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• W2034975029 abstract "The epidermal growth factor (EGF) receptor is located predominantly in the basolateral membrane of polarized epithelia, where it plays a pivotal role during organogenesis and tissue homeostasis. We have shown previously that a 22-amino acid sequence in the EGF receptor juxtamembrane domain contains autonomous sorting information necessary for basolateral localization using the Madin-Darby canine kidney epithelial cell model. The goal of this study was to determine the molecular basis of EGF receptor basolateral membrane expression using site-directed mutagenesis to modify specific residues in this region. We now show that this sequence has two different, functionally redundant basolateral sorting signals with distinct amino acid requirements: one dependent on residues658LL659 conforming to well-characterized leucine-based sorting signals, and a second containing a polyproline core comprising residues Pro667 and Pro670(667PXXP670). Our data also suggest that Arg662 contributes to the function of the proline-based signal. 667PXXP670was the dominant signal when both motifs were present and was more effective than 658LL659 at overriding strong apical sorting signals located in the same molecule. Site-directed mutations at Arg662, Pro667, and Pro670 were also associated with increased apical expression of full-length EGF receptors, demonstrating for the first time that the juxtamembrane region is necessary for accurate polarized expression of the native molecule. The epidermal growth factor (EGF) receptor is located predominantly in the basolateral membrane of polarized epithelia, where it plays a pivotal role during organogenesis and tissue homeostasis. We have shown previously that a 22-amino acid sequence in the EGF receptor juxtamembrane domain contains autonomous sorting information necessary for basolateral localization using the Madin-Darby canine kidney epithelial cell model. The goal of this study was to determine the molecular basis of EGF receptor basolateral membrane expression using site-directed mutagenesis to modify specific residues in this region. We now show that this sequence has two different, functionally redundant basolateral sorting signals with distinct amino acid requirements: one dependent on residues658LL659 conforming to well-characterized leucine-based sorting signals, and a second containing a polyproline core comprising residues Pro667 and Pro670(667PXXP670). Our data also suggest that Arg662 contributes to the function of the proline-based signal. 667PXXP670was the dominant signal when both motifs were present and was more effective than 658LL659 at overriding strong apical sorting signals located in the same molecule. Site-directed mutations at Arg662, Pro667, and Pro670 were also associated with increased apical expression of full-length EGF receptors, demonstrating for the first time that the juxtamembrane region is necessary for accurate polarized expression of the native molecule. glycophosphatidylinositol adaptor protein trans-Golgi network epidermal growth factor EGF receptor Madin-Darby canine kidney minimal essential medium fetal bovine serum Chinese hamster ovary cytomegalovirus fluorescein isothiocyanate confocal laser scanning microscopy decay accelerating factor The ability to establish and maintain plasma membrane asymmetry is a fundamental property of polarized epithelial cells that form physical barriers between various body compartments (reviewed in Refs. 1Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (815) Google Scholar and 2Matlin K.S. Caplan M.J. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. Raven Press, New York1992: 447-473Google Scholar). Epithelial cell plasma membranes are organized into distinct apical and basolateral domains, which face the lumen or underlying cells and connective tissue, respectively. Each domain's unique molecular composition facilitates a wide variety of organ-specific functions, including vectorial transport and compartmentalized cell signaling (1Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (815) Google Scholar, 2Matlin K.S. Caplan M.J. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. Raven Press, New York1992: 447-473Google Scholar, 3Kim S.K. Curr. Opin. Cell Biol. 1997; 9: 853-859Crossref PubMed Scopus (106) Google Scholar). The dynamic nature of plasma membrane asymmetry allows for epithelial cell plasticity and the ability to respond to a variety of physiological cues. Alterations in membrane polarity are often associated with epithelial cell dysfunction and pathophysiology, stressing the central role of spatial organization in normal cell function (reviewed in Ref. 4Fish E.M. Molitoris B.A. New Engl. J. Med. 1994; 330: 1580-1588Crossref PubMed Scopus (194) Google Scholar). Epithelial membrane asymmetry is due in part to signal-mediated domain-selective membrane protein sorting from intracellular compartments (reviewed in Refs. 5Mays R.W. Beck K.A. Nelson W.J. Curr. Biol. 1994; 6: 16-24Crossref Scopus (136) Google Scholar, 6Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (391) Google Scholar, 7Mostov K.E. Cardone M.H. Bioessays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar). A number of relatively diverse apical sorting signals have now been identified, including glycophosphatidylinositol (GPI)1 membrane anchors (8Brown D.A. Crise B. Rose J.K. Science. 1989; 245: 1499-1501Crossref PubMed Scopus (304) Google Scholar,9Lisanti M. Sargiacomo M. Graeve L. Saltiel A. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9557-9561Crossref PubMed Scopus (289) Google Scholar), ectodomain glycans (10Scheiffele P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar, 11Yeaman C., Le Gall A.H. Baldwin A.N. Monlauzeur L., Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (246) Google Scholar), and transmembrane or ectodomain amino acid sequences (12Alonzo M.A. Fan L. Alarcon B. J. Biol. Chem. 1997; 272: 30748-30752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Basolateral sorting signals are generally composed of relatively short cytoplasmic amino acid sequences (6Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (391) Google Scholar, 7Mostov K.E. Cardone M.H. Bioessays. 1995; 17: 129-138Crossref PubMed Scopus (118) Google Scholar), and many are related to tyrosine (13Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 14Thomas D.C. Brewer C.B. Roth M.G. J. Biol. Chem. 1993; 268: 3313-3320Abstract Full Text PDF PubMed Google Scholar)- or leucine (15Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2969Crossref PubMed Scopus (220) Google Scholar, 16Odorizzi G. Trowbridge I.S. J. Biol. Chem. 1997; 272: 11757-11762Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar)-based signals originally identified as clathrin-coated membrane localization signals (reviewed in Refs. 17Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (703) Google Scholar and 18Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar). Tyrosine- and leucine-based signals are linked to clathrin via interactions with specific clathrin adaptor protein (AP) subunits (reviewed in Refs. 19Robinson M.S. Trends Cell Biol. 1997; 7: 99-102Abstract Full Text PDF PubMed Scopus (123) Google Scholar and 20Marks M.S. Ohno H. Kirchhausen T. Bonifacino J.S. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). The three major classes of mammalian APs are AP-1 found predominantly at thetrans-Golgi network (TGN), AP-2 at the plasma membrane, and AP-3 at the TGN and endosomes (19Robinson M.S. Trends Cell Biol. 1997; 7: 99-102Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 21Pearse B. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar, 22Odorizzi G. Cowles C.R. Emr S.D. Trends Cell Biol. 1998; 8: 282-288Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Although a role for AP-facilitated transport in clathrin-dependent pathways is well-established (19Robinson M.S. Trends Cell Biol. 1997; 7: 99-102Abstract Full Text PDF PubMed Scopus (123) Google Scholar), it is only recently that these molecules have been implicated in basolateral transport at the TGN (23Futter C.E. Gibson A. Allchin E.H. Maxwell S. Ruddock L.J. Odorizzi G. Domingo D. Trowbridge I.S. Hopkins C.R. J. Cell Biol. 1998; 141: 611-623Crossref PubMed Scopus (193) Google Scholar, 24Orzech E. Schlessinger K. Weiss A. Okamoto C.T. Aroeti B. J. Cell Biol. 1999; 274: 2201-2215Scopus (40) Google Scholar). Other basolateral sorting signals have been identified, however, that bear no relation to known clathrin-coated membrane localization motifs. Other than clusters of essential charged amino acids, most of the signals in this category are devoid of recognizable motifs that might offer clues regarding physiologically relevant protein-protein interactions (13Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar,25Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell Biol. 1993; 123: 1149-1160Crossref PubMed Scopus (118) Google Scholar, 26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The EGF receptor is located in the basolateral membrane in many different epithelial cell types (reviewed in Refs. 1Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (815) Google Scholar and 27Simons K. Fuller S.D. Annu. Rev. Cell Biol. 1985; 1: 243-288Crossref PubMed Scopus (565) Google Scholar). Nowhere is the importance of restricted basolateral EGF receptor expression more apparent than in polycystic kidney disease (28Wilson P.D. Sherwood A.C. Kidney Int. 1991; 39: 450-463Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 29Orellana S.A. Avner E.D. Semin. Nephrol. 1995; 15: 341-352PubMed Google Scholar, 30Grantham J.J. Am. J. Kidney Dis. 1996; 28: 788-803Abstract Full Text PDF PubMed Scopus (121) Google Scholar), where apically mislocalized receptors have a major role in disease progression (31Richards W.G. Sweeney W.E. Yoder B.K. Wilkinson J.E. Woychik R.P. Avner E.D. J. Clin. Invest. 1998; 101: 935-939Crossref PubMed Scopus (173) Google Scholar, 32Sweeney W.E. Chen Y. Nakanishi K. Frost P. Avner E.D. Kidney Int. 2000; 57: 33-40Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). We have shown previously that newly synthesized EGF receptors are delivered directly to the basolateral surface in the MDCK cell model (33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar) and have identified sorting information located between juxtamembrane domain residues Lys652 and Ala674 necessary for domain-specific targeting of cytoplasmically truncated receptors (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) (see Fig. 1 A). Importantly, this same sequence mediates basolateral targeting when transplanted to a heterologous reporter molecule, proof that is an autonomous, dominant signal (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Residues Lys652 through Ala674 lack critical tyrosine residues (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and do not overlap any of the EGF receptor sorting signals responsible for clathrin-mediated internalization located in the carboxyl terminus (34Chang C.P. Lazar C. Walsh B. Kouro M. Wiley H.S. Gill G.N. J. Biol. Chem. 1993; 268: 19312-19320Abstract Full Text PDF PubMed Google Scholar). In addition to MDCK cells, EGF receptors also exhibit a predominantly basolateral localization in LLC-PK1 cells (35Mullin J.M. McGinn M.T. Cancer Res. 1988; 48: 4886-4891PubMed Google Scholar, 36Kuwada S.K. Lund K.A., Li, X.F. Cliften P. Amsler K. Opresko L.K. Wiley H.S. Am. J. Physiol. 1998; 275: C1419-C1428Crossref PubMed Google Scholar), which lack a novel epithelial cell-specific AP-1 subunit isoform necessary for correctly sorting basolateral membrane cargo with AP-dependent sorting signals (37Ohno H. Tomemori T. Nakatsu F. Okazaki Y. Aguilar R.C. Foelsch H. Mellman I. Saito T. Shirasawa T. Bonifacino J.S. FEBS Lett. 1999; 449: 215-220Crossref PubMed Scopus (215) Google Scholar). Hence, polarized EGF receptor sorting from internal compartments is likely mediated by novel protein-protein interactions. Data presented in this study show that the EGF receptor juxtamembrane domain has a hierarchy of functionally redundant basolateral membrane localization signals with distinct amino acid requirements. Madin-Darby canine kidney (MDCK) epithelial cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 2 mm glutamine. MDCK cells were seeded on polycarbonate Transwell filter inserts (0.4-μm pore size) (Costar Corp., Cambridge, MA) at a density of 5 × 105 cells per 12-mm filter, or 5 × 106cells per 75-mm filter, to generate electrically resistant monolayers with well-developed tight junctions suitable for domain-specific assays 4–6 days later (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar). Chinese hamster ovary (CHO) cells were maintained in the alpha formulation of MEM supplemented with 10% FBS and 2 mm glutamine. Procedures for replacing EGF receptor residues Lys652, Leu664, and Pro675 with premature stop codons followed by a restriction site compatible with the polylinker in the eukaryotic expression plasmid pCB6+ have been described previously (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar). Cytoplasmically truncated receptors are named based on the carboxyl-terminal amino acid in the EGF receptor-coding region (e.g. c′-674 has a P675STOP substitution). Amino acid substitutions in the context of a c′-674 receptor were made using PCR and a full-length EGF receptor cDNA/pCB6+ template (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar). The forward primer, 5′-TGCGTCTCTTGCCGGAATGTCA-3′, was designed to anneal upstream to a BsmI restriction site located at EGF receptor nucleotide 1792, and reverse mutagenic primers (listed below) to introduce specific amino acid substitutions (bold italics), a P675STOP (bold), and an EcoRV site (underlined) orXbaI site (double underlined) compatible with the pCB6+ polylinker. The mutagenic primers were: R662T, 5′-TCTCTGATATCATCAAGCTTCTCCACTGGGTGTTAGCGGCTCAACCAACTCCGTCTCCTGCAG-3′; E663A, 5′-TCTCTGATATCATCAAGCTTTCCACTGGGTGTTAGCGGCTCAACCAACGCCCTCTCCTGC-3′; E666A, 5′-CTCTCTGATATC TCAAGCCTCTCCACTGGGTGTAAGCGGCGCCACAAGCTCCC-3′; P667A, 5′-AGAATCGATATCATCAAGCCTCTCCACTGGGTGTAAGAGCCTCCACAAGC-3′; P670A, 5′-AGAATCGATATCATCAGGCTTCCCCACTGGCTGTAAGAGGCTC-3′; and P667A,P670A (also called P675STOP-PxxP-2xA), 5′-GTTCG TCTAGA¯¯ TCAAGCTTCTCCACTGGCTGTAAGAGCCTCCACAAG-3′. An L658A,L659A substitution in a L664STOP background (L664STOP-LL-2xA) was made using the same forward primer, a reverse mutagenic primer 5′-GTTCG TCTAGA¯¯ TCACTCCCTCTCCTG-3′, and a cDNA with a L658A, L659A substitution in c′-674 as a template (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A compound mutation with six alanine substitutions (L658A,L659A,L664A,V665A,P667A, P670A) in a P675STOP background (P675STOP-LL,LV,PxxP-6xA) was made using the same forward primer, a reverse mutagenic primer 5′-CTTCG TCTAGA¯¯ TCAAGCTTCTCCGCTGGCTGTAAGTGCCTCCGCTGC-3′, and an L658A,L659A,L664A,V665A substitution in a P675STOP background as a template (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). PCR products were gel-purified, digested atBsmI and EcoRV or XbaI sites incorporated at the ends, and ligated to pCB6+ containing a cDNA encoding cytoplasmically truncated c′-697 receptors digested with the same restriction enzymes. Additional EGF receptor mutations studied in the context of a c′-674 receptor have been described elsewhere (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Point mutations were introduced into the full-length EGF receptor using a cDNA template cloned in pBK-CMVΔlacΔDraIII. pBK-CMVΔlacΔDraIII is a derivative of a pBK-CMV phagemid (Stratagene Cloning Systems, La Jolla, CA), created by deleting nucleotides 1098–1299 in the inducible lac promoter (38Kil S.J. Hobert M.E. Carlin C. J. Biol. Chem. 1999; 274: 3141-3150Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and by eliminating a unique DraIII site at nucleotide 240 in the f1 (−) origin of replication. Neither modification affected kanamycin-resistant growth in Escherichia coli. Sequences were amplified using the same forward primer described in the previous paragraph and one of the following reverse mutagenic primers designed to introduce a specific amino acid substitution (bold italics), aDraIII site (underlined) to facilitate subcloning, and a silent SacI site (double underlined) to facilitate recombinant screening without changing amino acid sequence: R662T, 5′-CTTCTCCACTGGGTGTAAGTGGCTCCAC GAGCTC¯¯ AGTCTCCTGCAGC-3′; P667A, 5′-CTTCTCCACTGGGTGTAAGTGCCTCCAC GAGCTC¯¯CC-3′; and P670L, 5′-CTTCTCCACTGAGTGTAAGAGGCTCCAC GAGCTC¯¯CC-3′. PCR products were digested at an EcoNI site located at EGFR nucleotide 1876 and the DraIII site introduced at the 3′-end of the PCR products, and ligated to full-length EGF receptor sequences cloned in pBK-CMVΔlacΔDraIII digested with the same enzymes. When sequenced, the recombinant products had an internal deletion, due to a second previously unrecognized EcoNI site located 5′ to the nucleotide 1876 EcoNI site. To create full-length products, recombinant molecules were digested with BstXI andXbaI, liberating an 1836-nucleotide product encoding the carboxyl half of the full-length EGF receptor, including the mutation. These fragments were ligated to a 2100-nucleotideXhoI-BxtXI fragment encoding the amino half of the wild-type EGF receptor, including the sequence deleted in the initial recombinants, and pBK-CMVΔlacΔDraIII digested atXhoI and XbaI sites located at 5′ and 3′ sites, respectively, in the polylinker. An E673A substitution was made using a forward mutagenic primer 5′-CTCTTACACCCAGTGGAGCAGCACCCAACCAAGC-3′, designed to anneal to a DraIII site (underlined) located at EGF receptor nucleotide 2284 and to introduce the amino acid substitution (bold italics). The reverse primer, 5′-CAAACGGTCACCCCGTAGCTCCA GACGTC¯¯ACTCTCTGGT-3′, was designed to anneal to a BstEII site (underlined) located at EGF receptor nucleotide 2889, and to introduce a silentAatII site (double underlined) for recombinant screening. PCR products were digested with DraIII and BstEII and ligated to full-length EGF receptor sequences cloned in pBK-CMVΔlacΔDraIII digested with the same enzymes. cDNAs encoding the extracellular domain of decay accelerating factor (DAFex), and DAFex linked to EGF receptor transmembrane and cytoplasmic sequences to residue 651 (DAF-651) and to residue 674 (DAF-674), cloned in pCB6+ have been described previously. DAF-663 was made using a forward primer 5′-GTATCTCGAGGGCTGTCCAAC-3′, designed to anneal at the 5′-end of sequences encoding the EGF receptor transmembrane domain, and to incorporate a silent XhoI site (underlined) without altering the amino acid coding sequence for EGF receptor residues Leu609 and Glu610 (bold italics); and a reverse mutagenic primer 5′-GTTCG TCTAGA¯¯ TCACTCCCTCTCCTG-3′, incorporating a stop code in place of the code for Glu-663 (bold) and an XbaI site (double underlined) compatible with the pCB6+ polylinker. DAF-674 with a P667A substitution was made using the same forward primer and a reverse mutagenic primer, 5′-GCGATATC TCAAGCTTCTCCACTGGGTGTAAGTGCCTC-3′, which was designed to introduce a P667A substitution (bold italics), a Pro675STOP (bold), and an EcoRV site (underlined) compatible with the pCB6+ polylinker. PCR products were digested withXhoI and XbaI or EcoRV and ligated directly to pCB6+/DAFex digested with the same restriction enzymes. PCR primers were designed using the DNASTAR software package (DNASTAR, Inc., Madison, WI). PCR amplifications were carried out using a RoboCycler 40 temperature cycler (Stratagene Cloning Systems, La Jolla, CA). All PCR products and religated recombinant products were sequenced by automated DNA sequencing (Cleveland Genomics, Cleveland, OH). Sequences were verified by analysis of genomic DNA recovered from permanent clonal cell lines. CHO cells were seeded at a density of ∼2 × 106 cells/100-mm tissue culture dish and transfected using DNA mixed with FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) 5 h later. Filter-grown MDCK cells were transfected using DNA mixed with Lipofectin transfection reagent (Invitrogen, Rockville, MD) added to both sides of the filter. Both cell types were assayed for transient membrane domain-specific protein expression 40–48 h post-transfection. Exogenously expressed human EGF receptors are readily detected in both cell types in CHO cells, because they lack endogenous receptors (39Livneh E. Prywes R. Kashles O. Reiss N. Sasson I., Y. Mory A.U. Schlessinger J. J. Biol. Chem. 1986; 261: 12490-12497Abstract Full Text PDF PubMed Google Scholar), and in MDCK cells, because exogenously expressed human receptors can be distinguished from endogenous canine receptors with species-specific antibodies (33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar). To make permanent MDCK cell lines, cells seeded at a density of ∼7 × 105 cells/100-mm tissue culture dish were transfected with DNA-Lipofectin complexes as described previously (33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar). Cells that had been grown in selection medium containing G418 (0.8 mg/ml Geneticin, Invitrogen) for 10–14 days were labeled with the monoclonal antibody EGF-R1, specific for an external human EGF receptor peptide epitope (40Waterfield M.D. Mayes E.L.V. Stroobant P. Bennet P.L.P. Young S. Goodfellow P.N. Banting G.S. Ozanne B. J. Cell. Biochem. 1982; 20: 149-161Crossref PubMed Scopus (276) Google Scholar, 41Carlin C.R. Knowles B.B. J. Biol. Chem. 1984; 259: 7902-7908Abstract Full Text PDF PubMed Google Scholar), followed by FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), for enrichment by sterile sorting on a flow cytometer (Cytofluorograph IIs, Ortho Instruments, Westwood, MA). Cells were rinsed twice and then preincubated in methionine and cysteine-free medium for up to 1 h, before metabolic labeling with [35S]Express Protein Labeling Mix (1175 Ci/mmol, PerkinElmer Life Sciences, Wilmington, DE) diluted in the amino acid-deficient medium supplemented with 10% dialyzed FBS and 0.2% BSA. Filter-grown MDCK cells were labeled from the basolateral surface. Labeling medium was replaced with complete MEM supplemented with a 10-fold excess of non-radioactive methionine and cysteine (chase medium), and cells were incubated for additional periods of time. Cells were lysed with 1% (w/v) Triton X-100 in 0.1 m Tris, pH 7.4, supplemented with 2 mm EDTA, 1 mm EGTA, 0.2 mm phenylmethylsulfonyl fluoride, and 1 μm leupeptin, for immunoprecipitation with antibodies recognizing external epitopes in the human EGF receptor (EGF-R1) or DAF (gift of Ed Medof, Case Western Reserve University) absorbed to protein A-Sepharose CL-4B beads (Sigma Chemical Co., St. Louis, MO). After extensive washing, immunoprecipitates were solubilized with Laemmli buffer for SDS-PAGE (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207084) Google Scholar), and fluorographic detection. Radioactive quantitation was carried out by Phosphorstorage autoradiography (Molecular Dynamics Inc., Sunnyvale, CA). Filter-grown MDCK cells that had been metabolically labeled were rinsed twice with ice-cold MEM supplemented with 25 mm HEPES, pH 7.4, and 1% BSA (M/H/B) and then incubated with EGF-R1 ascites (10 μl/ml) added to one side of the filter for 1 h on ice. Cells were rinsed four times with PBS supplemented with 1% BSA and then lysed with 1% (w/v) Nonidet P-40 in 0.1 m Tris, pH 6.8, supplemented with 15% (w/v) glycerol, 2 mm EDTA, 1 mm EGTA, and protease inhibitors. Cell lysates were added directly to protein A-Sepharose beads to capture surface-exposed EGF receptors, and immunoprecipitates were washed, solubilized, and resolved by SDS-PAGE exactly as described in the previous section. Filter-grown cells were rinsed three times with ice-cold MEM supplemented with 0.2% BSA and then incubated with ∼10 nm125I-EGF for 2 h at 4 °C. Receptor-grade mouse EGF (Toyobo Biochemicals, Osaka, Japan) was labeled with 125I (carrier-free, >350 mCi/ml, PerkinElmer Life Sciences) using chloramine-T. Cells were rinsed three times with the MEM/BSA solution and then incubated with 2 mmdisuccinimidyl suberate (Pierce Chemical Co., Rockford, IL) in a solution of 0.1 m HEPES, pH 7.4, supplemented with 120 mm NaCl, 50 mm KCl, 8 mm glucose, and 1.2 mm MgSO4 for 15 min at room temperature. The reaction was quenched by a 5-min incubation with 0.05m Tris, pH 7.4, at room temperature. Cells were lysed with 1% Nonidet P-40 exactly as described above, and equal aliquots of total cell protein were separated by SDS-PAGE. Filter-grown cells were rinsed three times with PBS and fixed with 3% paraformaldehyde in PBS for 10 min at room temperature. Some cells were stained with antibodies to external epitopes added to the apical or basolateral surface of non-permeabilized cells. Others were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and then stained with the ZO-1-specific rat monoclonal R26.4C antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or a β-catenin-specific mouse monoclonal antibody (Transduction Laboratories, Lexington, KY). Secondary antibodies were fluorophore-conjugated, species-specific Fab fragments that had been solid-phase absorbed to prevent cross-reactivity with primary antibodies made in other species (Jackson ImmunoResearch Laboratories, Inc., Fort Washington, PA). Cells were stained with primary or secondary antibodies for 1 h each at 37 °C. Antibodies were diluted in PBS supplemented with 3% radioimmunoassay-grade bovine serum albumin, and cells were blocked with a solution containing 5% normal serum from the host animal used to generate the secondary antibody between incubations with primary and secondary antibodies. Filters were excised from plastic inserts and placed cell-side up on a glass slide. Coverslips were mounted on the cells using SlowFade anti-fade solution from Molecular Probes (Eugene, OR). Cells were examined with a Zeiss LSM 410 scanning laser confocal microscope (Zeiss, Gottingen, Germany) using the 488/568-nm wavelength lines of an argon-krypton laser. Cells were optically sectioned every 0.5 μm. Image resolution using a Zeiss 100× Neofluor objective and Zeiss LSM software was 512 × 512 pixels. The amino acid sequence of the originally identified a 22-amino acid juxtamembrane domain necessary for basolateral localization of cytoplasmically truncated EGF receptors is shown in Fig.1 A (26Hobert M.E. Kil S. Carlin C.R. J. Biol. Chem. 1997; 272: 32901-32909Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Hobert M. Carlin C. J. Cell. Physiol. 1995; 162: 434-446Crossref PubMed Scopus (51) Google Scholar). We sought to characterize the molecular basis of basolateral localization, by focusing on several consensus amino acid protein interaction motifs located in this region. Examination of the sequence revealed the existence of a two consensus leucine-based motifs, located at Leu658-Leu659 and Leu664-Val665 (Fig. 1 A). Although predominantly characterized in endocytic pathways (reviewed in Ref.18Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar), leucine-based motifs are implicated in basolateral sorting of at least two other membrane proteins (15Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2969Crossref PubMed Scopus (220) Google Scholar, 16Odorizzi G. Trowbridge I.S. J. Biol. Chem. 1997; 272: 11757-11762Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) (also see TableI). Because leucine-based signals are impaired by dialanine substitution (18Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar), we changed each of the leucine-based signals to alanines, either individually (P675STOP-LL-2xA or P675STOP-LV-2xA) or" @default.
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• W2034975029 date "2002-10-01" @default.
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