Abstract
Intracellular trafficking of G protein-coupled receptors (GPCRs) controls their localization and degradation, which affects a cell’s ability to adapt to extracellular stimuli. Although the perturbation of trafficking induces important diseases, these trafficking mechanisms are poorly understood. Herein, we demonstrate an optogenetic method using an optical dimerizer, cryptochrome (CRY) and its partner protein (CIB), to analyze the trafficking mechanisms of GPCRs and their regulatory proteins. Temporally controlling the interaction between β-arrestin and β2-adrenergic receptor (ADRB2) reveals that the duration of the β-arrestin-ADRB2 interaction determines the trafficking pathway of ADRB2. Remarkably, the phosphorylation of ADRB2 by G protein-coupled receptor kinases is unnecessary to trigger clathrin-mediated endocytosis, and β-arrestin interacting with unphosphorylated ADRB2 fails to activate mitogen-activated protein kinase (MAPK) signaling, in contrast to the ADRB2 agonist isoproterenol. Temporal control of β-arrestin-GPCR interactions will enable the investigation of the unique roles of β-arrestin and the mechanism by which it regulates β-arrestin-specific trafficking pathways of different GPCRs.
Highlights
Optogenetics, in combination with fluorescence and bioluminescence imaging techniques, is a useful technique for the spatiotemporal analysis of intracellular signaling
We demonstrate an optogenetic approach using the reversible interaction between CRY and cryptochrome-interacting basic-helix-loop-helix 1 (CIB) to investigate the significance of the interaction of β-arrestin with G protein-coupled receptors (GPCRs) in intracellular trafficking in living cells
We developed an optogenetic method for the analysis of intracellular trafficking of ADRB2 regulated by β-arrestin
Summary
Basic Strategy for Light-Induced Interactions between ADRB2 and β-Arrestin. ADRB2 is a GPCR that regulates cardiovascular and pulmonary functions[20,21]. Light irradiation did not induce an increase in the number of spots in the presence of Dyngo4a the colocalization of ArrestinCRY with ADRB2CIB was induced Considering these results, we concluded that the endocytosis of ADRB2CIB was triggered by light-induced interaction of β-arrestin with ADRB2, and the observed ADRB2CIB fluorescent spots were ADRB2CIB-containing vesicles produced by clathrin-mediated endocytosis. ADRB2CIB was ubiquitinated to a greater degree when the irradiation time was increased to 120 min (Fig. 4c,d) These results demonstrate that prolonged interaction of β-arrestin with ADRB2, stimulated by blue light, promotes the sorting of ADRB2CIB to lysosomes. Several fluorescent spots of Mdm[2] were observed in the cytosol after 15 min of light irradiation, and they colocalized with the ADRB2CIB-containing vesicles This result suggests that the cytosolic Mdm[2] is recruited to the light-induced ADRB2-β-arrestin complexes. The results suggest that β-arrestin regulates ERK1/2 phosphorylation in different manners depending on the GPCRs
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