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Demonstration of a direct interaction between ?2-adrenergic receptor and insulin receptor by BRET and bioinformatics.

ABSTRACT: Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and ?(2)-adrenergic receptor (?(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 ?(2)AR properties, we showed that IR coexpression had no effect on the ligand binding properties of ?(2)AR; however, IR reduced ?(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 ?(2)AR and IR, we employed quantitative BRET(2) saturation and competition assays. Saturation assay data suggested constitutive ?(2)AR and IR homo- and heteromerization. Calculated acceptor/donor (AD50) 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 BRET(2) signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET(2) data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of ?(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of ?(2)AR and a cytoplasmic part of the IR ? 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 ?(2)AR:IR oligomer formation, likely comprising heteromers of homodimers.

SUBMITTER: Mandic M

PROVIDER: S-EPMC4234468 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Demonstration of a direct interaction between β2-adrenergic receptor and insulin receptor by BRET and bioinformatics.

Mandić Maja M Drinovec Luka L Glisic Sanja S Veljkovic Nevena N Nøhr Jane J Vrecl Milka M

PloS one 20141117 11

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β(2)-adrenergic receptor (β(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 β(2)AR properties, we showed that IR coexpression had no effec ...[more]

PMID: 25401701

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Project description:G protein-coupled receptors are involved in many biological processes, relaying the extracellular signal inside the cell. Signaling is regulated by the interactions between receptors and their ligands, it can be stimulated by agonists, or inhibited by antagonists or inverse agonists. The development of a new drug targeting a member of this family requires to take into account the pharmacological profile of the designed ligands in order to elicit the desired response. The structure-based virtual screening of chemical libraries may prioritize a specific class of ligands by combining docking results and ligand binding information provided by crystallographic structures. The performance of the method depends on the relevance of the structural data, in particular the conformation of the targeted site, the binding mode of the reference ligand, and the approach used to compare the interactions formed by the docked ligand with those formed by the reference ligand in the crystallographic structure. Here, we propose a new method based on the conformational dynamics of a single protein-ligand reference complex to improve the biased selection of ligands with specific pharmacological properties in a structure-based virtual screening exercise. Interactions patterns between a reference agonist and the receptor, here exemplified on the β2 adrenergic receptor, were extracted from molecular dynamics simulations of the agonist/receptor complex and encoded in graphs used to train a one-class machine learning classifier. Different conditions were tested: low to high affinity agonists, varying simulation duration, considering or ignoring hydrophobic contacts, and tuning of the classifier parametrization. The best models applied to post-process raw data from retrospective virtual screening obtained by docking of test libraries effectively filtered out irrelevant poses, discarding inactive and non-agonist ligands while identifying agonists. Taken together, our results suggest that consistency of the binding mode during the simulation is a key to the success of the method.

Dataset Information

Demonstration of a direct interaction between ?2-adrenergic receptor and insulin receptor by BRET and bioinformatics.

Publications

Demonstration of a direct interaction between β2-adrenergic receptor and insulin receptor by BRET and bioinformatics.

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