title: Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor.
abstract: Agonist-induced endocytosis and processing of the G protein-coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)- or hemagglutinin epitope-tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5-GFP and Rab4-GFP in early endosomes, and subsequently with Rab11-GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand-receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa(R)-transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine-Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.
authors.affiliation: Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Hunyady
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Baukal
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Gaborik
authors.forename: Jesus A
authors.initials: JA
authors.lastname: Olivares-Reyes
authors.forename: Marta
authors.initials: M
authors.lastname: Bor
authors.forename: Marta
authors.initials: M
authors.lastname: Szaszak
authors.forename: Robert
authors.initials: R
authors.lastname: Lodge
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Catt
authors.forename: Tamas
authors.initials: T
authors.lastname: Balla
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D015415:Biomarkers; D002460:Cell Line; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D000077191:Wortmannin; D020695:rab4 GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism
chemical_list: D000730:Androstadienes; D015415:Biomarkers; D002867:Chromones; D009025:Morpholines; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D000804:Angiotensin II; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000077191:Wortmannin