Intracellular Trafficking Assays for Dopamine D2-Like Receptors

Protocol
Part of the Neuromethods book series (NM, volume 96)

Abstract

G protein-coupled receptors (GPCRs) follow two endocytic pathways: homologous pathway, which is GPCR kinase (GRK) mediated and agonist induced, and heterologous pathway mediated by second messenger-dependent protein kinases. Dopamine D2 receptor undergoes GRK-mediated and, to a lesser extent, protein kinase C (PKC)-mediated endocytosis. Dopamine D3 receptor almost totally undergoes PKC-mediated endocytosis. D3 receptor also uniquely undergoes pharmacological sequestration, which involves conformational changes of receptor proteins that abrogates hydrophilic agonist binding. Pharmacological sequestration does not involve an actual movement of receptor to the cytosol. Internalization of GPCRs can be determined using ligand binding, flow cytometry, ELISA, fluorescence, or cell surface biotinylation assays. Here, we review convenient and commonly used methodologies involved in the internalization of dopamine receptors focusing on D2 and D3 receptor.

Key words

Receptor internalization Ligand binding GRK PKC Clathrin Caveolae Fluorescence-activated cell sorting ELISA 

Notes

Acknowledgment

This study was financially supported by the Ministry of Knowledge Economy (MKE) and Korea Institute for Advancement of Technology (KIAT) through the Inter-ER Cooperation Project (R0002019).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Pharmacology, College of PharmacyChonnam National UniversityGwang-JuKorea

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