title: 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.
authors.affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Kasturi
authors.initials: K
authors.lastname: Pal
authors.affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Hemant
authors.initials: H
authors.lastname: Badgandi
authors.affiliation: Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.
authors.forename: Saikat
authors.initials: S
authors.lastname: Mukhopadhyay
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002923:Cilia; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000032:analysis*; D012243:Rhodopsin / Q000032:analysis*; D015398:Signal Transduction / Q000502:physiology; D000072086:Smoothened Receptor; D013194:Staining and Labeling / Q000379:methods
chemical_list: C533095:GPR161 protein, mouse; D043562:Receptors, G-Protein-Coupled; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D012243:Rhodopsin
keywords: G protein-coupled receptor; Immunoblotting; Immunoprecipitation; Phosphorylation; Primary cilia; Surface labeling