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Direct Interaction with a Kinesin-related Motor Mediates Transport of Mammalian Discs Large Tumor Suppressor Homologue in Epithelial Cells

2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês

10.1074/jbc.m210362200

1083-351X

Noriyuki Asaba, Toshihiko Hanada, Atsuko Takeuchi, Athar H. Chishti,

Cellular Mechanics and Interactions

Membrane-associated guanylate kinase homologues (MAGUKs) are generally found under the plasma membrane of cell-cell contact sites and function as scaffolding proteins by linking cytoskeletal and signaling molecules to transmembrane receptors. The correct targeting of MAGUKs is essential for their receptor-clustering function; however, the molecular mechanism of their intracellular transport is unknown. Here, we show that the guanylate kinase-like domain of human discs large protein binds directly within the amino acids 607–831 of the stalk domain of GAKIN, a kinesin-like protein of broad distribution. The primary structure of the binding segment, termed MAGUK binding stalk domain, is conserved inDrosophila kinesin-73 and some other motor and non-motor proteins. This stalk segment is not found in GKAP, a synaptic protein that interacts with the guanylate kinase-like domain, and unlike GKAP, the binding of GAKIN is not regulated by the intramolecular interactions within the discs large protein. The recombinant motor domain of GAKIN is an active microtubule-stimulated ATPase withk cat = 45 s−1,K 0.5 (MT) = 0.1 μm. Overexpression of green fluorescent protein-fused GAKIN in Madin-Darby canine kidney epithelial cells induced long projections with both GAKIN and endogenous discs large accumulating at the tip of these projections. Importantly, the accumulation of endogenous discs large was eliminated when a mutant GAKIN lacking its motor domain was overexpressed under similar conditions. Taken together, our results indicate that discs large is a cargo molecule of GAKIN and suggest a mechanism for intracellular trafficking of MAGUK-laden vesicles to specialized membrane sites in mammalian cells. Membrane-associated guanylate kinase homologues (MAGUKs) are generally found under the plasma membrane of cell-cell contact sites and function as scaffolding proteins by linking cytoskeletal and signaling molecules to transmembrane receptors. The correct targeting of MAGUKs is essential for their receptor-clustering function; however, the molecular mechanism of their intracellular transport is unknown. Here, we show that the guanylate kinase-like domain of human discs large protein binds directly within the amino acids 607–831 of the stalk domain of GAKIN, a kinesin-like protein of broad distribution. The primary structure of the binding segment, termed MAGUK binding stalk domain, is conserved inDrosophila kinesin-73 and some other motor and non-motor proteins. This stalk segment is not found in GKAP, a synaptic protein that interacts with the guanylate kinase-like domain, and unlike GKAP, the binding of GAKIN is not regulated by the intramolecular interactions within the discs large protein. The recombinant motor domain of GAKIN is an active microtubule-stimulated ATPase withk cat = 45 s−1,K 0.5 (MT) = 0.1 μm. Overexpression of green fluorescent protein-fused GAKIN in Madin-Darby canine kidney epithelial cells induced long projections with both GAKIN and endogenous discs large accumulating at the tip of these projections. Importantly, the accumulation of endogenous discs large was eliminated when a mutant GAKIN lacking its motor domain was overexpressed under similar conditions. Taken together, our results indicate that discs large is a cargo molecule of GAKIN and suggest a mechanism for intracellular trafficking of MAGUK-laden vesicles to specialized membrane sites in mammalian cells. membrane-associated guanylate kinase homologues Src homology guanylate kinase-like discs large protein guanylate kinase-associatedkinesin GUK-associated protein microtubule-associated protein Madin-Darby canine kidney glutathione S-transferase 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid green fluorescent protein MAGUK binding stalk synapse-associated protein of 97 kDa Membrane-associated guanylate kinase homologues (MAGUKs)1 are a family of proteins composed of one or more PDZ domains, an SH3 domain, and a guanylate kinase-like (GUK) domain (1Anderson J.M. Curr. Biol. 1996; 6: 382-384Google Scholar). They are thought to play scaffolding