid receptor (GABAB). Like other members of this family, CASR functions as an obligate homodimer with an N-terminal extracellular domain (ECD) responsible for ligand binding, linked to the seven-transmembrane (7TM) domain.

We have recently determined cryo-electron microscopy (cryoEM) structures of the near-full-length human CASR homodimer in active and inactive states, revealing how ECD rearrangement upon Ca2+ binding induces the activation of the 7TMs and how allosteric modulators engage the receptor. Our results illustrate an essential asymmetry in the active state where each CASR protomer is stabilized by a PAM molecule bound to each 7TM in two distinct conformations, leading to the activation of only one transmembrane region, priming it for G protein coupling.

Here, we propose to extend these studies in order to characterize the mechanism and specificity of G protein activation by CASR, its dynamics, as well as the detailed action of allosteric modulators with distinct pharmacological interest. Specifically, we seek to apply:

- Structure-based mutagenesis coupled with cell signaling assays that monitor the effects of allosteric modulators
- CryoEM structural studies of CASR alone and in complex with allosteric modulators and distinct G proteins in a near-native lipid environment
- Single-molecule fluorescence resonance energy transfer (SMFRET) complemented by double electron-electron resonance (DEER) spectroscopy to reveal the dynamics of receptor and G protein activation as well as its modulation by different allosteric ligands.

Collectively, the proposed structural, cellular, biochemical, and biophysical experiments aim to provide a full mechanistic framework for transmembrane signaling by CASR and will guide the future development of novel drugs targeting this receptor.

