Abstract
BackgroundChimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR) and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR.ResultsThe rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization.Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.ConclusionThe covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes, but is an essential requirement for the correct post-endocytic sorting and recycling of the two proteins.
Highlights
Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the Nterminus of the G protein α-subunit have been extensively used to investigate several aspects of G protein-coupled receptors (GPCRs) signalling
Regarding beta-subtypes of adrenergic receptors (β1, β2 and β3-AR), it is known that, unlike β2-adrenergic receptor (β2AR), β3AR does not internalize in response to agonist; likewise, human β1AR is more resistant to agonist-mediated down-regulation and, it uses the same endocytosis mechanism as human β2AR, it is sorted to different endosomal compartments [7,8,9]
Hynes et al recently employed strategies that allowed the simultaneous imaging of the α and βγ components of heterotrimeric G proteins in live cells. They showed that Gs and β2AR dissociate upon agonist stimulation, internalize via different mechanisms and traffic to distinct sub-cellular localizations [18]. To further address this question, we report here a study on agonistmediated internalization and intracellular sorting between the wild type adrenergic receptor, β2AR, compared to that of the chimeric receptor, β2AR-Gαs, resulting from the fusion of β2-adrenergic receptor with the aminoterminal of the G protein α-subunit [19]
Summary
Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the Nterminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling Both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR) and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. In response to agonist stimulation, β2ARs undergo rapid phosphorylation by both second messengerdependent protein kinases and G protein-coupled receptor kinases (GRKs) [3] This event targets receptors for the binding of arrestin proteins, which sterically uncouples receptors from their cognate heterotrimeric G proteins and favours the receptors endocytosis via clathrin-coated vesicles into endosomal compartments [1,4,5]. Mutations of this sequence inhibit efficient recycling and cause the missorting of internalized receptors to lysosomes, thereby increasing the down-regulation [13]
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