Protocol to characterize G(i/o) and G(s) protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements

Activation of G(s) or G(i/o) protein-coupled receptors (GPCRs) leads to changes of intracellular cyclic adenosine monophosphate (cAMP) levels. This protocol describes steps for cloning HA- and FLAG-tagged GPCRs, transient transfection of CHO-K1 or HEK293-T cells, and determination of basal and ligan...

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Detalles Bibliográficos
Autores principales: Liebing, Aenne-Dorothea, Krumbholz, Petra, Stäubert, Claudia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9958081/
https://www.ncbi.nlm.nih.gov/pubmed/36853674
http://dx.doi.org/10.1016/j.xpro.2023.102120
Descripción
Sumario:Activation of G(s) or G(i/o) protein-coupled receptors (GPCRs) leads to changes of intracellular cyclic adenosine monophosphate (cAMP) levels. This protocol describes steps for cloning HA- and FLAG-tagged GPCRs, transient transfection of CHO-K1 or HEK293-T cells, and determination of basal and ligand-induced changes in intracellular cAMP levels. We detail enzyme-linked immunosorbent assays to determine relative GPCR plasma membrane and total expression levels. For complete details on the use and execution of this protocol, please refer to Schulze et al. (2022).(1)

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