A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk.

The angiotensin AT receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET technique to monitor the effect of angiotensin II on the interaction between Rluc tagged insulin receptor and GFP tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc).

Real-time trafficking and signaling of the glucagon-like peptide-1 receptor.

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates.

A BRET assay for monitoring insulin receptor interactions and ligand pharmacology.

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.

Structural model and trans-interaction of the entire ectodomain of the olfactory cell adhesion molecule.

The ectodomain of olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) consists of five immunoglobulin (Ig) domains (IgI-V), followed by two fibronectin-type 3 (Fn3) domains (Fn3I-II). A complete structural model of the entire ectodomain of human OCAM has been assembled from crystal structures of six recombinant proteins corresponding to different regions of the ectodomain. The model is the longest experimentally based composite structural model of an entire IgCAM ectodomain.

Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique.

Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood.