Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Summary

GPCR-β-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-β-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Abstract

Interactions between G-protein coupled receptors (GPCRs) and β-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with β-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-β-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-β-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus ‘phosphorylation codes’ written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-β-arrestin complex.

Introduction

GPCRs represent the target of nearly 35% of current drugs in the market^1,2 and a clear understanding of their pharmacology is crucial in the development of novel therapeutic drugs³. One of the key aspects in GPCR drug discovery, particularly during the development of biased agonists is the characterization of novel ligands towards receptor-β-arrestin interactions⁴ and β-arrestin interactions with other cytosolic proteins such as clathrin⁵.

It has been documented that β-arrestin dependent signaling....

Protocol

1. Primer design strategy

1. Design primers to introduce genes of interest into pBiT1.1-C [TK/LgBiT], pBiT2.1-C [TK/SmBiT], pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] Vectors.
2. Select at least one of these three sites as one of the two unique restriction enzymes needed for directional cloning due to the presence of an in-frame stop codon that divides the multicloning site as shown in Figure 1¹¹.
3. Incorporate nucleotide sequence into the p.......

Discussion

Using the method presented here, interactions between any GPCR and β-arrestin1/2 can be monitored in real time living systems using this GPCR-β-arrestin structural complementation assay. In this regard, we were able to observe differential β-arrestin recruitment between the two β-arrestin isoforms by the GLP-1r (A prototypical Class B GPCR), we also observed a dissociation of the receptor-β-arrestin complex a few minutes after reaching the maximum luminescent signal.

I.......

Materials

Name	Company	Catalog Number	Comments
Antibiotics penicillin streptomycin	Welgene	LS202-02	Penicillin/Streptomycin
Bacterial Incubator	JEIO Tech	IB-05G	Incubator (Air-Jacket), Basic
Cell culture medium	Welgene	LM 001-05	DMEM Cell culture medium
Cell culture transfection medium	Gibco	31985-070	Optimem 1X cell culture medium
CO2 Incubator	NUAIRE	NU5720	Direct Heat CO2 Incubator
Digital water bath	Lab Tech	LWB-122D	Digital water bath lab tech
DNA Polymerase proof reading	ELPIS Biotech	EBT-1011	PfU DNA polymerase
DNA purification kit	Cosmogenetech	CMP0112	miniprepLaboPass Purificartion Kit Plasmid Mini
DNA Taq Polymerase	Enzynomics	P750	nTaq DNA polymerase
Enzyme restriction BglII	New England Biolabs	R0144L	BglII
Enzyme restriction buffer	New England Biolabs	B72045	CutSmart 10X Buffer
Enzyme restriction EcoRI	New England Biolabs	R3101L	EcoRI-HF
Enzyme restriction NheI	New England Biolabs	R01315	NheI
Enzyme restriction XhoI	New England Biolabs	R0146L	XhoI
Fetal Bovine Serum	Gibco Canada	12483020	Fetal Bovine Serum
Gel/PCR DNA MiniKit	Real Biotech Corporation	KH23108	HiYield Gel/PCR DNA MiniKit
Ligase	ELPIS Biotech	EBT-1025	T4 DNA Ligase
Light microscope	Olympus	CKX53SF	CKX53 Microscope Olympus
lipid transfection reagent	Invitrogen	11668-019	Lipofectamine 2000
Luminometer	Biotek/Fisher Scientific	12504386	Synergy 2 Multi-Mode Microplate Readers
NanoBiT System	Promega	N2014	NanoBiT PPI MCS Starter System
Nanoluciferase substrate	Promega	N2012	Nano-Glo Live Cell assay system
PCR Thermal cycler	Eppendorf	6336000015	Master cycler Nexus SX1
Poly-L-lysine	Sigma Aldrich	P4707-50ML	Poly-L-lysine solution
Trypsin EDTA	Gibco	25200-056	Trysin EDTA 10X
White Cell culture 96 well plates	Corning	3917	Assay Plate 96 well plate