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J Vis Exp. 2017 Jun 9;(124). doi: 10.3791/55561.

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

Author information

1
The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine.
2
The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine; Department of Neurology and neurosurgery, Johns Hopkins University School of Medicine.
3
The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine; Departments of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine; Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine; ssnyder@jhmi.edu.

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.

PMID:
28654029
DOI:
10.3791/55561
[Indexed for MEDLINE]

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