• Characterization of Proton Sensitive G protein-Coupled Receptors

      Nam, Alisha; Okasha, Najeah; Spencer, Angela; Lambert, Nevin; Biological Sciences; Department of Pharmacology and Toxicology; Chemistry and Physics; Spencer, Angela; Augusta University (1/31/2020)
      G protein-coupled receptors (GPCRs) are membrane-bound receptors that can stimulate an intracellular signaling pathway following activation by a ligand. According to the International Union of Basic and Clinical Pharmacology (IUPHAR) database, GPR4, GPR65, and GPR132 are Class A orphan GPCRs with protons reported as their putative endogenous ligand; however, these receptors are currently understudied. After confirming whether these receptors are pH-sensitive, the purpose of our study was to investigate the interactions between GPR4, GPR65 and GPR132 and G protein subtypes (G?s, G?i, G?q, and G?12) upon stimulation with an acidic solution. Using bioluminescence resonance energy transfer (BRET), we studied the coupling between luciferase-tagged GPR receptors and fluorescent protein (Venus)-tagged G proteins in response to pH changes. Data indicated that all three receptors responded to pH changes. Upon extracellular response to pH changes, the receptors activate different G protein subtypes and thus, different signaling pathways: GPR4 activates G?i, G?q, and G?12; GPR65 activates all four subtypes; and GPR132 activates G?i�and weakly activates G?q, and G?12. Identifying these receptors as true proton sensors leads the way in understanding the role they play in maintaining acid-base homeostasis and will be critical for the development of novel drugs combatting acid-base related disorders.
    • Investigating Signaling Pathways Involving the HCA Receptor Family

      Saj, Dalia; Spencer, Angela; Okashah, Najeah; Lambert, Nevin; Biological Sciences; Chemistry and Physics; Department of Pharmacology and Toxicology; Spencer, Angela; Augusta University (1/31/2020)
      Increasing obesity rates have put the American population at higher risk for developing obesity-related medical conditions such as hypertension, heart disease, and diabetes. The hydroxycarboxylic acid (HCA) receptor family is a family of G protein-coupled receptors (GPCRs) that are expressed in adipose tissue and function as metabolic sensors, making them potential pharmaceutical targets in the treatment of obesity and other metabolic disorders. The HCA receptor family consists of the HCA1, HCA2, and HCA3�receptors, which are activated by hydroxycarboxylic acids such as lactate and 3-hydroxybutyric acid. We utilized bioluminescence resonance energy transfer (BRET) to study agonist-induced coupling of luciferase-tagged HCA receptors to Venus fluorescent protein-tagged G protein heterotrimers or arrestins. Our results indicate that the three HCA receptors couple to the Gi/o�subfamily of G proteins. The data additionally confirms a lack of coupling to the other G protein subfamilies (Gs,�Gq,�and G12), and lacks evidence of arrestin recruitment to HCA receptors. Overall, our study highlights the use of BRET as a powerful tool for analysis of GPCR signaling and demonstrates its possible use for future studies to determine the potency of potential drugs targeting HCA receptors as a therapy for health-related problems such as obesity.