Visualizing differences in ligand-induced β-arrestin-GFP interactions and trafficking between three recently characterized G protein-coupled receptors

beta -Arrestin 1-GFP or beta -arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-l, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-l and apelin receptors were able to rapidly translocate both beta -arrestin l-GFP and beta -arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta -arrestin 2-GFP. beta -Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-l receptor, internalization of beta -arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Go-internalization of the orexin-l receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-l receptor antagonist resulted in cessation of incorporation of beta -arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta -arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta -arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta -arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.