The Mouse CD1d Cytoplasmic Tail Mediates CD1d Trafficking and Antigen Presentation by Adaptor Protein 3-Dependent and -Independent Mechanisms 1 Anna P. Lawton,* Theodore I. Prigozy, 2 * Laurent Brossay, 3 * Bo Pei,* Archana Khurana,* Donald Martin,* Tiancheng Zhu,* Kira Spa ¨te, † Megda Ozga, † Stefan Ho ¨ning, † Oddmund Bakke, ‡§ and Mitchell Kronenberg 4 * ¶ The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of V14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the  subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids. The Journal of Immunology, 2005, 174: 3179 –3186. T he CD1 family of proteins is a group of nonclassical, class I-like Ag-presenting molecules (1). Whereas MHC-en- coded class I and class II molecules present peptides, CD1 molecules present various lipid Ags (2). Group I CD1 molecules, which include human CD1a, CD1b, and CD1c, present mycobac- teria-derived and brain-derived glycolipids (2– 4). Group II CD1 molecules, e.g., CD1d, present glycolipids to V14i NKT cells in mice and their homologues in other species, and they regulate their development in mice (5). V14i NKT cells are a distinct sublineage of T lymphocytes, which may be involved in immune regulation and host defense (6). V14i NKT cells are autoreactive to mouse CD1d (mCD1d) 5 (7), and this response is enhanced greatly by a synthetic phytosphin- golipid, -D-galactosyl ceramide (GalCer) (8, 9). Current studies of the cellular requirements for lipid-Ag presentation by CD1 mol- ecules provide evidence of both endosomal and nonendosomal pathways for glycolipid-Ag presentation to T cells (10). Whereas the presentation of GalCer by mCD1d does not require internal- ization because even plate-bound recombinant mCD1d protein can present GalCer to V14i NKT cells (11), analogues of GalCer that have additional sugars such as the 2' and/or 3' carbon of the galactose have to be internalized and processed to generate the monosaccharide GalCer before they can be recognized by TCR. The processing of these analogues presumably occurs in the lyso- somes (11). Analyses of tail deletion mutations demonstrates that the cyto- plasmic tail of mCD1d is critical for its localization to low-pH endosomal compartments, Ag presentation, and the development of V14i NKT cells (12–15). The cytoplasmic tail of mCD1d con- tains a Tyr-based endosomal-targeting motif, YXX (X = any amino acid and = hydrophobic amino acid). This sequence is likely to govern the endosomal localization of mCD1d, based on its similarity to those in a number of other proteins, including human CD1b, CD1c, and CD1d. The Tyr motif is predicted to bind one or more of adaptor pro- tein complexes (AP), AP-1, AP-2, AP-3, and AP-4, which are involved in targeting integral membrane proteins, to intracellular compartments (16 –19). AP-1 and AP-2 are components of clathrin coats associated with the trans-Golgi network (TGN)/endosomes and the plasma membrane, respectively (20). AP-1 is important for the trafficking of proteins from the TGN to endosomes, and AP-2 is involved in cargo recruitment in endocytosis (21). AP-3 has been shown to be part of both clathrin and nonclathrin coats lo- calized to endosomes, and it is important in the localization of membrane proteins to lysosome-related vesicles (22). AP-4 is as- sociated with the TGN, transport vesicles, and endosomes, and it *Division of Developmental Immunology, Jolla Institute for Allergy and Immunol- ogy, San Diego, CA 92121; † Biochemistry II, University of Go ¨ttingen, Go ¨ttingen, Germany; ‡ Department of Biomedicine, University of Bergen, Bergen, Norway; § De- partment of Molecular Biosciences, University of Oslo, Oslo, Norway; and ¶ Division of Biology, University of California San Diego, La Jolla, CA 92093 Received for publication August 6, 2004. Accepted for publication December 1, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant RO1 AI40617 (to M.K.), a grant from the Human Frontiers of Science Program (to M.K.), and grants from the University of Oslo and the Research Council of Norway (to O.B.). A.P.L. is the recipient of a National Research Service Award from National Institutes of Health (Grant AI52552). 2 Current address: Tampa Bay Research Institute, St. Petersburg, FL 33712. 3 Current address: Department of Molecular Microbiology and Immunology and Graduate Program in Pathobiology, Division of Biology and Medicine, Brown Uni- versity, Providence, RI 02912. 4 Address correspondence and reprint requests to Dr. Mitchell Kronenberg, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: mitch@liai.org 5 Abbreviations used in this paper: mCD1d, mouse CD1d; GalCer, -D-galctosyl cer- amide; AP, adaptor protein complex; TGN, trans-Golgi network; Gal(132)GalCer, -2'-galactosyl-GalCer; TfR, transferrin receptor; SPR, surface plasmon resonance. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00