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 language | English (US) |
|---|---|
| Pages (from-to) | 303-322 |
| Number of pages | 20 |
| Journal | Methods in Cell Biology |
| Volume | 127 |
| DOIs | |
| State | Published - 2015 |
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Keywords
- G protein-coupled receptor
- Immunoblotting
- Immunoprecipitation
- Phosphorylation
- Primary cilia
- Surface labeling
ASJC Scopus subject areas
- Cell Biology
- Medicine(all)
Cite this
Studying G protein-coupled receptors : Immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols. / Pal, Kasturi; Badgandi, Hemant; Mukhopadhyay, Saikat.
In: Methods in Cell Biology, Vol. 127, 2015, p. 303-322.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Studying G protein-coupled receptors
T2 - Immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols
AU - Pal, Kasturi
AU - Badgandi, Hemant
AU - Mukhopadhyay, Saikat
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - G protein-coupled receptor
KW - Immunoblotting
KW - Immunoprecipitation
KW - Phosphorylation
KW - Primary cilia
KW - Surface labeling
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UR - http://www.scopus.com/inward/citedby.url?scp=84955414459&partnerID=8YFLogxK
U2 - 10.1016/bs.mcb.2014.12.003
DO - 10.1016/bs.mcb.2014.12.003
M3 - Article
C2 - 25837398
AN - SCOPUS:84955414459
VL - 127
SP - 303
EP - 322
JO - Methods in Cell Biology
JF - Methods in Cell Biology
SN - 0091-679X
ER -