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

Kasturi Pal, Hemant Badgandi, Saikat Mukhopadhyay

Research output: Contribution to journalArticle

6 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)303-322
Number of pages20
JournalMethods in Cell Biology
Volume127
DOIs
StatePublished - 2015

Keywords

  • G protein-coupled receptor
  • Immunoblotting
  • Immunoprecipitation
  • Phosphorylation
  • Primary cilia
  • Surface labeling

ASJC Scopus subject areas

  • Cell Biology
  • Medicine(all)

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