Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• G-protein-coupled receptors (GPCRs) are crucial cell surface receptors and significant drug targets.
• Existing cell-based assays for GPCR profiling are often time-consuming and expensive.
• GPCR internalization, often mediated by β-arrestin, is a key functional indicator.

Purpose of the Study:

• To develop a novel, rapid, and cost-effective assay for quantifying GPCR internalization.
• To establish a versatile cell-based system for GPCR characterization and drug discovery screening.

Main Methods:

• Utilized DnaE intein-mediated reconstitution of fragmented luciferase (Renilla or Firefly).
• Assay detects interactions between activated GPCRs and β-arrestin2 or Rab5, indicating internalization.
• Validated the assay using functionally diverse GPCRs and known agonists/antagonists.

Main Results:

• The novel assay accurately quantifies GPCR internalization.
• EC50 values for known compounds closely matched previous reports, demonstrating assay sensitivity.
• The system proved to be cost-effective, rapid, and easy to manipulate compared to conventional methods.

Conclusions:

• The developed assay provides a sensitive, efficient, and economical method for GPCR functional analysis.
• This assay system has broad applicability for GPCR characterization and high-throughput drug screening.
• It offers a valuable tool for advancing GPCR-targeted drug discovery efforts.