The Cytoplasmic Tail of CD1d Contains Two Overlapping Basolateral Sorting Signals

Abstract

CD1d is a member of the CD1 polypeptide family that represents a new arm of host defense against invading pathogens. In our previous work (Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P., and Bakke, O. (1999) J. Immunol. 162, 1488–1495) we have shown that CD1d contained a classic tyrosine-based internalization signal (YQGV) in its short cytoplasmic tail. CD1d is expressed in polarized epithelial cells, and we found that the cytoplasmic tail of CD1d also contained information for basolateral sorting. Interestingly, a mutation of the critical tyrosine residue of the endosomal sorting signal did not result in the loss of basolateral targeting of the mutant CD1d. To search for a basolateral sorting signal we have constructed a full set of alanine mutants, but no single alanine substitution inactivated the signal. However, deletions or mutations of either the C-terminal valine/leucine pair or the critical tyrosine residue from the internalization signal and either residue from the C-terminal valine/leucine pair inactivated basolateral sorting. Our data thus suggest that the cytoplasmic tail contains two overlapping basolateral signals, one tyrosine- and the other leucine-based, each being sufficient to direct CD1d to the basolateral membrane of polarized Madin-Darby canine kidney cells. CD1d is a member of the CD1 polypeptide family that represents a new arm of host defense against invading pathogens. In our previous work (Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P., and Bakke, O. (1999) J. Immunol. 162, 1488–1495) we have shown that CD1d contained a classic tyrosine-based internalization signal (YQGV) in its short cytoplasmic tail. CD1d is expressed in polarized epithelial cells, and we found that the cytoplasmic tail of CD1d also contained information for basolateral sorting. Interestingly, a mutation of the critical tyrosine residue of the endosomal sorting signal did not result in the loss of basolateral targeting of the mutant CD1d. To search for a basolateral sorting signal we have constructed a full set of alanine mutants, but no single alanine substitution inactivated the signal. However, deletions or mutations of either the C-terminal valine/leucine pair or the critical tyrosine residue from the internalization signal and either residue from the C-terminal valine/leucine pair inactivated basolateral sorting. Our data thus suggest that the cytoplasmic tail contains two overlapping basolateral signals, one tyrosine- and the other leucine-based, each being sufficient to direct CD1d to the basolateral membrane of polarized Madin-Darby canine kidney cells. Madin-Darby canine kidney polymerase chain reaction adaptor protein complex Dulbecco's modified Eagle's medium trans-Golgi network CD1 polypeptides, evolutionarily related to the major histocompatibility complex class I molecules, represent a new class of antigen-presenting molecules that bind and present lipids and glycolipids rather than peptide antigens and are implicated in host defense against invading pathogens (for review, see Refs. 1.Porcelli S.A. Brenner M.B. Curr. Biol. 1997; 7: R508-R511Abstract Full Text Full Text PDF PubMed Google Scholar, 2.Nordeng T.W. Gorvel J.-P. Bakke O. Curr. Top. Microbiol. Immunol. 1998; 232: 179-215PubMed Google Scholar, 3.Sugita M. Moody D.B. Jackman R.M. Grant E.P. Rosat J.P. Behar S.M. Peters P.J. Porcelli S.A. Brenner M.B. Clin. Immunol. Immunopathol. 1998; 87: 8-14Crossref PubMed Scopus (33) Google Scholar, 4.Jackman R.M. Moody D.B. Porcelli S.A. Crit. Rev. Immunol. 1999; 19: 49-63PubMed Google Scholar). CD1d has been reported to present glycolipid antigens such as α-galactosylceramide to the CD1d-restricted natural killer T cells (5.Brossay L. Chioda M. Burdin N. Koezuka Y. Casorati G. Dellabona P. Kronenberg M. J. Exp. Med. 1998; 188: 1521-1528Crossref PubMed Scopus (560) Google Scholar, 6.Spada F.M. Koezuka Y. Porcelli S.A. J. Exp. Med. 1998; 188: 1529-1534Crossref PubMed Scopus (402) Google Scholar, 7.Eberl G. Lees R. Smiley S.T. Taniguchi M. Grusby M.J. MacDonald H.R. J. Immunol. 1999; 162: 6410-6419PubMed Google Scholar, 8.Singh N. Hong S. Scherer D.C. Serizawa I. Burdin N. Kronenberg M. Koezuka Y. Van Kaer L. J. Immunol. 1999; 163: 2373-2377PubMed Google Scholar), but recent data demonstrate that CD1d may interact with a broader array of T cells (9.Behar S.M. Podrebarac T.A. Roy C.J. Wang C.R. Brenner M.B. J. Immunol. 1999; 162: 161-167PubMed Google Scholar). We have previously studied the mechanisms of intracellular trafficking of CD1d in MDCK1 cells and found that the short cytoplasmic tail of CD1d was important for its internalization and basolateral sorting (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar). Our results showed that CD1d contained a classical tyrosine-based internalization signal in its cytoplasmic tail. Replacing either the tyrosine or the hydrophobic valine residue in the +3 position from the tyrosine residue with alanine resulted in a loss of active internalization. However, alanine substitution of neither the critical tyrosine nor the valine disrupted the basolateral distribution of CD1d. Nonetheless, basolateral sorting information in the cytoplasmic tail of CD1d was sufficient to redirect the otherwise apically distributed protein CD8 to the basolateral surface, indicating that the tail contained sufficient information for basolateral sorting (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar). Although the mechanisms for apical sorting remain largely undefined, a number of basolateral sorting signals have been identified. Basolateral sorting signals are currently subdivided into two major classes: signals that are either co-linear or not co-linear with the signals for coated pit localization. Signals that are co-linear with the signals for coated pit localization can be further subdivided into the tyrosine-based basolateral sorting signals, such as those of lysosomal associated membrane protein-1 (11.Höning S. Hunziker W. J. Cell Biol. 1995; 128: 321-332Crossref PubMed Scopus (116) Google Scholar), lysosomal acid phosphatase (12.Peters C. Braun M. Weber B. Wendland M. Schmidt B. Pohlmann R. Waheed A. von Figura K. EMBO J. 1990; 9: 3497-3506Crossref PubMed Scopus (140) Google Scholar), and TGN38 (13.Wong S.H. Hong W. J. Biol. Chem. 1993; 268: 22853-22862Abstract Full Text PDF PubMed Google Scholar) and leucine-based signals found, for example, in the invariant chain (14.Simonsen A. Stang E. Bremnes B. Roe M. Prydz K. Bakke O. J. Cell Sci. 1997; 110: 597-609Crossref PubMed Google Scholar), Fc receptor II (15.Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2969Crossref PubMed Scopus (218) Google Scholar), and furin (16.Simmen T. Nobile M. Bonifacino J.S. Hunziker W. Mol. Cell. Biol. 1999; 19: 3136-3144Crossref PubMed Scopus (67) Google Scholar). Signals that are not co-linear with the signals for the coated pit internalization may be either tyrosine-dependent, such as signals in the vesicular stomatitis virus G protein (17.Thomas D.C. Roth M.G. J. Biol. Chem. 1994; 269: 15732-15739Abstract Full Text PDF PubMed Google Scholar) and the low density lipoprotein receptor proximal signal (18.Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar), or tyrosine-independent such as in polyimmunoglobulin receptor (19.Okamoto C.T. Song W. Bomsel M. Mostov K.E. J. Biol. Chem. 1994; 269: 15676-15682Abstract Full Text PDF PubMed Google Scholar) and the transferrin receptor (20.Odorizzi G. Trowbridge I.S. J. Cell Biol. 1997; 137: 1255-1264Crossref PubMed Scopus (120) Google Scholar). Tyrosine- and leucine-based sorting signals are believed to interact with one or more of the adaptor complexes, AP1 at the TGN, AP2 at the plasma membrane, and AP3 intracellularly (reviewed in Refs.21.Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-298Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 22.Kirchhausen T. Bonifacino J.S. Riezman H. Curr. Opin. Cell Biol. 1997; 9: 488-495Crossref PubMed Scopus (351) Google Scholar, 23.Marks M.S. Ohno H. Kirchhausen T. Bonifacino S.J. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 24.Bonifacino J.S. Dell'Angelica E.C. J. Cell Biol. 1999; 145: 923-926Crossref PubMed Scopus (366) Google Scholar), and it has recently been reported that interaction with AP1B may be a part of the polarization machinery (25.Fölsch H. Ohno H. Bonifacino J.S. Mellman I. Cell. 1999; 99: 189-198Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 26.Ohno 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 (213) Google Scholar). In this study, we sought to identify the basolateral sorting signal in the cytoplasmic tail of CD1d. Our data indicate that there are two overlapping signals within the very last five C-terminal amino acids of CD1d, one tyrosine- and one leucine-based, each being sufficient for its basolateral sorting. Amino acid composition of the cytoplasmic tails of constructs used in this study is given in Fig. 1. Mutations in the cytoplasmic tail of CD1d were created by PCR using the wild-type CD1d cDNA as a template. All constructs were subcloned into the pMEP4 vector (Invitrogen) and sequenced. MDCK (strain II) cells were grown in full growth medium (DMEM supplemented with 10% fetal calf serum, 2 mm glutamine, 25 units/ml penicillin, and 25 μg/ml streptomycin) in 5% CO2 in a 37 °C incubator. MDCK cells were stably transfected by the calcium phosphate procedure as described elsewhere (27.Wigler M. Sweet R. Sim G.K. Wold B. Pellicer A. Lacy E. Maniatis T. Silverstein S. Axel R. Cell. 1979; 16: 777-785Abstract Full Text PDF PubMed Scopus (860) Google Scholar). Clones expressing DNA constructs under control of the inducible metallothionein promoter in the pMEP4 vector were selected in the presence of hygromycin B (0.3 mg/ml). Resistant clones were isolated and incubated with 25 mmCdCl2 overnight to induce expression of the protein of interest. Clones expressing constructs of interest were identified by screening with the D5 antibody (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar). D5 antibody was labeled with Na125I using IODO-BEADs (Sigma) as described previously (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar). Briefly, the antibody (100 μg) was incubated with 1 mCi of Na125I and IODO-BEADs for 10 min on ice. Iodinated antibody was then separated from free Na125I on Sephadex G-25M columns (Amersham Pharmacia Biotech). The specific activity of the labeled antibody was determined by trichloroacetic acid precipitation. The amount of soluble radioactivity was generally less than 5% of total radioactivity. Stably transfected MDCK cells were grown on Transwell polycarbonate filter units (Costar; 106 cells/filter) for 4 days prior to experiments. Expression of the constructs of interest was induced by overnight incubation with 0–25 μm CdCl2. To avoid possible artifacts because of the selection of a single clone, different clones were chosen and tested at varying concentrations of CdCl2. Care was taken not to overexpress CD1d constructs to levels that might induce missorting. Positive controls (cells that expressed basolaterally sorted wild-type CD1d at same or higher levels as constructs in question) were included in each set of experiments. Cells were then cooled on ice and incubated with 125I-D5 (2–10 μg/ml) added apically or basolaterally. The integrity of tight junctions was followed by monitoring the leakage of radioactivity from the basolateral to the apical side. Unbound antibodies were removed by extensive washing with PBS++ and filters were excised and counted with a CobraTM Auto-Gamma counter. Nonspecific binding was determined in parallel experiments with non-transfected MDCK cells and corrected for. Using the same cell line and growth condition we have shown earlier that the apical:basolateral surface membrane is 1:1 (14.Simonsen A. Stang E. Bremnes B. Roe M. Prydz K. Bakke O. J. Cell Sci. 1997; 110: 597-609Crossref PubMed Google Scholar) and a molecule is distributed polarized when it is significantly different from this. DMEM and fetal calf serum were obtained from BioWhittaker. Oligonucleotides for PCR mutagenesis were synthesized by Medprobe (Norway). Materials for PCR amplification, restriction, ligation, and sequencing were from New England Biolabs. All other materials (unless specified otherwise) were purchased from Sigma. In a previous study (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar), we reported that a deletion of the last six amino acids from the CD1d cytoplasmic tail (Fig.1 delta SYQGLVconstruct) abolished basolateral sorting of CD1d in MDCK cells (Fig.2). Furthermore, fusing the last eight CD1d cytoplasmic amino acids to the transmembrane and extracellular domains of the CD8 molecule re-routed this otherwise apical protein to the basolateral surface of MDCK cells. Interestingly, mutation in the tyrosine residue critical for internalization did not impair basolateral sorting of CD1d. We therefore undertook an alanine scan of the CD1d cytoplasmic tail (Fig. 1) to identify possible important amino acid residue(s) for its basolateral sorting. The alanine scan did not reveal any single critical residue important for basolateral sorting of CD1d (Fig. 2). However, all individual alanine substitutions somewhat increased the amount of CD1d routed to the apical side as compared with the wild-type molecule. The largest effect (over 20% sorted apically compared with less then 5% for the wild-type molecule) was registered when either of the two most C-terminal residues, valine and leucine, was mutated to alanine (Fig.2). This led us to investigate in more detail the role of the C-terminal valine and leucine residues in basolateral sorting of CD1d. As internalization and endosomal sorting were abolished by mutation of the tyrosine alone, we concluded that these residues were not essential for endosomal sorting of CD1d. A single mutation of the valine or the leucine was not sufficient to abolish basolateral sorting, and we therefore made mutants with a dual substitution or deletion of both residues (Fig. 1, constructs VA, LA, anddelta VL). As shown in Fig.3, these mutants were no longer sorted basolaterally, indicating that valine and leucine residues were in fact involved in basolateral sorting. It is well established that leucine-based endosomal sorting signals are not necessarily recognized for basolateral sorting (11.Höning S. Hunziker W. J. Cell Biol. 1995; 128: 321-332Crossref PubMed Scopus (116) Google Scholar, 28.Lehmann L.E. Eberle W. Krull S. Prill V. Schmidt B. Sander C. von Figura K. Peters C. EMBO J. 1992; 11: 4391-4399Crossref PubMed Scopus (53) Google Scholar, 29.Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 30.Prill V. Lehmann L. von Figura K. Peters C. EMBO J. 1993; 12: 2181-2193Crossref PubMed Scopus (87) Google Scholar, 31.Matter K. Yamamoto E.M. Mellman I. J. Cell Biol. 1994; 126: 991-1004Crossref PubMed Scopus (213) Google Scholar, 32.Simonsen A. Bremnes B. Nordeng T.W. Bakke O. Eur. J. Cell Biol. 1998; 76: 25-32Crossref PubMed Scopus (25) Google Scholar), and our results document the reverse: a basolateral sorting signal that is not active in internalization. Previous studies have shown that single point mutations within a leucine-based sorting motif were sufficient to abolish basolateral sorting (e.g. Refs. 14.Simonsen A. Stang E. Bremnes B. Roe M. Prydz K. Bakke O. J. Cell Sci. 1997; 110: 597-609Crossref PubMed Google Scholar and 15.Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2969Crossref PubMed Scopus (218) Google Scholar). This was clearly not the case for the VL-based signal of CD1d, as both residues had to be mutated to impair the basolateral sorting. We, however, noticed that the valine was also a part of the tyrosine-based internalization signal. We therefore decided to reinvestigate the role of the cytoplasmic tyrosine residue in basolateral sorting of CD1d. Two constructs were made; either valine or leucine was mutated to alanine in addition to the tyrosine (Fig. 1, constructs YA,VA and YA, LA). Both constructs were no longer sorted basolaterally. From this we conclude that the tyrosine is also involved in targeting CD1d to the basolateral surface. Curiously the YA, LA, delta VL, and the VA, LA constructs were distributed predominantly to the apical membrane (Fig. 3). This might in principle indicate the presence of apical sorting information in these mutants as a truncation of the last eight cytoplasmic residues of CD1d led to truly non-polarized sorting (50/50 apical/basolateral distribution, Fig. 2). However, we cannot draw any conclusion until more is known. As it was required to mutate residues both within the tyrosine-based internalization signal in addition to the residues in the putative leucine-based basolateral sorting signal in order to disrupt basolateral sorting of CD1d, it is tempting to conclude that the cytoplasmic tail of CD1d contains two overlapping basolateral sorting signals. Efficient basolateral sorting by the tyrosine-based signal required an intact tyrosine residue and one of the bulky hydrophobic residues at the position +3 or +4. A basolateral tyrosine sorting signal with a hydrophobic residue in position +4 has to our knowledge not been reported before, but in most studies the context of tyrosine signals was not in Our data thus suggest that we have identified a new context for a tyrosine-based basolateral signal. It is that endosomal sorting by the same tyrosine-based signal was by single mutation of the +3 valine (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar). This that the context for internalization and basolateral sorting by the signal are The leucine-based basolateral signal is also as this of sorting signal has not been found at the very of However, it is reported that internalization of molecules was and on a leucine-based signal when all residues C-terminal to the signal were This has been for example, for the signal in J. J. N. C. J. Cell Biol. 1997; PubMed Scopus Google Scholar) and in the signal E. C.R. L. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is therefore not that the leucine-based basolateral sorting signal at the very C-terminal of the CD1d molecule is We to investigate the distribution of CD1d constructs because our previous studies have shown that synthesized CD1d molecules to the cell surface (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar), and levels of were of of surface of the synthesized CD1d molecules be to and and possible of CD1d molecules at the cell surface might also be to the We that CD1d molecules are sorted from the to the cell surface most synthesized in the MDCK cells A. C. K. J. Cell Biol. 1990; PubMed Scopus Google Scholar). for this is that and mutants have internalization signal (10.Rodionov D.G. Nordeng T.W. Pedersen K. Balk S.P. Bakke O. J. Immunol. 1999; 162: 1488-1495PubMed Google Scholar) but were sorted basolaterally (Fig. 2). the synthesized CD1d molecules been to the apical membrane and and to the basolateral membrane, a mutation in the internalization signal have led to a predominantly apical or non-polarized distribution of the and mutants, is not the tyrosine- and leucine-based sorting signals have been shown to interact with adaptor molecules (e.g. Refs. H. J. H. I. S. Saito T. A. Kirchhausen T. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) the leucine signals have been reported to bind the chain of AP2 I. P. Kirchhausen T. EMBO J. 1998; PubMed Scopus Google Scholar) the medium of AP1 and AP2 D.G. Bakke O. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. D. B. von Figura K. Bakke O. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A study by Ohno 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 (213) Google Scholar) has identified a medium chain that is expressed in polarized cells. It was recently shown that this chain is to in may polarized sorting in a cell line this molecule (25.Fölsch H. Ohno H. Bonifacino J.S. Mellman I. Cell. 1999; 99: 189-198Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Furthermore, the adaptor may be involved in basolateral sorting of molecules both tyrosine- and leucine-based sorting signals. and W. This to the that and basolateral sorting signals may be recognized by different Our that the context of the CD1d tyrosine signal is different for internalization and basolateral sorting and that its leucine-based signal is for polarized sorting to the of adaptor for internalization and basolateral sorting. this we can there are two basolateral signals within the CD1d molecule. This is not as other molecules also more than one polarization signal. two basolateral signals have been found in the low density lipoprotein receptor K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (303) Google Scholar), and the complex of major histocompatibility complex class II and invariant chain contains no less than different basolateral signals (14.Simonsen A. Stang E. Bremnes B. Roe M. Prydz K. Bakke O. J. Cell Sci. 1997; 110: 597-609Crossref PubMed Google Scholar). sorting signals in these molecules may in principle bind more than one adaptor at the same In the two basolateral sorting signals in CD1d are and most not that the but not the leucine-based is involved in internalization of CD1d, it is that the signals are to interact with different of the intracellular but the mechanisms of such remain to be We Balk the D5 antibody used for this We also for