Measuring g-protein-coupled receptor signaling via radio-labeled GTP binding

Chirag Vasavda, Nicholas W. Zaccor, Paul C. Scherer, Charlotte Sumner, Solomon H Snyder

Research output: Contribution to journalArticle

Abstract

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPγS), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTPγS binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the μ-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Original languageEnglish (US)
Article numbere55561
JournalJournal of Visualized Experiments
Volume2017
Issue number124
DOIs
StatePublished - Jun 9 2017

Keywords

  • Agonist
  • Antagonist
  • Biochemistry
  • Cellular fractionation
  • GPCR
  • GTPγS binding
  • Issue 124
  • Membrane preparation
  • Pharmacology
  • μ-opioid receptor

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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