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Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding.

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]GTP?S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP?S 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.

SUBMITTER: Vasavda C

PROVIDER: S-EPMC5608332 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding.

Vasavda Chirag C Zaccor Nicholas W NW Scherer Paul C PC Sumner Charlotte J CJ Snyder Solomon H SH

Journal of visualized experiments : JoVE 20170609 124

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 t ...[more]

PMID: 28654029

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Dataset Information

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding.

Publications

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding.

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