Functional characterization of β2-adrenergic and insulin receptor heteromers.

This study aimed to functionally characterize β2-adrenergic (β2AR) and insulin receptor (IR) heteromers in regard to β-arrestin 2 (βarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR β chain in heteromerization with β2AR. Evidence for β2AR:IR:βarr2 complex formation and the specificity of the IR:βarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of β2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-βarr2 recruitment to the β2AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-βarr2 1-185 mutant lacking the proposed IR binding domain. β2AR:IR heteromerization also influenced the pharmacological phenotype of β2AR, i.e., its efficacy in recruiting βarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR β chain in the interaction with β2AR was provided by BRET2 saturation and HIT assays using an IR 1-1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1-1271-RLuc8:β2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1-1271 and β2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of βarr2 recruitment to the IR 1-1271:β2AR heteromer. Complementary 3D visualization of β2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β2AR:IR heteromerization. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.