Protocol to characterize Gi/o and Gs protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements

Aenne-Dorothea Liebing; Petra Krumbholz; Claudia Stäubert

doi:10.1016/j.xpro.2023.102120

Protocol to characterize G_i/o and G_s protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements

STAR Protoc. 2023 Mar 17;4(1):102120. doi: 10.1016/j.xpro.2023.102120. Epub 2023 Feb 14.

Authors

Aenne-Dorothea Liebing¹, Petra Krumbholz¹, Claudia Stäubert²

Affiliations

¹ Rudolf-Schönheimer Institute for Biochemistry, Faculty of Medicine, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany.
² Rudolf-Schönheimer Institute for Biochemistry, Faculty of Medicine, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany. Electronic address: claudia.staeubert@medizin.uni-leipzig.de.

Abstract

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.

Keywords: Cell Biology; Cell Culture; Cell Membrane; Cell-based Assays; Molecular Biology; Signal Transduction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Membrane / metabolism
Cyclic AMP* / analysis
Cyclic AMP* / metabolism
Enzyme-Linked Immunosorbent Assay
HEK293 Cells
Humans
Receptors, G-Protein-Coupled* / genetics
Receptors, G-Protein-Coupled* / metabolism

Substances

Cyclic AMP
Receptors, G-Protein-Coupled