Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40 ☆

摘要: Abstract G protein-coupled receptor 40 (GPR40) is believed to be an attractive target enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found couple Gq protein, leading the activation of phospholipase C and subsequent increases intracellular Ca2 + level. However, underlying mechanisms that regulate internalization desensitization remain elucidated. In present study, a construct fused enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging localization by confocal microscopy. stably transfected HEK-293 cells, receptors underwent rapid agonist-induced constitutive ligand-independent internalization. Our data demonstrated agonist-mediated significantly blocked hypertonic sucrose treatment siRNA mediated depletion heavy chain clathrin. contrast, not affected or knock-down clathrin expression, but it methyl-β-cyclodextrin (MβCD) nystatin. Furthermore, our results using arrestin-3-EGFP redistribution assay siRNA-mediated arrestin-3 GRK2 expression revealed play essential role regulation internalization, are involved Additionally, observation showed upon agonist, internalized were rapidly recycled back plasma membrane via Rab4/Rab5 positive endosomes, whereas constitutively cell surface through Rab5 endosomes. Because FFA exhibit high level homology, observations could applicable other members this family.