• Profiling the HCA Receptor Family through BRET Analysis of GPCR-G-Protein and GPCR-Arrestin Interactions

      Saj, Dalia; Department of Biological Sciences; Department of Pharmacology & Toxicology (Augusta University, 2020-05)
      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 Gαi/o subfamily of G proteins. The data additionally confirms a lack of coupling to the other G protein subfamilies (Gαs, Gαq, and Gα12/13), 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.
    • The Study of 5ht-1d and 5ht-1f Receptor Interactions with Mini G Proteins via Bret Analysis

      Trang, Amy; Department of Chemistry & Physics; Department of Pharmacology and Toxicology; Spencer, Angela; Lambert, Nevin; Augusta University (2019-02-13)
      G protein-coupled receptors (GPCRs) are receptors involved in signal transduction, a process for converting extracellular signals into internal messages to elicit a cellular response. Signal transduction pathways involve activating various G protein subtypes (Gs, Gi/o, Gq/11 and G12/13) which typically lead to second messenger production. Traditionally, second messenger concentration assays are used to identify GPCR coupling with G protein(s), but they are not efficient in profiling GPCRs since they compare the concentrations from different downstream signals. Instead, novel tools, such as Bioluminescence Resonance Energy Transfer (BRET) and mini G (mG) proteins, can be used to profile GPCRs. BRET is a technique that provides quantitative data when protein-protein interaction occurs and requires the proteins of interest to be fused with either a bioluminescent protein or fluorescent protein. In this study, we used mG proteins representing each G protein subtype to identify 5-hydroxytryptamine (5-HT; serotonin) receptor coupling upon serotonin stimulation. Through BRET assays, we determined that both the 5-HT1D and 5-HT1F receptors couple primarily with the mGsiand mGo classes of mG proteins. This supports previous studies that these receptors couple to Gi/o proteins and suggests that the use of mG proteins in BRET assays is an effective tool for GPCR profiling.
    • Unconventional Coupling of 5HT7 receptors to Gs heterotrimers

      Adams, Elizabeth; Department of Pharmacology and Toxicology; Lambert, Nevin; Augusta University (2019-02-13)
      GPCRs play a major role in cell signaling through their interactions with heterotrimeric G proteins. In conventional models of GPCR-G protein coupling, agonist binding promotes a conformational change within the receptor, which then associates with G proteins, facilitating the exchange of GDP for GTP. GTP-bound G proteins dissociate from the receptor and exert their effects on downstream signaling molecules. Previous studies suggest that serotonin 5HT7 receptors associate with Gs�heterotrimers prior to agonist binding, and that 5HT7-Gs�complexes dissociate after the G protein is activated. Here we study this unconventional mode of coupling using bioluminescence resonance energy transfer (BRET) between luciferase-tagged 5HT7 receptors and Gs�heterotrimers labeled with Venus. Our results confirm that 5HT7 receptors interact with inactive (GDP-bound) Gs�heterotrimers in the absence of an agonist, and that this interaction is stabilized by the inverse agonist methiothepin. Stimulation with the endogenous agonist serotonin (5HT) decreased BRET between 5HT7 receptors and Gs, indicating that the activation of the receptor leads to 5HT7-Gscomplex dissociation. Interestingly, Gs�activation was not required for complex dissociation. These results are consistent with the hypothesis that 5HT7 receptors couple to Gs�heterotrimers via an unconventional mechanism involving ligand-sensitive complexes of receptors and inactive Gs.