title: Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B₂ receptor cycling.
abstract: The bradykinin (BK) B₂ receptor (B₂R) is G protein coupled and phosphorylated upon agonist stimulation; its endocytosis and recycling are documented. We assessed the effect of drugs that affect the cytoskeleton on B2R cycling. These drugs were targeted to tubulin (paclitaxel, or the novel combretastatin A-4 mimetic 3,4,5-trimethoxyphenyl-4-(2-oxoimidazolidin-1-yl)benzenesulfonate [IMZ-602]) and actin (cytochalasin D). Tubulin ligands did not alter agonist-induced receptor endocytosis, as shown using antibodies reactive with myc-tagged B₂Rs (microscopy, cytofluorometry), but rather reduced the progression of the ligand-receptor-β-arrestin complex from the cell periphery to the interior. The 3 fluorescent probes of this complex (B2R-green fluorescent protein [B2R-GFP], the fluorescent agonist fluorescein-5-thiocarbamoyl-D-Arg-[Hyp³, Igl⁵, Oic⁷, Igl⁸]-BK and β-arrestin2-GFP) were condensed in punctuate structures that remained close to the cell surface in the presence of IMZ-602. Cytochalasin D selectively inhibited the recycling of endocytosed B₂R-GFP (B₂R-GFP imaging, [³H]BK binding). Dominant negative (GDP-locked)-Rab5 and -Rab11 reproduced the effects of inhibitors of tubulin and actin, respectively, on the cycling of B₂R-GFP. GDP-locked-Rab4 also inhibited B₂R-GFP recycling to the cell surface. Consistent with the displacement of cargo along specific cytoskeletal elements, Rab5-associated progression of the endocytosed BK B₂R follows microtubules toward their (-) end, while its recycling progresses along actin fibers to the cell surface. However, tubulin ligands do not suppress the tested desensitization or resensitization mechanisms of the B₂R.
authors: Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Québec, QC G1V 4G2, Canada.
authors: Xavier
authors: X
authors: Charest-Morin
authors: Sébastien
authors: S
authors: Fortin
authors: Robert
authors: R
authors: Lodge
authors: Caroline
authors: C
authors: Roy
authors: Lajos
authors: L
authors: Gera
authors: René C
authors: RC
authors: Gaudreault
authors: François
authors: F
authors: Marceau
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors; D001920:Bradykinin / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects*; D004705:Endocytosis / Q000187:drug effects; D006153:Guanosine Diphosphate / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D021381:Protein Transport / Q000187:drug effects; D043782:Receptor, Bradykinin B2 / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology*; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D000199:Actins; D001920:Bradykinin; D015638:Cytochalasin D; D006153:Guanosine Diphosphate; D043782:Receptor, Bradykinin B2; D050257:Tubulin Modulators; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins