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

  • ️Wed Jan 01 2014

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

Maja Mandić et al. PLoS One. 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.

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Conflict of interest statement

Competing Interests: It is true that two coauthors are employed by commercial companies; however this does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The competing interests and financial disclosures of both co-authors employed by commercial companies are as follows: Luka Drinovec is employed by the company Aerosol d.o.o. This company produces instruments for the measurement of aerosolized soot (black carbon) and some accessories for aerosol measurement. Aerosol d.o.o. does not have any connection with molecular biology, development of medicines, etc. Thus, the authors declare that there are no competing interests for Luka Drinovec coauthoring the article “Demonstration of a direct interaction between β2-adrenergic receptor and insulin receptor by BRET and bioinformatics”. Response to Reviewers: Jane Nøhr is employed by Novo Nordisk A/S and owns Novo Nordisk A/S company stocks. The work on this paper was done as a part of collaboration with academia, i.e., co-supervision of Maja Mandic at the Veterinary faculty, University of Ljubljana. Therefore, the authors declare that there are no competing interests for Jane Nohr coauthoring the article, “Demonstration of a direct interaction between β2-adrenergic receptor and insulin receptor by BRET and bioinformatics”.

Figures

Figure 1
Figure 1

Effect of IR coexpression on β2AR surface expression and internalization in HEK-293 cells. (A) ELISA was performed on intact HEK-293 cells transiently transfected with HA-β2AR-RLuc8 alone (open bars) or HA-β2AR-RLuc8 together with IR-GFP2 (hatched bars) at a 1∶1 cDNA ratio using an antibody directed against the HA epitope. Antibody binding, as an index of receptor surface expression, was determined by measuring the absorbance at 450 nm. HA-β2AR-RLuc8 and IR-GFP2 total relative expression was determined by luminescence and fluorescence measurement, respectively. Total luminescence was measured after the addition of the RLuc substrate coelenterazine 400a. Total fluorescence was measured with an excitation filter at 380 nm and an emission filter at 515 nm. Fold change is relative to open bars for surface expression and total luminescence, and relative to hatched bars for total fluorescence. Data are expressed as the means ± S.E. of three independent experiments performed in triplicate. *, p<0.05 as compared with HA-β2AR-RLuc8 transfected cells. (B, C) Effect of IR coexpression on the time-course of β2AR internalization. HEK-293 cells were transiently transfected with either β2AR (dotted line) or β2AR together with the IR (solid line) at a 1∶1 cDNA ratio. β2AR internalization was first induced by (B) isoproterenol (10 µM) or (C) the combination of isoproterenol (10 µM) and insulin (0.1 µM) for the indicated time intervals. Receptor sequestration was then defined as the decrease in specific [125I]-iodopindolol binding compared with the total binding obtained in untreated cells. The amount of internalized receptors as a function of time was fitted using a one-site binding (hyperbola) curve fit (GraphPad Prism 5.0). Data are expressed as the means ± S.E. from three independent experiments performed in triplicate.

Figure 2
Figure 2. Visualization of β2AR and IR cellular localization by confocal microscopy.

IR-GFP2 and HA-β2AR-RLuc8 cellular localization is shown in untreated (control) cells (upper panels) and cells concomitantly treated with isoproterenol (10 µM) and insulin (0.1 µM) for 10 min at 37°C (lower panels). The green color indicates IR-GFP2; red indicates HA-β2AR-RLuc8; yellow/orange is the overlapping region indicating colocalization of IR-GFP2 and HA-β2AR-RLuc8. Note that both receptors have comparable localization in untreated and agonist-stimulated cells and that they exhibit a high degree of colocalization. However, in agonist-stimulated cells a proportion of intracellular receptors did not colocalize. Objective 40× and zoom factor 4 apply for all images.

Figure 3
Figure 3. BRET2 saturation curves of β2AR and IR homo- and heteromers.

HEK-293 cells were transiently cotransfected with a constant amount of RLuc8-tagged and increasing amounts of GFP2-tagged receptor encoding constructs. BRET2 values were plotted as a function of the ratio between the acceptor and donor fusion proteins (A/D ratio). A dataset is composed of 3–5 independent saturation experiments that were fitted using a nonlinear regression equation assuming a single binding site using GraphPad Prism 5.0 (Fitting results are found in Table 3).

Figure 4
Figure 4. Homologous and heterologous BRET2 competition assay.

(A, B) HEK-293 cells were cotransfected with a constant amount of RLuc8- and GFP2-tagged receptor while increasing the amount of untagged receptor. In the homologous competition assay (dotted line), BRET2 signals decreased with an increasing amount of WT receptor confirming the competition effect. In the heterologous BRET2 assay (solid line), where different WT receptors were used to compete with the tagged homomer receptor pair, a transient increase in the BRET2 signal with a subsequent hyperbolical decrease was observed. BRET0 is the BRET2 signal obtained in the absence of competitor. Data are expressed as the means ± S.E. from three independent experiments performed in triplicate. (C, D) Receptor-GFP2/Receptor-RLuc8 expression ratio (GFP2/RLuc8 ratio) in each sample was evaluated for total luminescence and total fluorescence. Total luminescence and total fluorescence was measured as described under Material and Methods. Note that GFP2/RLuc8 ratio was roughly constant in the absence or presence of increasing concentrations of competitor (untagged β2AR or IR). Data are expressed as the means ± S.E. from three independent experiments performed in triplicate.

Figure 5
Figure 5. Informational spectrum (IS) of (A) β2AR, (B) IR and (C) cross-spectrum (CS) of β2AR and IR.

CS of β2AR and IR revealed common information corresponding to the IS frequency F(0.216) (panel C).

Figure 6
Figure 6. Mapping of the domains with maximal contribution to the frequency component F(0.216) in the informational spectrum of (A) β2AR and (B) IR.

Peptide scanning of the β2AR and IR identified regions encompassing residues at the end of the 7th TM domain and C-terminal tail of β2AR and a cytoplasmic part of the IR β chain as prospective interaction domains. The regions encompassing residues 325–364 in β2AR (A) and 1269–1314 IR (B) are essential for the information represented by the frequency F(0.216). The position of the first amino acid (aa) in the domain is shown. Panel B; the amino acid position denote the positions in IR β subunit starting from amino acid 763.

Figure 7
Figure 7. Consensus informational spectrum (CIS) of β2AR:IR tetramers with the characteristic peak at F(0.216).

(A) β2AR:β2AR:β2AR:IR, (B) β2AR:β2AR:IR:IR and (C) β2AR:IR:IR:IR tetramers. Note that the interaction affinities among the receptors decreased in the order β2AR:β2AR:β2AR:IR≥β2AR:β2AR:IR:IR>β2AR:IR:IR:IR.

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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.

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