functions at specialized membrane sites, such as synaptic membrane, tight junction, and adherens junction (2Fanning A.S. Anderson J.M. Curr. Opin. Cell. Biol. 1999; 11: 432-439Google Scholar, 3Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Google Scholar, 4Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Google Scholar).Drosophila Dlg is a MAGUK encoded by lethal (1Anderson J.M. Curr. Biol. 1996; 6: 382-384Google Scholar) discs large-1 tumor-suppressor gene (dlg), and mutations ofdlg cause neoplastic overgrowth of the imaginal discs (5Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Google Scholar). The Dlg protein localizes to the septate junctions in epithelial cells where it regulates cell proliferation, apical-basal cell polarity, and the organization of junctional structure (6Woods D.F. Hough C. Peel D. Callaini G. Bryant P.J. J. Cell Biol. 1996; 134: 1469-1482Google Scholar, 7Hough C.D. Woods D.F. Park S. Bryant P.J. Genes Dev. 1997; 11: 3242-3253Google Scholar, 8Budnik V. Koh Y.H. Guan B. Hartmann B. Hough C. Woods D. Gorczyca M. Neuron. 1996; 17: 627-640Google Scholar). The human homologue ofDrosophila Dlg, termed hDlg, and its rat counterpart SAP97, localize at the pre- and postsynaptic membrane sites as well as the basolateral membrane of epithelial cells (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 10Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar) and are proposed to perform scaffolding functions by linking cytoskeletal components to the transmembrane proteins (11Marfatia S.M. Morais Cabral J.H. Lin L. Hough C. Bryant P.J. Stolz L. Chishti A.H. J. Cell Biol. 1996; 135: 753-766Google Scholar). In addition to the scaffolding function, mounting evidence now indicates that hDlg regulates cell proliferation and could be involved in tumorigenesis. For example, hDlg interacts with viral oncoproteins such as high-risk human papillomavirus E6 and human T-cell leukemia virus type 1 (HTLV-1) Tax (12Lee S.S. Weiss R.S. Javier R.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6670-6675Google Scholar, 13Kiyono T. Hiraiwa A. Fujita M. Hayashi Y. Akiyama T. Ishibashi M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11612-11616Google Scholar, 14Suzuki T. Ohsugi Y. Uchida-Toita M. Akiyama T. Yoshida M. Oncogene. 1999; 18: 5967-5972Google Scholar). Similarly, hDlg forms a complex with adenomatous polyposis coli (APC) tumor suppressor gene product and negatively regulates cell cycle progression (15Matsumine A. Ogai A. Senda T. Okumura N. Satoh K. Baeg G.H. Kawahara T. Kobayashi S. Okada M. Toyoshima K. Akiyama T. Science. 1996; 272: 1020-1023Google Scholar, 16Ishidate T. Matsumine A. Toyoshima K. Akiyama T. Oncogene. 2000; 19: 365-372Google Scholar). The mechanism of how hDlg regulates cell proliferation is still largely unknown.Recently, we identified GAKIN (guanylate kinase-associated kinesin), which is also classified as human KIF13B (17Miki H. Setou M. Kaneshiro K. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7004-7011Google Scholar), from Jurkat T lymphoma cells as a binding partner for the GUK domain of hDlg (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). In T cells, hDlg interacts with tyrosine kinase Lck and potassium channel Kv1.3 (19Hanada T. Lin L. Chandy K.G. Oh S.S. Chishti A.H. J. Biol. Chem. 1997; 272: 26899-26904Google Scholar) and translocates to the immune synapse-like structure upon cross-linking of cell surface CD2 molecules (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). These observations suggest that hDlg might play a role in the formation of physical contacts between T cells and antigen-presenting cells and regulate activation of T cells during immune response. An intriguing possibility emerges suggesting the role of GAKIN in the transport of hDlg to the immune synapse upon activation of T cells. Since transcripts of GAKIN and hDlg are ubiquitously expressed, it is conceivable that GAKIN-dependent trafficking is a widespread mechanism across species and tissues for the transport of hDlg and other MAGUKs. Consistent with this paradigm is the recent evidence indicating a role of soluble adaptor proteins in the transport of cargo vesicles via kinesin-like motors. For example, KIF17 motor interacts with mLin-10, which in turn mediates its interaction with the cargo vesicles containing NMDA receptor subunits (20Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Google Scholar). Similarly, KIF13A motor binds to a subunit