Functional characterization of β2-adrenergic and insulin receptor heteromers

Abstract This study aimed to functionally characterize β 2 -adrenergic (β 2 AR) 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 β 2 AR. Evidence for β 2 AR: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 β 2 AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP 2 -βarr2 recruitment to the β 2 AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRET const ) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP 2 -βarr2 1–185 mutant lacking the proposed IR binding domain. β 2 AR:IR heteromerization also influenced the pharmacological phenotype of β 2 AR, 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 β 2 AR was provided by BRET 2 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:β 2 AR-GFP 2 , saturation was not reached, most likely reflecting random collisions between IR 1–1271 and β 2 AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET 2 signal was detected that would be indicative of βarr2 recruitment to the IR 1–1271:β 2 AR heteromer. Complementary 3D visualization of β 2 AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β 2 AR:IR heteromerization.