Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα Protein-Specific Scintillation Proximity Assays.

Abstract

G protein-coupled receptors (GPCRs) are important regulators of cellular signaling functions and therefore are a major target for drug discovery. The CB1 cannabinoid receptor is among the most highly expressed GPCRs in neurons, where it regulates many differentiated neuronal functions. One model system for studying the biochemistry of neuronal responses is the use of neuroblastoma cells originating from the C1300 tumor in the A/J mouse, including cloned cell lines NS20, N2A, N18TG2, N4TG1, and N1E-115, and various immortalized hybrids of neurons with N18TG2 cells. GPCR signal transduction is mediated through interaction with multiple types and subtypes of G proteins that transduce the receptor stimulus to effectors. The [35S]GTPɣS assay provides a valuable pharmacological method to evaluate efficacy and potency in the first step in GPCR signaling. Here, we present detailed protocols for the [35S]GTPɣS-binding assay to measure the total G protein binding and the antibody-targeted scintillation proximity assay to measure specific Gα proteins in neuroblastoma cell membrane preparations. This chapter presents step-by-step methods from cell culture, membrane preparation, assay procedures, and data analysis.