of AP-1 complex mediating its interaction with the vesicles containing mannose-6-phosphate receptor (21Nakagawa T. Setou M. Seog D. Ogasawara K. Dohmae N. Takio K. Hirokawa N. Cell. 2000; 103: 569-581Google Scholar). Conventional kinesin, via its light chain, binds to c-Jun N-terminal kinase-interacting proteins that mediate interaction with specific cargo vesicles (22Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Google Scholar). Since hDlg is a soluble scaffolding protein, it can in principle link cargo vesicles to an intracellular motor, and therefore this property fits well with the common paradigm of being a motor-cargo adaptor molecule.The guanylate kinase-like domains of MAGUKs exhibit little or no guanylate kinase activity (23Kuhlendahl S. Spangenberg O. Konrad M. Kim E. Garner C.C. Eur. J. Biochem. 1998; 252: 305-313Google Scholar), and their principal function seems to serve as a protein-protein interaction motif (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar, 24Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Google Scholar). Besides GAKIN, several proteins are reported to interact with the GUK domains of MAGUKs. These GUK-binders include GKAP (GUK-associated protein) (24Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Google Scholar), MAP1A (microtubule-associated protein 1A) (25Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Google Scholar), BEGAIN (brain-enriched guanylate kinase-associated protein) (26Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Google Scholar), a Rap specific GTPase-activating protein SPAR (27Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Google Scholar), and protein kinase A-anchoring protein AKAP79 (28Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar). At this stage, it is not clear how a single GUK domain binds to so many seemingly non-related proteins and how these interactions are regulated. Recent determination of the crystal structures of PSD-95 revealed the existence of a novel mode of intramolecular interactions between the SH3 and GUK domains, providing a new perspective on the functional regulation of hDlg interactions (29McGee A.W. Dakoji S.R. Olsen O. Bredt D.S. Lim W.A. Prehoda K.E. Mol. Cell. 2001; 8: 1291-1301Google Scholar, 30Tavares G.A. Panepucci E.H. Brunger A.T. Mol. Cell. 2001; 8: 1313-1325Google Scholar, 31McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Google Scholar, 32Shin H. Hsueh Y.P. Yang F.C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Google Scholar). Interestingly, the binding of GKAP to the GUK domain of SAP97 is regulated by a series of intramolecular interactions between the SH3 and GUK domains (33Wu H. Reissner C. Kuhlendahl S. Coblentz B. Reuver S. Kindler S. Gundelfinger E.D. Garner C.C. EMBO J. 2000; 19: 5740-5751Google Scholar). The PDZ domains regulate MAP1A binding to the GUK domain intramolecularly, although their mechanism of regulation seems distinct from that of GKAP (25Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Google Scholar). In this manuscript, we provide evidence for the existence of a novel protein-binding domain that links GAKIN to the GUK domain of hDlg. The binding mode between GAKIN and hDlg appears to be distinct from that of GKAP binding to hDlg/SAP97. Our results also suggest that GAKIN mediates intracellular trafficking of Dlg in epithelial cells.DISCUSSIONHuman Dlg protein and its rat orthologue SAP97 are localize at specialized membrane regions where cells form contacts such as the synaptic membrane in neuronal cells (10Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar), adherens junctions of epithelial cells (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 43Reuver S.M. Garner C.C. J. Cell Sci. 1998; 111: 1071-1080Google Scholar), and contact sites between T lymphocyte and antigen-presenting cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). In contrast, hDlg/SAP97 is present predominantly in the cytoplasm and appears to attach to intracellular membranes in cells that do not display cell-cell contacts (43Reuver S.M. Garner C.C. J. Cell Sci. 1998; 111: 1071-1080Google Scholar, 44Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.