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

Mandić M, Drinovec L, Glisic S, Veljkovic N, Nøhr J, Vrecl M - PLoS ONE (2014)

Bottom Line: 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.In heterologous competition assays, a transient increase in the BRET(2) signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation.In summary, our data suggest direct interaction and higher-order β(2)AR:IR oligomer formation, likely comprising heteromers of homodimers.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia.

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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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.
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pone-0112664-g006: 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.

Mentions: Computational peptide scanning of β2AR and IR was performed to identify the regions of proteins essential for information corresponding to the frequency F(0.216). The computer-assisted peptide scanning survey of the primary structure of β2AR with overlapping windows of different lengths revealed that the region encompassing residues 325–364 is essential for the information represented by the frequency F(0.216) (Fig. 6A). Further peptide scanning of IR identified three principal regions as important for the information represented by the frequency F(0.216), however region encompassing residues 1269–1314 represents the most probable domain involved in this interaction (Fig. 6B).


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

Mandić M, Drinovec L, Glisic S, Veljkovic N, Nøhr J, Vrecl M - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234468&req=5

pone-0112664-g006: 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.
Mentions: Computational peptide scanning of β2AR and IR was performed to identify the regions of proteins essential for information corresponding to the frequency F(0.216). The computer-assisted peptide scanning survey of the primary structure of β2AR with overlapping windows of different lengths revealed that the region encompassing residues 325–364 is essential for the information represented by the frequency F(0.216) (Fig. 6A). Further peptide scanning of IR identified three principal regions as important for the information represented by the frequency F(0.216), however region encompassing residues 1269–1314 represents the most probable domain involved in this interaction (Fig. 6B).

Bottom Line: 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.In heterologous competition assays, a transient increase in the BRET(2) signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation.In summary, our data suggest direct interaction and higher-order β(2)AR:IR oligomer formation, likely comprising heteromers of homodimers.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia.

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.

Show MeSH
Related in: MedlinePlus