Visualizing differences in ligand-induced beta-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-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-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-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 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.
Evans
N A
NA
SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK. Nicholas_A_Evans@sbphrd.com
Groarke
D A
DA
Warrack
J
J
Greenwood
C J
CJ
Dodgson
K
K
Milligan
G
G
Wilson
S
S
D000818:Animals; D000073861:Apelin; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002352:Carrier Proteins / Q000819:agonists / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins; D006801:Humans; D007028:Hypothalamic Hormones / Q000819:agonists / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology; D008024:Ligands; D008164:Luminescent Proteins / Q000032:analysis; D008543:Melanins / Q000819:agonists / Q000378:metabolism*; D064446:Orexin Receptors; D010907:Pituitary Hormones / Q000819:agonists / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012235:Rhodamines / Q000032:analysis; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C114557:5-carboxytetramethylrhodamine succinimidyl ester; C115436:APLN protein, human; C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D000073861:Apelin; D019390:Arrestins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D007028:Hypothalamic Hormones; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008164:Luminescent Proteins; D008543:Melanins; D064446:Orexin Receptors; D010907:Pituitary Hormones; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; C019463:melanin-concentrating hormone; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases