Demonstration of a Direct Interaction between beta(2)-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics
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Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and beta(2)-adrenergic receptor (beta(2)AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on beta(2)AR properties, we showed that IR coexpression had no effect on the ligand binding properties of beta(2)AR; however, IR reduced beta(2)AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between beta(2)AR and IR, we employed quantitative BRET 2 saturation and competition assays. Saturation assay data suggested constitutive beta(2)AR and IR homo-and heteromerization. Calculated acceptor/donor ...(AD(50)) values as a measure of the relative affinity for homo-and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET2 signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET2 data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of beta(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of beta(2)AR and a cytoplasmic part of the IR beta chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order beta(2)AR: IR oligomer formation, likely comprising heteromers of homodimers.
Source:PLoS One, 2014, 9, 11
- Application of the EIIP/ISM bioinformatics platform in discovery of novel therapeutic targets and potential therapeutic molecules (RS-173001)
- Basileus S Program, Slovenian Research Agency program [P4-0053]