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