Endocrine Abstracts

Functional interplay between different classes of receptors has emerged as a notable factor of cellular response in health and disease. One of the most prominent examples of cross-talk involves the regulation of glucose metabolism, a major regulatory function under the influence of both insulin receptor (IR) and β₂-adrenergic receptor (β₂-AR) representing the receptor tyrosine kinases (RTKs) and the seven transmembrane receptors (7TMRs) respectively. The cross-talk between these different classes of receptors may occur at the RTK/7TMR level or via various intracellular effector/adaptor molecules such as β-arrestins. First we employed BRET² proximity assays to detect possible heterodimerization between β₂-AR and IR in live HEK-293 cells. BRET² saturation assay results suggested a constitutive IR and β₂-AR homodimerization as well as heterodimerization between the IR and β₂-AR. The existence of heterodimerization was further tested by BRET² competition assay, where unexpectedly, a transient increase in the BRET signal was observed when untagged β₂-AR was coexpressed with a constant amount of tagged IR suggesting that these complexes possibly constitute of trimmers or higher oligomers. Further BRET evidence for the IR/β₂-AR heterodimerization was provided by the receptor-heteromer investigation technology (HIT) showing isoproterenol- but not insulin-induced GFP²-β-arr2 recruitment to the heteromer complex consisting of IR-Rluc8 and untagged β₂-AR; IR/β-arr2 interaction was only found to be constitutive. Next we applied informational spectrum method (ISM), a virtual spectroscopy method for investigation of protein-protein interactions. Computational peptide scanning of the β₂-AR and IR identified domains encompassing residues at the end of 7^th TM domain and C-terminal tail of β₂-AR and cytoplasmic part of IR beta chain as prospective interaction domains. In summary our data suggest direct interaction between β₂-AR and IR and existence of multiprotein complexes consisting of IR, β₂-AR and β-arr2.