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Study of Dopamine D1 Receptor Regulation by G Protein-Coupled Receptor Kinases Using Whole-Cell Phosphorylation and Cross-Linking Methods

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Dopamine Receptor Technologies

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

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Abstract

Cells utilize receptor desensitization to prevent sustained receptor signaling and potential cellular injuries through diminishing the receptor’s responsiveness toward agonists. One of the key facilitators for desensitization among G protein-coupled receptors (GPCR) is the family of G protein-coupled receptor kinases (GRK). In the agonist-activated state, the receptor is phosphorylated by GRKs to allow binding of cytosolic arrestins, which leads to uncoupling from the G protein. Yet there are many nuances when studying GRK-mediated phosphorylation, which include basal phosphorylation by GRKs, the various GRK isoforms, and structural targets of GRKs on the receptor. To address questions stemming from such examples as well as to pursue other avenues concerning GRK-mediated phosphorylation, we focus on the dopamine D1 receptor subtype (D1R) and detail two assays: whole-cell phosphorylation and coimmunoprecipitation using the cross-linker dithiobis(succinimidyl propionate) (DSP) to demonstrate potential interactions between D1R and GRK isoforms. In addition, we provide an overview of past studies concerning the desensitization properties of D1R and a brief protocol for indirect immunofluorescence confocal microscopy to visualize the co-localization between D1R and GRK isoforms.

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Acknowledgments

This research was supported by an operating grant from the Canadian Health Research Institutes (MOP-81341) to MT. Boyang Zhang holds an Ontario Graduate Scholarship (OGS). Caroline Lefebvre is the recipient of scholarships from OGS and Fonds de recherche du Québec-Santé.

Author information

Authors and Affiliations

  1. Neuroscience Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada, K1H 8M5

    Keyvan Sedaghat Ph.D., Boyang Zhang & Xiaodi Yang

  2. Departments of Medicine, Cellular and Molecular Medicine, and Psychiatry, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada, K1H 8M5

    Keyvan Sedaghat Ph.D., Boyang Zhang & Xiaodi Yang

  3. Ottawa Hospital Research Institute (Neuroscience Program), Departments of Medicine, Cellular and Molecular Medicine, and Psychiatry, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada, K1H 8M5

    Caroline Lefebvre

  4. Ottawa Hospital Research Institute and University of Ottawa, Ottawa, ON, Canada, K1H 8M5

    Mario Tiberi

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  1. Keyvan Sedaghat Ph.D.
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  2. Boyang Zhang
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Correspondence to Keyvan Sedaghat Ph.D. .

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  1. Ottawa Hospital Research Institute and University of Ottawa, Ottawa, Ontario, Canada

    Mario Tiberi

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Sedaghat, K., Zhang, B., Yang, X., Lefebvre, C., Tiberi, M. (2015). Study of Dopamine D1 Receptor Regulation by G Protein-Coupled Receptor Kinases Using Whole-Cell Phosphorylation and Cross-Linking Methods. In: Tiberi, M. (eds) Dopamine Receptor Technologies. Neuromethods, vol 96. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2196-6_7

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  • DOI: https://doi.org/10.1007/978-1-4939-2196-6_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2195-9

  • Online ISBN: 978-1-4939-2196-6

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