title: Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels.
abstract: Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Tome
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Charles P
authors.initials: CP
authors.lastname: Schaefer
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Leigh M
authors.initials: LM
authors.lastname: Jacobs
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Herndon
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Fabian O
authors.initials: FO
authors.lastname: Matty
authors.affiliation: Department of Medical Pharmacology, University of Arizona, Tucson, Arizona, USA.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Davis
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism*; D001921:Brain / Q000098:blood supply / Q000378:metabolism*; D002199:Capillary Permeability / Q000502:physiology*; D021941:Caveolae / Q000378:metabolism; D005260:Female; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D055806:Microvessels / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D021381:Protein Transport; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D050883:HSC70 Heat-Shock Proteins; D019704:Protein Disulfide-Isomerases
keywords: ATP synthase β subunit; P-glycoprotein; Rab5; blood-brain barrier; caveolae; protein disulfide isomerase
references.citation: Biochim Biophys Acta. 2004 Jun 1;1699(1-2):35-44
references.pmid: 15158710
references.citation: Front Physiol. 2012 Jan 06;2:120
references.pmid: 22232608
references.citation: Biochim Biophys Acta. 1998 Apr 22;1391(3):367-76
references.pmid: 9555096
references.citation: Am J Physiol Cell Physiol. 2013 Oct 1;305(7):C776-87
references.pmid: 23885065
references.citation: J Clin Invest. 1999 Feb;103(3):393-9
references.pmid: 9927500
references.citation: Pharmacol Rev. 2008 Jun;60(2):196-209
references.pmid: 18560012
references.citation: J Biol Chem. 1995 Sep 15;270(37):21839-44
references.pmid: 7545169
references.citation: J Biol Chem. 2000 Jan 21;275(3):1920-9
references.pmid: 10636893
references.citation: Proteomics. 2011 Dec;11(23):4514-28
references.pmid: 21932443
references.citation: J Endocrinol. 2004 Nov;183(2):353-63
references.pmid: 15531723
references.citation: J Cereb Blood Flow Metab. 2010 Sep;30(9):1593-7
references.pmid: 20628400
references.citation: NeuroRx. 2005 Jan;2(1):27-43
references.pmid: 15717055
references.citation: Curr Drug Targets. 2006 Aug;7(8):911-21
references.pmid: 16918320
references.citation: Nature. 2014 May 15;509(7500):310-7
references.pmid: 24828189
references.citation: J Neurochem. 2007 Dec;103(6):2540-55
references.pmid: 17931362
references.citation: Mol Pharmacol. 2004 Nov;66(5):1332-9
references.pmid: 15308763
references.citation: J Neurochem. 2003 Nov;87(4):1010-23
references.pmid: 14622130
references.citation: Biochim Biophys Acta. 2014 Feb;1838(2):532-45
references.pmid: 23899502
references.citation: Cancer Control. 2003 Mar-Apr;10(2):159-65
references.pmid: 12712010
references.citation: Neuron. 2011 Aug 11;71(3):406-24
references.pmid: 21835339
references.citation: Trends Pharmacol Sci. 2010 Jun;31(6):246-54
references.pmid: 20417575
references.citation: Electrophoresis. 2012 Aug;33(16):2527-36
references.pmid: 22899260
references.citation: Biochemistry. 2008 Apr 29;47(17):4955-63
references.pmid: 18393449
references.citation: FEMS Microbiol Lett. 2010 Feb;303(2):156-62
references.pmid: 20067530
references.citation: J Histochem Cytochem. 2006 Oct;54(10):1159-67
references.pmid: 16801529
references.citation: Mol Cell Proteomics. 2011 Oct;10(10):M110.007146
references.pmid: 21753190
references.citation: J Neurochem. 2012 Sep;122(5):962-75
references.pmid: 22716933
references.citation: Nat Rev Mol Cell Biol. 2007 Aug;8(8):622-32
references.pmid: 17637737
references.citation: Proteomics. 2004 Nov;4(11):3536-48
references.pmid: 15378739
references.citation: J Neurochem. 2007 Sep;102(5):1677-90
references.pmid: 17697052
references.citation: FEBS Lett. 2000 Jan 28;466(2-3):219-24
references.pmid: 10682831
references.citation: J Biol Chem. 2013 Jul 12;288(28):20326-33
references.pmid: 23733192
references.citation: Proteome Sci. 2012 Aug 02;10(1):47
references.pmid: 22857383
references.citation: Eur J Biochem. 2000 Oct;267(20):6102-9
references.pmid: 11012661
references.citation: Mol Biol Cell. 1995 Nov;6(11):1591-603
references.pmid: 8589458
references.citation: Biochem Soc Trans. 2012 Dec 1;40(6):1360-7
references.pmid: 23176481
references.citation: Biol Chem. 2009 Aug;390(8):753-9
references.pmid: 19361275
references.citation: PLoS One. 2014;9(4):e93439
references.pmid: 24747515
references.citation: Physiol Rev. 2011 Jan;91(1):119-49
references.pmid: 21248164
references.citation: Nat Commun. 2013;4:1831
references.pmid: 23652019
references.citation: Int J Biochem Cell Biol. 2012 Mar;44(3):461-4
references.pmid: 22212176
references.citation: Cell Mol Life Sci. 2009 Aug;66(16):2623-42
references.pmid: 19381436
references.citation: Nat Rev Mol Cell Biol. 2013 Feb;14(2):98-112
references.pmid: 23340574
references.citation: J Biol Chem. 2001 Jul 20;276(29):26980-7
references.pmid: 11356825
references.citation: PLoS One. 2010;5(4):e10189
references.pmid: 20422004
references.citation: Cell. 2008 Jan 11;132(1):113-24
references.pmid: 18191225
references.citation: J Physiol Pharmacol. 2009 Oct;60 Suppl 4:105-9
references.pmid: 20083858
references.citation: Curr Pharm Des. 2014;20(10):1463-71
references.pmid: 23789954
references.citation: Epilepsia. 2010 Aug;51(8):1333-47
references.pmid: 20477844
references.citation: Med Sci Monit. 2004 Jan;10(1):RA5-14
references.pmid: 14704647
references.citation: Biochim Biophys Acta. 2006 Oct;1761(10):1182-90
references.pmid: 16996794
references.citation: Neurobiol Dis. 2010 Jan;37(1):13-25
references.pmid: 19664713
references.citation: Mol Membr Biol. 2003 Jan-Mar;20(1):27-33
references.pmid: 12745923
references.citation: Annu Rev Biochem. 2002;71:537-92
references.pmid: 12045106
references.citation: Science. 2013 Jul 5;341(6141):74-6
references.pmid: 23704371
references.citation: Nature. 2010 Nov 25;468(7323):557-61
references.pmid: 20944627
references.citation: Int J Cancer. 2000 Jan 15;85(2):281-8
references.pmid: 10629090
references.citation: Nat Neurosci. 2006 Apr;9(4):487-8
references.pmid: 16565717
references.citation: Neurobiol Dis. 2004 Jun;16(1):1-13
references.pmid: 15207256
references.citation: J Neurochem. 2007 Apr;101(1):1-8
references.pmid: 17326770
references.citation: J Neurochem. 2004 May;89(3):788-800
references.pmid: 15086534
references.citation: J Biol Chem. 2007 Sep 28;282(39):28721-32
references.pmid: 17636261
references.citation: Curr Med Chem. 2013;20(1):56-63
references.pmid: 23151003