TY - JOUR
T1 - Measuring g-protein-coupled receptor signaling via radio-labeled GTP binding
AU - Vasavda, Chirag
AU - Zaccor, Nicholas W.
AU - Scherer, Paul C.
AU - Sumner, Charlotte J.
AU - Snyder, Solomon H.
N1 - Funding Information:
This work was supported by National Institutes of HealthNIH grant DA-000266 and the Medical Scientist Training ProgramT32 grant (C.V., N.W.Z., and P.C.S.). The authors would also like to acknowledge somersault18:24 (somersault1824.com) for the Library of Science & Medical Illustrations.
Publisher Copyright:
© 2017 Journal of Visualized Experiments.
PY - 2017/6/9
Y1 - 2017/6/9
N2 - 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.
AB - 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.
KW - Agonist
KW - Antagonist
KW - Biochemistry
KW - Cellular fractionation
KW - GPCR
KW - GTPγS binding
KW - Issue 124
KW - Membrane preparation
KW - Pharmacology
KW - μ-opioid receptor
UR - http://www.scopus.com/inward/record.url?scp=85021199457&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85021199457&partnerID=8YFLogxK
U2 - 10.3791/55561
DO - 10.3791/55561
M3 - Article
C2 - 28654029
AN - SCOPUS:85021199457
SN - 1940-087X
VL - 2017
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 124
M1 - e55561
ER -