Abstract
Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β2-adrenergic receptor (β2AR), 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 β2AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β2AR; however, IR reduced β2AR 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 β2AR and IR, we employed quantitative BRET2 saturation and competition assays. Saturation assay data suggested constitutive β2AR 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 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 β2AR and IR identified intracellular domains 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. 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 β2AR:IR oligomer formation, likely comprising heteromers of homodimers.
Highlights
The functional interplay between different classes of receptors represents a means of fine-tuning the control of cellular functions that could be fundamental for understanding pathologic conditions and responses to therapeutic agents that interact with cellsurface receptors
Assay data evaluation The BRET2 values were fitted using the following equation for dimers: BRET = BRETmax(1/(1+AD50/X)), where X is the ratio of acceptor (A; Receptor-GFP2) to donor ((D; Receptor-Renilla luciferase 8 (RLuc8)) molecules
BRET2 data from saturation assays were fitted using the following equation for dimers: BRET = BRETmax(1/(1+AD50/X)) where X is the ratio of acceptor (A; Receptor-GFP2) to donor (D; Receptor-RLuc8) molecules
Summary
The functional interplay between different classes of receptors represents a means of fine-tuning the control of cellular functions that could be fundamental for understanding pathologic conditions and responses to therapeutic agents that interact with cellsurface receptors. Glucose metabolism is under the cooperative regulation of the RTK insulin receptor (IR) and b2-adrenergic receptor (b2AR), a representative 7TMR. Catecholamines act in opposition to insulin, stimulating glycogen breakdown, gluconeogenesis, and lipolysis [2]. They play an important role in counter-regulation of insulininduced hypoglycemia. The ability of insulin to counter regulate catecholamine action at the tissue/cellular level is crucial for the ‘‘tight’’ regulation of serum glucose levels, and interaction between IR and b2AR could be a required action in this process
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