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

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

doi:10.3791/55561

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Method Article

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

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DOI:

10.3791/55561

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June 9th, 2017

Chirag Vasavda^*¹ , Nicholas W. Zaccor^*¹ , Paul C. Scherer¹ , Charlotte J. Sumner¹^,² , Solomon H. Snyder¹^,³^,⁴

¹The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, ²Department of Neurology and neurosurgery, Johns Hopkins University School of Medicine, ³Departments of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, ⁴Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine

^* These authors contributed equally

Summary

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [³⁵S]guanosine-5'-O-(3-thio)triphosphate ([³⁵S]GTPγS), in response to a ligand of interest.

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.

Introduction

G-Protein-Coupled Receptors (GPCRs) are a large family of cell-surface receptors responsible for a remarkable array of physiological processes, including analgesia, olfaction, and behavior¹. GPCRs act by sensing specific external signals and subsequently stimulating intracellular signaling. They therefore mark a key junction between the external and internal environments of a cell. Due to the critical role GPCRs play in biology, they have become major targets for both basic research and drug discovery²^,³.

Unlike other receptor families that bind dis....