Glycoprotein-glycoprotein Receptor Binding Detection Using Bioluminescence Resonance Energy Transfer.

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Endocrinology

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses to hormones.
• Traditional ligand-binding assays for hormone-receptor interactions lack information on localization and activity.
• Existing methods often require purified membranes and radiolabeling, posing safety and cost concerns.

Purpose of the Study:

• To develop and validate a novel bioluminescence resonance energy transfer (BRET) assay for studying hormone-receptor interactions in living cells.
• To assess the functionality of G protein-coupled receptors and their ligands using BRET.
• To provide a safe, cost-efficient, and effective tool for characterizing mutations in hormone-receptor systems.

Main Methods:

• Application of bioluminescence resonance energy transfer (BRET) in living cells.
• Construction of fusion proteins: Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and enhanced green fluorescent protein (EGFP)-ligands (human chorionic gonadotropin or LH).
• Measurement of luminescence activity to determine receptor expression and hormone-receptor interactions.

Main Results:

• The Gluc-LHCGR fusion protein is expressed on the plasma membrane and is functional.
• EGFP-ligands are secreted, bind, and activate both wild-type and Gluc-LHCGR.
• BRET successfully determined interactions between mutated hormones and the LHCGR, demonstrating assay utility.

Conclusions:

• Bioluminescence resonance energy transfer in living cells provides a sensitive and functional assay for hormone-receptor interactions.
• This BRET assay is a fast, effective, safe, and cost-efficient tool for molecular characterization of mutations in receptors and ligands.
• The assay is compatible with downstream functional assays, enabling comprehensive analysis of receptor activation.