Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5.
Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)--green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 microM) and a dominant-negative form of the enzyme (p85alpha DeltaiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R-green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.
Houle
Steeve
S
Centre de recherche en cancérologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, Québec, Canada G1R 2J6.
Marceau
François
F
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007601:Jugular Veins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018942:Macrolides; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects; D011817:Rabbits; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D000077191:Wortmannin; D020696:rab5 GTP-Binding Proteins / Q000502:physiology*
D000730:Androstadienes; D000900:Anti-Bacterial Agents; D002867:Chromones; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D018942:Macrolides; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D000077191:Wortmannin; D020696:rab5 GTP-Binding Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C040929:bafilomycin A1