Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β
2-adrenergic receptor (β
2AR), 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
2AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β
2AR; however, IR reduced β
2AR 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
2AR and IR, we employed quantitative BRET
2 saturation and competition assays. Saturation assay data suggested constitutive β
2AR and IR homo- and heteromerization. Calculated acceptor/donor (AD
50) 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
2AR and IR identified intracellular domains encompassing residues at the end of the 7
th TM domain and C-terminal tail of β
2AR 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
2AR:IR oligomer formation, likely comprising heteromers of homodimers.
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