title: A BRET assay for monitoring insulin receptor interactions and ligand pharmacology.
abstract: The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.
authors.affiliation: Incretin Biology, Hagedorn Research Institute, Gentofte, Denmark.
authors.forename: Nikolaj
authors.initials: N
authors.lastname: Kulahin
authors.forename: Samra J
authors.initials: SJ
authors.lastname: Sanni
authors.forename: Rita
authors.initials: R
authors.lastname: Slaaby
authors.forename: Jane
authors.initials: J
authors.lastname: Nøhr
authors.forename: Steen
authors.initials: S
authors.lastname: Gammeltoft
authors.forename: Jakob Lerche
authors.initials: JL
authors.lastname: Hansen
authors.forename: Rasmus
authors.initials: R
authors.lastname: Jorgensen
mesh_terms: D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000032:analysis*; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects; D011972:Receptor, Insulin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
chemical_list: D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin