Interaction with PDZK1 Is Required for Expression of Organic Anion Transporting Protein 1A1 on the Hepatocyte Surface
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m503969200
ISSN1083-351X
AutoresPijun Wang, Jin J. Wang, Yansen Xiao, John W. Murray, Phyllis M. Novikoff, Ruth Hogue Angeletti, George A. Orr, Debin Lan, David L. Silver, Allan W. Wolkoff,
Tópico(s)Ion Transport and Channel Regulation
ResumoAlthough many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function. Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function. A major function of the hepatocyte is the removal of various xenobiotic and endogenous organic anionic compounds from the circulation. Sulfobromophthalein (BSP) 1The abbreviations used are: BSP, sulfobromophthalein; Oatp, organic anion transporting protein; SR-BI, scavenger receptor B type I; aa, amino acids; PBS, phosphate-buffered saline; MALDI, matrix-assisted laser desorption ionization; MS, mass spectroscopy; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is a model organic anion that circulates bound avidly to albumin and is extracted rapidly and efficiently by the hepatocyte (1Gartner U. Stockert R.J. Levine W.G. Wolkoff A.W. Gastroenterology. 1982; 83: 1163-1169Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 2Stollman Y.R. Gartner U. Theilmann L. Ohmi N. Wolkoff A.W. J. Clin. Investig. 1983; 72: 718-723Crossref PubMed Scopus (75) Google Scholar, 3Gartner U. Goeser T. Wolkoff A.W. Gastroenterology. 1997; 113: 1707-1713Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Establishment of a method to synthesize [35S]BSP of high specific activity (4Kurisu H. Nilprabhassorn P. Wolkoff A.W. Anal. Biochem. 1989; 179: 72-74Crossref PubMed Scopus (37) Google Scholar) facilitated studies to identify its hepatocyte transporter(s). Studies performed in a Xenopus laevis oocyte expression system (5Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. J. Clin. Investig. 1991; 88: 2146-2149Crossref PubMed Scopus (63) Google Scholar) identified a candidate transporter that was initially termed organic anion transporting polypeptide (Oatp). Since this initial description more than 20 additional members of the Oatp family have been described (6Hagenbuch B. Meier P.J. Biochim. Biophys. Acta. 2003; 1609: 1-18Crossref PubMed Scopus (744) Google Scholar, 7Hagenbuch B. Meier P.J. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 653-665Crossref PubMed Scopus (845) Google Scholar). The original protein was termed Oatp1 and, subsequently, Oatp1a1 (see Table I) in a proposal for standardization of nomenclature (7Hagenbuch B. Meier P.J. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 653-665Crossref PubMed Scopus (845) Google Scholar). Studies utilizing antisense knock-out of Oatp1a1 expression in Xenopus oocytes that had been injected with rat liver mRNA suggested that this protein is responsible for a substantial fraction of organic anion transport by the liver (8Hagenbuch B. Scharschmidt B.F. Meier P.J. Biochem. J. 1996; 316: 901-904Crossref PubMed Scopus (112) Google Scholar), although this remains to be validated by other methods.Table IMembers of the Oatp family that are found in rat, mouse, or human liverSpeciesOriginal nameNew nameNCBI accession numberC-terminal sequencePotential PDZ consensusPlasma membraneRatOatp1Oatp1a1NM_017111KTKLYesYesRatOatp2Oatp1a4NM_131906VTEDNoYesRatOatp4Oatp1b2NM_031650ETPLYesYesRatOatp9Oatp2b1NM_080786LQELNoNDaND, not determined.MouseOatp1Oatp1A1NM_013797KTKLYesYesMouseOatp2Oatp1a4NM_030687KTKLYesNDMouseOatp4Oatp1b2NM_020495ETPLYesNDHumanOATP-AOATP1A2NM_021094KTKLYesYesHumanOATP-COATP1B1NM_006446ETHCNoYesHumanOATP8OATP1B3NM_019844AAANNoYesHumanOATP-BOATP2B1NM_007256DSRVYesYesa ND, not determined. Open table in a new tab The family of organic anion transport proteins (Oatps) is characterized by a high degree of amino acid similarity as well as overlap of transported substrates, although their tissue distributions are varied (6Hagenbuch B. Meier P.J. Biochim. Biophys. Acta. 2003; 1609: 1-18Crossref PubMed Scopus (744) Google Scholar, 7Hagenbuch B. Meier P.J. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 653-665Crossref PubMed Scopus (845) Google Scholar). In addition, they have similar predicted membrane topologies and biochemical characteristics. Although evidence suggests that the Oatps are important in clearance of drugs from the circulation (6Hagenbuch B. Meier P.J. Biochim. Biophys. Acta. 2003; 1609: 1-18Crossref PubMed Scopus (744) Google Scholar, 7Hagenbuch B. Meier P.J. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 653-665Crossref PubMed Scopus (845) Google Scholar, 9Hata S. Wang P. Eftychiou N. Ananthanarayanan M. Batta A. Salen G. Pang K.S. Wolkoff A.W. Am. J. Physiol. Gastrointest. Liver Physiol. 2003; 285: 829-G83Crossref PubMed Scopus (79) Google Scholar), little is known regarding the mechanism by which they act, their oligomerization state, or mechanisms for subcellular trafficking. Of note is the fact that all of the Oatps that have been examined have distinct plasma membrane distributions, except for the prostaglandin transporters in which intracellular localization appears to predominate (10Bao Y. Pucci M.L. Chan B.S. Lu R. Ito S. Schuster V.L. Am. J. Physiol. Renal Physiol. 2002; 282: 1103-1110Crossref PubMed Scopus (97) Google Scholar). Examination of their C-terminal sequences reveals that many of the known members of the Oatp family have PDZ consensus binding sites (see Table I). The prostaglandin transporters are among the group of Oatps that lack a putative PDZ binding domain (11Kanai N. Lu R. Satriano J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (350) Google Scholar, 12Lu R. Kanai N. Bao Y. Schuster V.L. J. Clin. Investig. 1996; 98: 1142-1149Crossref PubMed Scopus (206) Google Scholar). Generally PDZ consensus binding sites are established by the sequence of the C-terminal four amino acids (13Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar, 14Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 15Kim E. Sheng M. Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1243) Google Scholar). Three classes of PDZ consensus binding sites have been described, relating these peptide sequences to the crystal structures of known PDZ domains to which they bind (14Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). The PDZ consensus sites that are present in the hepatic Oatps are all of Class I, defined by the sequence X(S/T)XΦ, where X is any amino acid, and Φ is a hydrophobic amino acid (14Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). A relatively large number of PDZ proteins have been described (14Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 15Kim E. Sheng M. Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1243) Google Scholar), although there is as yet no way to predict which if any will bind a particular protein with a PDZ consensus binding site. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of the Oatp family to examine whether interaction with a PDZ domain-containing protein provides a mechanism governing its subcellular localization. Oatp1a1 is located on the basolateral plasma membrane of the hepatocyte (16Bergwerk A.J. Shi X. Ford A.C. Kanai N. Jacquemin E. Burk R.D. Bai S. Novikoff P.M. Stieger B. Meier P.J. Schuster V.L. Wolkoff A.W. Am. J. Physiol. 1996; 271: G231-G238PubMed Google Scholar) as well as on the apical plasma membranes of the epithelial cells of the choroid plexus and the S3 segment of the renal proximal tubule (16Bergwerk A.J. Shi X. Ford A.C. Kanai N. Jacquemin E. Burk R.D. Bai S. Novikoff P.M. Stieger B. Meier P.J. Schuster V.L. Wolkoff A.W. Am. J. Physiol. 1996; 271: G231-G238PubMed Google Scholar, 17Angeletti R.H. Novikoff P.M. Juvvadi S. Fritschy J.-M. Meier P.J. Wolkoff A.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 283-286Crossref PubMed Scopus (128) Google Scholar, 18Angeletti R.H. Bergwerk A.J. Novikoff P.M. Wolkoff A.W. Am. J. Physiol. 1998; 275: C882-C887Crossref PubMed Google Scholar). Its terminal four amino acids (KTKL) are consistent with a type I PDZ binding motif (14Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar). There is also a mouse homolog of this protein (NM_013797) that is 81% identical to the rat Oatp1a1, and the C-terminal 11 amino acids of these two proteins are identical. Antibodies and Reagents—The antibody against the N terminus of Oatp1a1 (MEETEKKIATQEGRC) linked to KLH was prepared in rabbits by Covance Research Products Inc. (Denver, PA) as previously described (19Glavy J.S. Wu S.M. Wang P.J. Orr G.A. Wolkoff A.W. J. Biol. Chem. 2000; 275: 1479-1484Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This antibody recognizes Oatp1a1 in rat and mouse liver and was used for immunoblots at a 1:1000 dilution. An antibody specific to rat Oatp1a1, used in immunoblots at a dilution of 1:2500, was raised in rabbits to a KLH-linked peptide corresponding to 13 amino acids near the C terminus of rat Oatp1a1 (aa 646–658) as described previously (16Bergwerk A.J. Shi X. Ford A.C. Kanai N. Jacquemin E. Burk R.D. Bai S. Novikoff P.M. Stieger B. Meier P.J. Schuster V.L. Wolkoff A.W. Am. J. Physiol. 1996; 271: G231-G238PubMed Google Scholar). A rabbit antibody to Oatp1a4, used in immunoblots at a dilution of 1:10,000, was prepared to a KLH-linked peptide corresponding to the 11 C-terminal amino acids (aa 650–661) of the protein. Rabbit antibodies to scavenger receptor B type I (SR-BI) and PDZK1 were as previously described (20Silver D.L. J. Biol. Chem. 2002; 277: 34042-34047Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and were used in immunoblots at dilutions of 1:1000. A rabbit polyclonal antibody that recognizes the mouse asialoglycoprotein receptor was kindly provided by Dr. Richard Stockert for immunofluorescence studies. ECL reagent for Western blot analysis was obtained from PerkinElmer Life Sciences. Horseradish peroxidase (HRP)-conjugated affinity-purified goat anti-rabbit IgG and HRP-conjugated affinity-purified goat anti-mouse IgG were obtained from Jackson ImmunoResearch (West Grove, PA) and were used in immunoblots at dilutions of 1:50,000 and 1:10,000, respectively. 293T cells were obtained from Dr. Robert Burk (21Mohan S. Burk R.D. Oncogene. 2003; 22: 5270-5280Crossref PubMed Scopus (12) Google Scholar). All other reagents were obtained from Sigma unless otherwise noted. All animal procedures were approved by the university committees on animal use. Preparation of C-terminal Peptide Affinity Gel—The peptides CHGSPQVENDGELKTKL corresponding to the C-terminal 16 amino acids of rat Oatp1a1 and CHGSPQVENDGEL in which the last 4 amino acids were deleted were synthesized in The Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine. An additional cysteine residue was included at the N terminus to facilitate coupling to Ultralink Iodoacetyl Gel (Pierce) according to the manufacturer's instructions. Isolation of Peptide-binding Proteins from Rat Liver Cytosol—The liver was surgically removed from a rat under pentobarbital anesthesia and was immediately infused through the portal vein with 30 ml of ice-cold PBS. It was quickly weighed and Dounce-homogenized in PBS (10 ml/3 g of liver) containing protease inhibitors (Sigma, catalog # P-8340). After filtration through 3 layers of cheesecloth, the homogenate was centrifuged at 4 °C at 100,000 × g for 1 h. Supernatant (5 ml) was mixed with 1 ml of the peptide-coupled gel in a column and rotated overnight at 4 °C. The gel was then washed successively with 200 ml of ice-cold PBS, 10 ml of 0.5 m NaCl, 10 ml of 1 m NaCl, and 20 ml of PBS. Proteins remaining bound to the washed gel were eluted with sample buffer and subjected to 10% SDS-PAGE after which they were identified by Coomassie Blue or silver-staining. Mass Spectrometry Analysis—Coomassie Blue-stained bands were excised from SDS-polyacrylamide gels, destained with 0.2 m ammonium bicarbonate in 50% acetonitrile, and reduced using 20 mm tris (2-carboxyethyl)-phosphine-HCl, and free cysteine residues were alkylated with 55 mm iodoacetamide. After digestion for 16 h at 37 °C with 25 ng/μl sequencing grade modified trypsin (Promega), products were cleaned and concentrated using a C18 ZipTip (Millipore), mixed with 0.5 μl of 10 mg/ml 1-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% (v/v) trifluoroacetic acid, and applied onto a matrix-assisted laser desorption/ionization (MALDI) plate. Spectra were recorded with a PerSeptive Voyager-DE STR MALDI time-of-flight mass spectrometer operated in the reflection mode. The mass measurement accuracy with internal calibration was better than 100 ppm. The measured peptide masses were used for data base searching with ProFound algorithm (ProteoMetrics, NY) and Matrix Science (Mascot). For electrospray ionization MS/MS analysis, an APIQSTAR LC/MS/MS system (Applied Biosystems, Foster City, CA) was used. Fragment ion (tandem) mass spectra were obtained using collision-induced dissociation and analyzed using Matrix Science (Mascot) software. Immunoprecipitation of Rat Liver Membrane Extracts—Antiserum against the N-terminal peptide of Oatp1a1 was immunopurified with peptide coupled to Sulfolink-agarose (Pierce) according to the manufacturer's instructions. This purified antibody was covalently coupled to immobilized protein A-agarose (Sigma) by incubating for 1 h at room temperature in 40 mm dimethyl pimelimidate (Pierce) in 0.2 m triethanolamine buffer, pH 8.2. A 0.1 m Na2CO3 extracted rat liver pellet, highly enriched in Oatp1a1 (22Shi X. Bai S. Ford A.C. Burk R.D. Jacquemin E. Hagenbuch B. Meier P.J. Wolkoff A.W. J. Biol. Chem. 1995; 270: 25591-25595Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), was resuspended in PBS containing 1% Triton X-100 and protease inhibitors (0.1 mm leupeptin, 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.01 mm pepstatin A, 1 mm EDTA, and 0.01% sodium azide). After centrifugation at 100,000 × g for 1 h at 4 °C, the supernatant was immunoprecipitated with 50 μl of anti-Oatp1a1-protein A gel and washed with 1% Triton X-100 in PBS, and 200-μl fractions were eluted with 0.2 m glycine, pH 2.3, directly into 1 m Tris-base to neutralize the pH. The eluates were then subjected to Western blot analysis. Co-immunoprecipitation of Oatp1a1 and PDZK1 after Coexpression in 293T Cells—Oatp1a1 cDNA was excised from pSPORT-Oatp1a1 using the KpnI and NotI multicloning restriction enzyme sites (23Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (550) Google Scholar) and cloned into pCDNA3.1(+) (Invitrogen). PDZK1 cDNA was cloned into pFLAG-CMV-5c (Sigma) after PCR amplification from a pCDNA3.1/hygro-PDZK1 plasmid (20Silver D.L. J. Biol. Chem. 2002; 277: 34042-34047Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), resulting in a plasmid encoding PDZK1 with FLAG at its N terminus. Transient co-transfection of 293T cells with pCDNA3.1-Oatp1a1 and pFLAG-CMV/PDZK1 was performed using PolyFect transfection reagent (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Cells were harvested 2 days after transfection, washed with PBS, and incubated for 30–60 min on ice with PBS containing 1% CHAPS and protease inhibitors (Sigma #P-8340). The lysate was centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was incubated overnight at 4 °C with Oatp1a1-protein A affinity gel or anti-FLAG M2 affinity gel (Sigma) (25 μl of gel/4 × 106 cells). Each gel was washed with 1% CHAPS in PBS, incubated with SDS-PAGE sample buffer, and centrifuged, and the supernatant was subjected to Western blot analysis. A control study was also performed in which Oatp1a4 and FLAG-PDZK1 were cotransfected into 293T cells, which were then subjected to FLAG immunoprecipitation as described above. For these studies, Oatp1a4 cDNA was excised from a pCR2.1-Oatp1a4 plasmid that was provided by Dr. Richard Kim (24Cvetkovic M. Leake B. Fromm M.F. Wilkinson G.R. Kim R.B. Drug Metab. Dispos.. 1999; 27: 866-871PubMed Google Scholar). The Oatp1a4 insert was excised using KpnI- and XhoI-multicloning restriction enzyme sites and cloned into the pCDNA3.1/Zeo(–) plasmid. Identification of Oatp1a1-interacting PDZK1 Domains—As previously described, PDZK1 has four independent PDZ binding domains (25Ikemoto M. Arai H. Feng D. Tanaka K. Aoki J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar, 26Gisler S.M. Madjdpour C. Bacic D. Pribanic S. Taylor S.S. Biber J. Murer H. Kidney Int. 2003; 64: 1746-1754Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). pGEX6p-1 glutathione S-transferase expression plasmids (Amersham Biosciences) containing cDNAs encoding each of these domains were prepared using the pCDNA3.1/hygro-PDZK1 plasmid as the PCR template. For amplification of intact PDZK1 a sense primer (5′-ACTTAAGGATCCACAGAAATGGCCTCCACCTTCAAC-3′) and antisense primer (5′-ACGGGCCCTCTAGACTCGAG-3′) encoding BamHI and XhoI restriction sites, respectively, were used. For the first domain (aa 1–110) sense (5′-CGCGGATCCATGGCCTCCACCTTCAACCCCAGAGAGTGT-3′) and antisense (5′-CCGCTCGAGCTACCTCTGGCTTTGATCCAGCTCTTTCAA-3′) primers encoding BamHI and XhoI restriction sites, respectively, were used, and for domains 2–4 (aa 113–235, 221–343, or 356–519, respectively) the sense and antisense primers encoded EcoRI and XhoI restriction sites, respectively. The primers used for domain 2 are 5′-CCGGAATTCGCTCTGAATGATAAGAAACCGGGCCCTGGG-3′ (sense) and 5′-CCGCTCGAGTTACAAACTGGCTGTCTCCCTCTTGAATTGTGT-3′ (antisense). The primers used for domain 3 are 5′-CCGCAATTCAGTGAACAGAAGACACAATTCAAGAGGGAG-3′ (sense) and 5′-CCGCTCGAGTTATTCTTGACTTTGGCAGTAAAGAAGTGGAGA-3′ (antisense). The primers used for domain 4 are 5′-CCGGAATTCATCCCTGCTCCTCTGGAGGCCACAGGCTCA-3′ (sense) and 5′-CCGCTCGAGTCACATCTCCGTGTCTTCAGAGTTAGACGAAGA-3′ (antisense). The glutathione S-transferase fusion proteins were expressed in Escherichia coli (DH5α, Invitrogen) and affinity-purified using GSH-agarose gel (Sigma). Briefly, the bacteria were grown overnight in 5 ml of LB/ampicillin medium at 37 °C. The culture was then diluted into 50 ml of LB/ampicillin medium and grown at 37 °C until the A600 was 0.6–1 absorption units. Expression of fusion proteins was induced by the addition of 0.1 mm isopropyl 1-thio-β-d-galactopyranoside (Sigma) followed by an additional 4 h of incubation. The bacteria were harvested, resuspended in 1% Triton X-100 in PBS (5 ml/50 ml bacterial culture) containing protease inhibitor (Sigma catalog #P-8340), and sonicated on ice. The bacterial lysate was centrifuged at 12,000 rpm (Sorvall RC-5B centrifuge) for 15 min at 4 °C, and the supernatant was rotated with GSH-agarose gel overnight at 4 °C. The gel was washed several times with 1% Triton X-100 in PBS, resuspended in extracted rat liver membrane containing protease inhibitors as prepared above (5 mg membrane/15 μl of gel), and rotated overnight at 4 °C. The gel was washed with 1% Triton X-100 in PBS 5 times before the addition of SDS-PAGE sample buffer for Western blot analysis. Generation of PDZK1 Gene-targeted Mice—Mice in which exon 1 and part of intron 1 of the PDZK1 allele were replaced by the Neo cassette were prepared and bred in the Columbia University Transgenic Facility as described previously (27Lan D. Silver D.L. J. Biol. Chem. 2005; 280: 23390-23396Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Immunofluorescence Localization of PDZK1 in Liver—Male wild type or PDZK1 knock-out mice were anesthetized with ether, and the livers were removed and fixed by immersion for 3 h at 4 °C with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4, containing 7.5% sucrose and frozen sections (∼30 μm thick) were prepared (16Bergwerk A.J. Shi X. Ford A.C. Kanai N. Jacquemin E. Burk R.D. Bai S. Novikoff P.M. Stieger B. Meier P.J. Schuster V.L. Wolkoff A.W. Am. J. Physiol. 1996; 271: G231-G238PubMed Google Scholar). Sections were exposed overnight at 4 °C to anti-Oatp1a1, anti-asialoglycoprotein receptor, or anti-PDZK1 diluted 1:100 in PBS and, after rinsing in PBS, were exposed overnight at 4 °C to a 1:400 dilution of a Cy3-labeled donkey antibody to rabbit IgG (Jackson ImmunoResearch). Controls included examination of sections for autofluorescence after exposure to nonspecific primary antiserum. Wide field immunofluorescence images were captured with a 60× Olympus objective (1.4 NA) on an Olympus IX71 microscope. Rhodamine fluorescence was excited with a DG-4 (Sutter Instruments) xenon light source, and digital images were recorded on a Photometrics CoolSnap HQ CCD camera controlled by Metamorph imaging software (Universal Imaging Corp). For deconvolution, liver was optically sectioned every 0.25 μm using an MS-2000 automated piezoelectric x, y, z stage (Applied Scientific Instruments) for a total of 72 optical sections. Image stacks were deconvolved using the Metamorph "measured deconvolution" algorithm. For this, suboptical resolution fluorescent beads (PS-Spec, Molecular Probes) were sectioned in the z dimension under identical conditions as the liver. Bead stacks were assembled to generate a measured "point spread function," and this point spread function was used to optically deconvolve the stacks of liver fluorescence images. [35S]BSP Plasma Disappearance—[35S]BSP (1000 μCi/μmol) was synthesized as previously described (4Kurisu H. Nilprabhassorn P. Wolkoff A.W. Anal. Biochem. 1989; 179: 72-74Crossref PubMed Scopus (37) Google Scholar). Male wild type and PDZK1 knock-out mice were anesthetized with ketamine and injected retroorbitally with ∼140,000 cpm of [35S]BSP in 0.1 ml of PBS. Timed blood samples (∼40 μl) were obtained from the contralateral retroorbital sinus in heparinized capillary tubes, which were then centrifuged, and radioactivity in an aliquot of plasma was quantified. Plasma disappearance of radioactivity as a fraction of the injected dose was fit by a non-linear least squares algorithm (SigmaPlot v 6.1, SPSS, Inc., Chicago, IL) to the single exponential equation C(t) = ae–bt, where C(t) is the fraction of injected [35S]BSP/ml plasma at time t, a is the fraction of injected [35S]BSP/ml plasma at time 0, and b is the fractional disappearance rate in s–1. The volume of distribution of [35S]BSP was calculated as 1/a, and the serum half-life of [35S]BSP (t½) was calculated as ln (2)/b. Isolation and Identification of Rat Liver Cytosolic Proteins That Bind to the C-terminal Tail of Oatp1a1—For these experiments, a peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was covalently coupled to agarose gel. This peptide minus the last 4 amino acids that comprise the PDZ binding consensus domain was also coupled to agarose gel and used as a control. The columns were washed extensively with PBS as well as 1 m NaCl. Proteins bound to the washed gel were eluted with SDS-PAGE sample buffer and detected after SDS-PAGE by silver stain or staining with Coomassie Blue. A representative silver-stained SDS-PAGE gel is seen in Fig. 1a. A major protein band of ∼70 kDa was detected only in the material that was bound to the intact peptide (lane 1) and not to the peptide lacking the terminal 4 amino acids (lane 2). This band was visualized after Coomassie Blue staining of replicate gels and was excised. After reduction and alkylation, the gel slice was incubated overnight with trypsin. The resulting tryptic peptides were identified by MALDI mass spectrometry. A representative MALDI mass spectrum is shown in Fig. 1b. Data base analysis revealed a high correspondence of the observed peptide masses to those that would be obtained by tryptic digestion of rat PDZK1. These 64 possible tryptic peptides are indicated in Fig. 1c by alternating shading that has been applied to the PDZK1 sequence. After analysis of multiple MALDI spectra, 50 peptides corresponding to 66% of the protein sequence of PDZK1 were identified as indicated by the solid underlines in Fig. 1c. Identification as PDZK1 was confirmed by tandem MS/MS analysis. A representative MS/MS spectrum is shown in Fig. 1d, in which the sequence corresponding to the fifth PDZK1 tryptic peptide was identified. Masses in this figure are annotated using standard nomenclature as described (28Addona T. Clauser K. Coligan J.E. Dunn B.M. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley & Sons, Inc., Hoboken, New Jersey2005Google Scholar). Utilizing tandem MS/MS, the sequences of 27 peptides corresponding to 54% of the PDZK1 sequence were identified and are indicated by the broken underlines in Fig. 1c. Together, the two methods identified 93% of the PDZK1 sequence (Fig. 1c). In addition, identification of this protein as PDZK1 was confirmed by immunoblot using a peptide-specific antibody (data not shown). Interaction of Oatp1a1 and PDZK1 in Cells and Rat Liver— These studies showed that the C-terminal four amino acids of Oatp1a1 are necessary for interaction with PDZK1. However, these results are not predictive as to whether this interaction between Oatp1a1 and PDZK1 actually occurs in vivo. Although several proteins have been shown to bind to PDZK1 under in vitro conditions, the functional significance of this interaction has not always been clear (29Kato Y. Yoshida K. Watanabe C. Sai Y. Tsuji A. Pharm. Res. (N. Y.). 2004; 21: 1886-1894Crossref PubMed Scopus (63) Google Scholar, 30Capuano P. Bacic D. Stange G. Hernando N. Kaissling B. Pal R. Kocher O. Biber J. Wagner C.A. Murer H. Pfluegers Arch. Eur. J. Physiol. 2005; 449: 392-402Crossref PubMed Scopus (69) Google Scholar). To assess whether the interaction found with the C-terminal peptide occurs with full-length proteins expressed in cells, 293T cells were transfected with expression plasmids encoding Oatp1a1 and FLAG-PDZK1. Immunoprecipitation was performed with FLAG antibody or an antibody raised to the N terminus of Oatp1a1 that would not be expected to interfere with interaction of the C terminus with PDZK1. Western blot analysis of the FLAG immunoprecipitate with anti-Oatp1a1 revealed the presence of Oatp1a1 (Fig. 2a, left panel), and Western blot analysis of the Oatp1a1 immunoprecipitate revealed the presence of FLAG-PDZK1 (Fig. 2a, right panel). There was no product detected in the immunoprecipitate after cotransfection of either expression plasmid with
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