Real-time optical monitoring of ligand-mediated internalization of alpha1b-adrenoceptor with green fluorescent protein.
The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional alphalb-adrenoceptor tagged with the green fluorescent protein (alphalbAR/GFP) can be used to determine the molecular mechanism of intemalization of alphalbAR/ GFP in living cells. In mouse alphaT3 cells, alpha1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha1bAR/ GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alphalbAR/GFP redistribution. Agonist-promoted internalization of alphalbAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alphalbAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.
Department of Molecular and Cell Pharmacology, National Children's Medical Research Center, Tokyo, Japan.
T
T
Awaji
A
A
Hirasawa
M
M
Kataoka
H
H
Shinoura
Y
Y
Nakayama
T
T
Sugawara
S
S
Izumi
G
G
Tsujimoto
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009638:Norepinephrine / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D000316:Adrenergic alpha-Agonists; D004963:Estrenes; D008164:Luminescent Proteins; D011760:Pyrrolidinones; D018340:Receptors, Adrenergic, alpha-1; D011993:Recombinant Fusion Proteins; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D049452:Green Fluorescent Proteins; D007987:Gonadotropin-Releasing Hormone; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D009638:Norepinephrine