-P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-157Google Scholar). In addition to the PDZ domains that link hDlg to the cytoplasmic domains of transmembrane receptors (4Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Google Scholar), the primary structure of hDlg contains multiple protein-protein interaction domains that interact with cytoskeletal components and signaling molecules (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 19Hanada T. Lin L. Chandy K.G. Oh S.S. Chishti A.H. J. Biol. Chem. 1997; 272: 26899-26904Google Scholar). These multiple protein interactions presumably permit hDlg to function as a scaffolding protein by forming large protein complexes at the interface of the membrane-cytoskeleton (11Marfatia S.M. Morais Cabral J.H. Lin L. Hough C. Bryant P.J. Stolz L. Chishti A.H. J. Cell Biol. 1996; 135: 753-766Google Scholar). A major remaining issue of fundamental importance pertains to the mechanism of hDlg trafficking and its delivery to specialized sites. Our original identification of GAKIN was made by virtue of its association with the GUK domain of hDlg in the context of whole cell lysate (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar), therefore a possibility remained that the GAKIN-hDlg interaction might not be direct. In this manuscript, we demonstrate that GAKIN interacts directly with the GUK domain of hDlg (Fig. 1). A unique feature of this interaction is the unusual location of the hDlg-binding region within the stalk domain of GAKIN. The traditional view of kinesin motors implies that their globular C-terminal tails usually serve as the cargo binding modules (45Klopfenstein D.R. Vale R.D. Rogers S.L. Cell. 2000; 103: 537-540Google Scholar). However, in the case of GAKIN, the MBS domain that binds to hDlg is located within the N-terminal half of GAKIN downstream of its N-terminal motor domain (Fig. 2). Thus, the C-terminal half of GAKIN with a single copy of CAP-Gly domain either serves a regulatory region for cargo binding and/or binds to distinct cargo molecules. It is noteworthy here other intracellular motors, such as the Rab6-binding kinesin Rab6-KIFL, have been speculated to harbor cargo-binding domains in their coiled-coil regions (46Hill E. Clarke M. Barr F.A. EMBO J. 2000; 19: 5711-5719Google Scholar). In any case, the presence of the MBS domain in a variety of motor and non-motor proteins suggests that this region might represent a novel cargo-binding motif with implications in linking soluble adaptors to motor proteins within the scaffolding complex. For example, the KIF13A motor transports cargo vesicles via its C-terminal tail that interacts with β1-adaptin and mannose-6-phosphate receptor (21Nakagawa T. Setou M. Seog D. Ogasawara K. Dohmae N. Takio K. Hirokawa N. Cell. 2000; 103: 569-581Google Scholar). Our identification of the MBS domain in KIF13A raises the possibility that these motors could potentially bind additional cargoes within their long stalk domains. Similarly, the presence of two MBS domains in RIM-BP1 also implicates recruitment of additional proteins in the assembly of RIM and Rab3-based trafficking machinery in the brain (38Wang Y. Sugita S. Sudhof T.C. J. Biol. Chem. 2000; 275: 20033-20044Google Scholar). In summary, our mapping data on the direct interaction between hDlg and GAKIN reveals a novel protein-binding domain that could mediate similar interactions with a large number of GUK domain proteins.The MBS domain of GAKIN does not share any sequence similarity with proteins that bind to the GUK domains of MAGUKs. Indeed, our results indicate that binding of GAKIN to the GUK domain of hDlg is not regulated by intramolecular interactions of the SH3 and GUK domains (Fig. 3). Based on our observation that GKAP competes with the MBS domain of GAKIN for binding to the GUK domain of hDlg (Fig. 4), we speculate on a model that offers an explanation for at least one function of the intramolecular interactions of MAGUKs. According to this model, the MBS domain of GAKIN interacts with a "folded" state of hDlg in the cytoplasm and transports it to specialized membrane sites. This folded and thus closed state of hDlg does not permit binding with other GUK domain binders such as GKAP. Once the hDlg cargo reaches the target membrane sites, other membrane and protein interactions "unfold" the closed SH3-GUK conformation thus permitting the transfer of hDlg to another GUK domain binder such as GKAP. The final assembly of the mature scaffolding complex occurs at this site by recruitment of additional binding partners to the "open" conformation of hDlg. Further experimental verification of this model would require identification of other cargo molecules of the GAKIN-hDlg complex and further investigation as