St. Jude Children's Research Hospital

(1) TO PROMOTE EXTRAMURAL BASIC AND CLINICAL BIOMEDICAL RESEARCH THAT IMPROVES THE UNDERSTANDING OF THE MECHANISMS UNDERLYING DISEASE AND LEADS TO IMPROVED PREVENTIONS, DIAGNOSIS, AND TREATMENT OF DIABETES, DIGESTIVE, AND KIDNEY DISEASES. PROGRAMMATIC AREAS WITHIN THE NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES INCLUDE DIABETES, DIGESTIVE, ENDOCRINE, HEMATOLOGIC, LIVER, METABOLIC, NEPHROLOGIC, NUTRITION, OBESITY, AND UROLOGIC DISEASES. SPECIFIC PROGRAMS AREAS OF INTEREST INCLUDE THE FOLLOWING: (A) FOR DIABETES, ENDOCRINE, AND METABOLIC DISEASES AREAS: FUNDAMENTAL AND CLINICAL STUDIES INCLUDING THE ETIOLOGY, PATHOGENESIS, PREVENTION, DIAGNOSIS, TREATMENT AND CURE OF DIABETES MELLITUS AND ITS COMPLICATIONS; NORMAL AND ABNORMAL FUNCTION OF THE PITUITARY, THYROID, PARATHYROID, ADRENAL, AND OTHER HORMONE SECRETING GLANDS; HORMONAL REGULATION OF BONE, ADIPOSE TISSUE, AND LIVER; ON FUNDAMENTAL ASPECTS OF SIGNAL TRANSDUCTION, INCLUDING THE ACTION OF HORMONES, COREGULATORS, AND CHROMATIN REMODELING PROTEINS; HORMONE BIOSYNTHESIS, SECRETION, METABOLISM, AND BINDING; AND ON HORMONAL REGULATION OF GENE EXPRESSION AND THE ROLE(S) OF SELECTIVE RECEPTOR MODULATORS AS PARTIAL AGONISTS OR ANTAGONISTS OF HORMONE ACTION; AND FUNDAMENTAL STUDIES RELEVANT TO METABOLIC DISORDERS INCLUDING MEMBRANE STRUCTURE, FUNCTION, AND TRANSPORT PHENOMENA AND ENZYME BIOSYNTHESIS; AND BASIC AND CLINICAL STUDIES ON THE ETIOLOGY, PATHOGENESIS, PREVENTION, AND TREATMENT OF INHERITED METABOLIC DISORDERS (SUCH AS CYSTIC FIBROSIS). (B) FOR DIGESTIVE DISEASE AND NUTRITION AREAS: GENETICS AND GENOMICS OF THE GI TRACT AND ITS DISEASES; GENETICS AND GENOMICS OF LIVER/PANCREAS AND DISEASES; GENETICS AND GENOMICS OF NUTRITION; GENETICS AND GENOMICS OF OBESITY; BARIATRIC SURGERY; CLINICAL NUTRITION RESEARCH; CLINICAL OBESITY RESEARCH; COMPLICATIONS OF CHRONIC LIVER DISEASE; FATTY LIVER DISEASE; GENETIC LIVER DISEASE; HIV AND LIVER; CELL INJURY, REPAIR, FIBROSIS AND INFLAMMATION IN THE LIVER; LIVER CANCER; LIVER TRANSPLANTATION; PEDIATRIC LIVER DISEASE; VIRAL HEPATITIS AND INFECTIOUS DISEASES; GASTROINTESTINAL AND NUTRITION EFFECTS OF AIDS; GASTROINTESTINAL MUCOSAL AND IMMUNOLOGY; GASTROINTESTINAL MOTILITY; BASIC NEUROGASTROENTEROLOGY; GASTROINTESTINAL DEVELOPMENT; GASTROINTESTINAL EPITHELIAL BIOLOGY; GASTROINTESTINAL INFLAMMATION; DIGESTIVE DISEASES EPIDEMIOLOGY AND DATA SYSTEMS; NUTRITIONAL EPIDEMIOLOGY AND DATA SYSTEMS; AUTOIMMUNE LIVER DISEASE; BILE, BILIRUBIN AND CHOLESTASIS; BIOENGINEERING AND BIOTECHNOLOGY RELATED TO DIGESTIVE DISEASES, LIVER, NUTRITION AND OBESITY; CELL AND MOLECULAR BIOLOGY OF THE LIVER; DEVELOPMENTAL BIOLOGY AND REGENERATION; DRUG-INDUCED LIVER DISEASE; GALLBLADDER DISEASE AND BILIARY DISEASES; EXOCRINE PANCREAS BIOLOGY AND DISEASES; GASTROINTESTINAL NEUROENDOCRINOLOGY; GASTROINTESTINAL TRANSPORT AND ABSORPTION; NUTRIENT METABOLISM; PEDIATRIC CLINICAL OBESITY; CLINICAL TRIALS IN DIGESTIVE DISEASES; LIVER CLINICAL TRIALS; OBESITY PREVENTION AND TREATMENT; AND OBESITY AND EATING DISORDERS. (C) FOR KIDNEY, UROLOGIC AND HEMATOLOGIC DISEASES AREAS: STUDIES OF THE DEVELOPMENT, PHYSIOLOGY, AND CELL BIOLOGY OF THE KIDNEY; PATHOPHYSIOLOGY OF THE KIDNEY; GENETICS OF KIDNEY DISORDERS; IMMUNE MECHANISMS OF KIDNEY DISEASE; KIDNEY DISEASE AS A COMPLICATION OF DIABETES; EFFECTS OF DRUGS, NEPHROTOXINS AND ENVIRONMENTAL TOXINS ON THE KIDNEY; MECHANISMS OF KIDNEY INJURY REPAIR; IMPROVED DIAGNOSIS, PREVENTION AND TREATMENT OF CHRONIC KIDNEY DISEASE AND END-STAGE RENAL DISEASE; IMPROVED APPROACHES TO MAINTENANCE DIALYSIS THERAPIES; BASIC STUDIES OF LOWER URINARY TRACT CELL BIOLOGY, DEVELOPMENT, PHYSIOLOGY, AND PATHOPHYSIOLOGY; CLINICAL STUDIES OF BLADDER DYSFUNCTION, INCONTINENCE, PYELONEPHRITIS, INTERSTITIAL CYSTITIS, BENIGN PROSTATIC HYPERPLASIA, UROLITHIASIS, AND VESICOURETERAL REFLUX; DEVELOPMENT OF NOVEL DIAGNOSTIC TOOLS AND IMPROVED THERAPIES, INCLUDING TISSUE ENGINEERING STRATEGIES, FOR UROLOGIC DISORDERS;RESEARCH ON HEMATOPOIETIC CELL DIFFERENTIATION; METABOLISM OF IRON OVERLOAD AND DEFICIENCY; STRUCTURE, BIOSYNTHESIS AND GENETIC REGULATION OF HEMOGLOBIN; AS WELL AS RESEARCH ON THE ETIOLOGY, PATHOGENESIS, AND THERAPEUTIC MODALITIES FOR THE ANEMIA OF INFLAMMATION AND CHRONIC DISEASES.(2) TO ENCOURAGE BASIC AND CLINICAL RESEARCH TRAINING AND CAREER DEVELOPMENT OF SCIENTISTS DURING THE EARLY STAGES OF THEIR CAREERS. THE RUTH L. KIRSCHSTEIN NATIONAL RESEARCH SERVICE AWARD (NRSA) FUNDS BASIC AND CLINICAL RESEARCH TRAINING, SUPPORT FOR CAREER DEVELOPMENT, AND THE TRANSITION FROM POSTDOCTORAL BIOMEDICAL RESEARCH TRAINING TO INDEPENDENT RESEARCH RELATED TO DIABETES, DIGESTIVE, ENDOCRINE, HEMATOLOGIC, LIVER, METABOLIC, NEPHROLOGIC, NUTRITION, OBESITY, AND UROLOGIC DISEASES. (3) TO EXPAND AND IMPROVE THE SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM. THE SBIR PROGRAM AIMS TO INCREASE AND FACILITATE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT; TO ENHANCE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT; AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. (4) TO UTILIZE THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM. THE STTR PROGRAM INTENDS TO STIMULATE AND FOSTER SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS; TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS; TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT; AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION.

93.847 - Diabetes, Digestive, and Kidney Diseases Extramural Research

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [HHS - NIH]

Memphis, Tennessee 38105 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 422% from $602,652 to $3,143,834.