Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols.

Abstract

Primary cilia are signaling organelles that have been shown to coordinate cellular responses to extracellular cues during physiological processes ranging from organ patterning to cell cycle regulation. A variety of receptors, including G protein-coupled receptors (GPCRs), downstream effectors (adenylyl cyclases), and second messengers, such as calcium, accumulate in the ciliary compartment. Isolation of GPCRs is essential for studying posttranslational modifications, intracellular trafficking, and protein-protein interactions that are important in downstream signaling. However, the presence of multiple hydrophobic transmembrane domains, and the inherent conformational flexibility of GPCRs make their extraction from membranes and solubilization particularly challenging. Here, we describe detailed methods for immunoblotting and immunoprecipitation of GPCRs from whole cell extracts. These methods are applicable for studying other multipass transmembrane proteins (such as adenylyl cyclases). We also describe methods for determining GPCR phosphorylation, surface labeling by biotinylation, and cross-linking to detect transient interactions with other proteins. These methods are amenable for studying both ciliary and nonciliary GPCRs in the context of cellular signaling pathways.