to whether novel segments of GAKIN regulate protein-protein interactions of the scaffolding complex at or during the assembly process.The data presented in this manuscript suggest that human GAKIN is a biologically active kinesin motor, providing a molecular basis for the intracellular trafficking of hDlg in mammalian cells. Our results also suggest that direct binding of GAKIN to the GUK domain of hDlg could permit transport of a soluble multiprotein complex to specialized cell-cell contact sites. Alternatively, the GAKIN-hDlg interaction may also allow trafficking of the scaffolding complex attached to the intracellular vesicles. The hDlg-bearing vesicles are then delivered to the plus end of microtubules by GAKIN. The proposed model of GAKIN-dependent transport of intracellular vesicles is also consistent with the observed punctate and vesicular distribution of hDlg/SAP97 in neuronal, epithelial, and lymphoid cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar, 44Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.-P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-157Google Scholar, 47McLaughlin M. Hale R. Ellston D. Gaudet S. Lue R.A. Viel A. J. Biol. Chem. 2002; 277: 6406-6412Google Scholar). In addition to hDlg, the GAKIN-dependent trafficking could also play a pivotal role for the transport of PSD-95, based on the fact that GAKIN binds to the GUK domain of PSD-95 and is expressed abundantly in neuronal cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar).The proposed model adds a new example to the emerging general paradigm that scaffolding adaptor proteins act as molecular links between specific motor proteins and cargo vesicles. Recent examples supporting this paradigm include the mLin-10 adaptor that links KIF17 motor to vesicles via mLin-2, mLin-7, andN-methyl-d-aspartic acid receptor subunit complex (20Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Google Scholar), the role of JIPs in connecting the conventional kinesin to cargo vesicles via the Reelin receptor (22Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Google Scholar), and also the GRIP1 mediated linkage of kinesin heavy chains to vesicles via an AMPA receptor subunit (48Setou M. Seog D.H. Tanaka Y. Kanai Y. Takei Y. Kawagishi M. Hirokawa N. Nature. 2002; 417: 83-87Google Scholar). A more recent demonstration of SAP97 binding to the minus end-directed actin motor myosin VI also suggests a mechanism for the reversible translocation of MAGUKs in vivo (49Wu H. Nash J.E. Zamorano P. Garner C.C. J. Biol. Chem. 2002; 277: 30928-30934Google Scholar). In conclusion, the identity of various components of the hDlg and/or PSD-95 cargo complex transported by GAKIN could open an area of considerable interest for the assembly, transport, and regulation of this complex and more importantly could provide insights into the mechanism of dynamic regulation of the cell-cell contact structure in normal and disease states. Membrane-associated guanylate kinase homologues (MAGUKs)1 are a family of proteins composed of one or more PDZ domains, an SH3 domain, and a guanylate kinase-like (GUK) domain (1Anderson J.M. Curr. Biol. 1996; 6: 382-384Google Scholar). They are thought to play scaffolding functions at specialized membrane sites, such as synaptic membrane, tight junction, and adherens junction (2Fanning A.S. Anderson J.M. Curr. Opin. Cell. Biol. 1999; 11: 432-439Google Scholar, 3Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Google Scholar, 4Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Google Scholar).Drosophila Dlg is a MAGUK encoded by lethal (1Anderson J.M. Curr. Biol. 1996; 6: 382-384Google Scholar) discs large-1 tumor-suppressor gene (dlg), and mutations ofdlg cause neoplastic overgrowth of the imaginal discs (5Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Google Scholar). The Dlg protein localizes to the septate junctions in epithelial cells where it regulates cell proliferation, apical-basal cell polarity, and the organization of junctional structure (6Woods D.F. Hough C. Peel D. Callaini G. Bryant P.J. J. Cell Biol. 1996; 134: 1469-1482Google Scholar, 7Hough C.D. Woods D.F. Park S. Bryant P.J. Genes Dev. 1997; 11: 3242-3253Google Scholar, 8Budnik V. Koh Y.H. Guan B. Hartmann B. Hough C. Woods D. Gorczyca M. Neuron. 1996; 17: 627-640Google Scholar). The human homologue ofDrosophila Dlg, termed hDlg, and its rat counterpart SAP97, localize at the pre- and postsynaptic membrane sites as well as the basolateral membrane of epithelial cells (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 10Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar) and are proposed to perform scaffolding functions by linking cytoskeletal components to the transmembrane proteins (11Marfatia S.M. Morais Cabral J.H. Lin L. Hough C. Bryant P.J. Stolz L. Chishti A.H. J. Cell Biol. 1996; 135: 753-766Google Scholar). In addition to the scaffolding function, mounting evidence now indicates that hDlg regulates cell proliferation and could be involved in tumorigenesis. For example, hDlg interacts with viral oncoproteins such as high-risk human papillomavirus E6 and human T-cell leukemia virus type 1 (HTLV-1) Tax (12Lee S.S. Weiss R.S. Javier R.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6670-6675Google Scholar, 13Kiyono T. Hiraiwa A. Fujita M. Hayashi Y. Akiyama T. Ishibashi M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11612-11616Google Scholar, 14Suzuki T. Ohsugi Y. Uchida-Toita M. Akiyama T. Yoshida M. Oncogene. 1999; 18: 5967-5972Google Scholar). Similarly, hDlg forms a complex with adenomatous polyposis coli (APC) tumor suppressor gene product and negatively regulates cell cycle progression (15Matsumine A. Ogai A. Senda T. Okumura N. Satoh K. Baeg G.H. Kawahara T. Kobayashi S. Okada M. Toyoshima K. Akiyama T. Science. 1996; 272: 1020-1023Google Scholar, 16Ishidate T. Matsumine A. Toyoshima K. Akiyama T. Oncogene. 2000; 19: 365-372Google Scholar). The mechanism of how hDlg regulates cell proliferation is still largely unknown. Recently, we identified GAKIN (guanylate kinase-associated kinesin), which is also classified as human KIF13B (17Miki H. Setou M. Kaneshiro K. Hirokawa N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7004-7011Google Scholar), from Jurkat T lymphoma cells as a binding partner for the GUK domain of hDlg (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). In T cells, hDlg interacts with tyrosine kinase Lck and potassium channel Kv1.3 (19Hanada T. Lin L. Chandy K.G. Oh S.S. Chishti A.H. J. Biol. Chem. 1997; 272: 26899-26904Google Scholar) and translocates to the immune synapse-like structure upon cross-linking of cell surface CD2 molecules (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). These observations suggest that hDlg might play a role in the formation of physical contacts between T cells and antigen-presenting cells and regulate activation of T cells during immune response. An intriguing possibility emerges suggesting the role of GAKIN in the transport of hDlg to the immune synapse upon activation of T cells. Since transcripts of GAKIN and hDlg are ubiquitously expressed, it is conceivable that GAKIN-dependent trafficking is a widespread mechanism across species and tissues for the transport of hDlg and other MAGUKs. Consistent with this paradigm is the recent evidence indicating a role of soluble adaptor proteins in the transport of cargo vesicles via kinesin-like motors. For example, KIF17 motor interacts with mLin-10, which in turn mediates its interaction with the cargo vesicles containing NMDA receptor subunits (20Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Google Scholar). Similarly, KIF13A motor binds to a subunit of AP-1 complex mediating its interaction with the vesicles containing mannose-6-phosphate receptor (21Nakagawa T. Setou M. Seog D. Ogasawara K. Dohmae N. Takio K. Hirokawa N. Cell. 2000; 103: 569-581Google Scholar). Conventional kinesin, via its light chain, binds to c-Jun N-terminal kinase-interacting proteins that mediate interaction with specific cargo vesicles (22Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Google Scholar). Since hDlg is a soluble scaffolding protein, it can in principle link cargo vesicles to an intracellular motor, and therefore this property fits well with the common paradigm of being a motor-cargo adaptor molecule. The guanylate kinase-like domains of MAGUKs exhibit little or no guanylate kinase activity (23Kuhlendahl S. Spangenberg O. Konrad M. Kim E. Garner C.C. Eur. J. Biochem. 