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

PLoS One. 2014 Nov 17;9(11):e112664. doi: 10.1371/journal.pone.0112664. eCollection 2014.

Abstract

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 effect on the ligand binding properties of β(2)AR; however, IR reduced β(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 β(2)AR and IR, we employed quantitative BRET(2) saturation and competition assays. Saturation assay data suggested constitutive β(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 β(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of β(2)AR and a cytoplasmic part of the IR β 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 β(2)AR:IR oligomer formation, likely comprising heteromers of homodimers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Cell Line
  • Cell Membrane / metabolism
  • Computational Biology* / methods
  • Gene Expression
  • Humans
  • Models, Biological*
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Multimerization
  • Protein Transport
  • Receptor, Insulin / chemistry
  • Receptor, Insulin / genetics
  • Receptor, Insulin / metabolism*
  • Receptors, Adrenergic, beta-2 / chemistry
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Receptors, Adrenergic, beta-2
  • Recombinant Fusion Proteins
  • Receptor, Insulin

Grants and funding

The authors acknowledge funding from the Basileus S Program to M. Mandić, the Slovenian Research Agency program (P4-0053) to M. Vrecl, and the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 173001) to S. Glisic and N Veljkovic. M. Vrecl, S. Glisic and N. Veljkovic participate in the European COST Action CM1207 (GLISTEN). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.