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Glycoprotein-glycoprotein receptor binding detection using bioluminescence resonance energy transfer.

Endocrinology 2024 April 30
The glycoprotein receptors, members of the large G protein-coupled receptors (GPCRs) family, are characterized by a large extracellular domains responsible of binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity as these assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer (BRET) in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or luteinizing hormone) fused to the enhanced green fluorescent protein (eGFP). The Gluc-LHCGR, as well as other Gluc-GPCRs such as the somatostatin and the C-X-cytokine 4 receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric eGFP-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, BRET was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR, and show that this bioassay provides a fast and effective, safe and cost efficient tool to assist the molecular characterization of mutations in either the receptor or ligand, and it is compatible with downstream cellular assays to determine receptor activation/function.

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