Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors

Remi Janicot; Mikel Garcia-Marcos

doi:10.1016/j.xpro.2024.103355

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors

STAR Protoc. 2024 Dec 20;5(4):103355. doi: 10.1016/j.xpro.2024.103355. Epub 2024 Oct 1.

Authors

Remi Janicot¹, Mikel Garcia-Marcos²

Affiliations

¹ Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA.
² Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA; Department of Biology, College of Arts & Sciences, Boston University, Boston, MA 02115, USA. Electronic address: [email protected].

Abstract

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (G_s, G_i/o, G_q/11, G_12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.¹.

Keywords: Biotechnology and bioengineering; Cell Biology; Cell culture; Cell-based Assays; Molecular Biology; Signal Transduction.

MeSH terms

Animals
Bioluminescence Resonance Energy Transfer Techniques / methods
Biosensing Techniques* / methods
HEK293 Cells
Humans
Lentivirus / genetics
Primary Cell Culture / methods
Receptors, G-Protein-Coupled* / metabolism

Substances

Receptors, G-Protein-Coupled

Abstract

MeSH terms

Substances

Grants and funding