Direct Activity Measurement of Heterotrimeric Gi Proteins and Gq Protein By Effector Pulldown.

Abstract

Studying G protein-coupled receptor (GPCR) activation of heterotrimeric G proteins is crucial for understanding diverse physiological processes and developing novel therapeutics. Traditional methods to assay GPCR activation of G proteins, including assays of second messengers and biosensors, involve complex or indirect procedures. However, second messengers like cAMP and calcium are not direct readouts of GPCR activity due to signaling crosstalk, while biosensors can have undesired consequences due to structural alteration caused by fluorescent protein insertion. Here, we present a streamlined protocol employing GST-tagged bait proteins and epitope-embedded Gα subunits to achieve direct monitoring of Gα activity within cells. This method involves purification of GST-tagged bait constructs from bacteria and subsequent direct interaction studies with GluGlu-tagged Gα proteins expressed in any human cells of interest by including GST-tagged bait proteins in the cell lysis buffer. The approach enables sensitive detection of activated Gα within cells following extracellular stimulation. Advantages of this protocol include high sensitivity, enhanced monitoring of GPCR signaling dynamics under physiologically relevant conditions with minimum alteration in Gα, and the ability to distinguish between highly homologous isoforms within the same Gα family. Key features • Improved analysis of GPCR-Gα signaling: Establishes a novel effector pulldown method to monitor Gα activation within cells. • Endogenous GPCR activity measurement with increased sensitivity: Enables assays of endogenous physiological GPCR activities with improved sensitivity by increasing sample sizes. • Epitope Incorporation without affecting functions: Uses epitope tag sequence with a few amino acid substitutions functioning like wild-type proteins, adaptable for any endogenous Gα assay if suitable antibodies are available. • Isoform distinction: Distinguishes Gα isoforms by using embedded epitope.