1998; 252: 305-313Google Scholar), and their principal function seems to serve as a protein-protein interaction motif (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar, 24Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Google Scholar). Besides GAKIN, several proteins are reported to interact with the GUK domains of MAGUKs. These GUK-binders include GKAP (GUK-associated protein) (24Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Google Scholar), MAP1A (microtubule-associated protein 1A) (25Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Google Scholar), BEGAIN (brain-enriched guanylate kinase-associated protein) (26Deguchi M. Hata Y. Takeuchi M. Ide N. Hirao K. Yao I. Irie M. Toyoda A. Takai Y. J. Biol. Chem. 1998; 273: 26269-26272Google Scholar), a Rap specific GTPase-activating protein SPAR (27Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Google Scholar), and protein kinase A-anchoring protein AKAP79 (28Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar). At this stage, it is not clear how a single GUK domain binds to so many seemingly non-related proteins and how these interactions are regulated. Recent determination of the crystal structures of PSD-95 revealed the existence of a novel mode of intramolecular interactions between the SH3 and GUK domains, providing a new perspective on the functional regulation of hDlg interactions (29McGee A.W. Dakoji S.R. Olsen O. Bredt D.S. Lim W.A. Prehoda K.E. Mol. Cell. 2001; 8: 1291-1301Google Scholar, 30Tavares G.A. Panepucci E.H. Brunger A.T. Mol. Cell. 2001; 8: 1313-1325Google Scholar, 31McGee A.W. Bredt D.S. J. Biol. Chem. 1999; 274: 17431-17436Google Scholar, 32Shin H. Hsueh Y.P. Yang F.C. Kim E. Sheng M. J. Neurosci. 2000; 20: 3580-3587Google Scholar). Interestingly, the binding of GKAP to the GUK domain of SAP97 is regulated by a series of intramolecular interactions between the SH3 and GUK domains (33Wu H. Reissner C. Kuhlendahl S. Coblentz B. Reuver S. Kindler S. Gundelfinger E.D. Garner C.C. EMBO J. 2000; 19: 5740-5751Google Scholar). The PDZ domains regulate MAP1A binding to the GUK domain intramolecularly, although their mechanism of regulation seems distinct from that of GKAP (25Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Google Scholar). In this manuscript, we provide evidence for the existence of a novel protein-binding domain that links GAKIN to the GUK domain of hDlg. The binding mode between GAKIN and hDlg appears to be distinct from that of GKAP binding to hDlg/SAP97. Our results also suggest that GAKIN mediates intracellular trafficking of Dlg in epithelial cells. DISCUSSIONHuman Dlg protein and its rat orthologue SAP97 are localize at specialized membrane regions where cells form contacts such as the synaptic membrane in neuronal cells (10Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar), adherens junctions of epithelial cells (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 43Reuver S.M. Garner C.C. J. Cell Sci. 1998; 111: 1071-1080Google Scholar), and contact sites between T lymphocyte and antigen-presenting cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). In contrast, hDlg/SAP97 is present predominantly in the cytoplasm and appears to attach to intracellular membranes in cells that do not display cell-cell contacts (43Reuver S.M. Garner C.C. J. Cell Sci. 1998; 111: 1071-1080Google Scholar, 44Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.-P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-157Google Scholar). In addition to the PDZ domains that link hDlg to the cytoplasmic domains of transmembrane receptors (4Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Google Scholar), the primary structure of hDlg contains multiple protein-protein interaction domains that interact with cytoskeletal components and signaling molecules (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 19Hanada T. Lin L. Chandy K.G. Oh S.S. Chishti A.H. J. Biol. Chem. 1997; 272: 26899-26904Google Scholar). These multiple protein interactions presumably permit hDlg to function as a scaffolding protein by forming large protein complexes at the interface of the membrane-cytoskeleton (11Marfatia S.M. Morais Cabral J.H. Lin L. Hough C. Bryant P.J. Stolz L. Chishti A.H. J. Cell Biol. 1996; 135: 753-766Google Scholar). A major remaining issue of fundamental importance pertains to the mechanism of hDlg trafficking and its delivery to specialized sites. Our original identification of GAKIN was made by virtue of its association with the GUK domain of hDlg in the context of whole cell lysate (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar), therefore a possibility remained that the GAKIN-hDlg interaction might not be direct. In this manuscript, we demonstrate that GAKIN interacts directly with the GUK domain of hDlg (Fig. 1). A unique feature of this interaction is the unusual location of the hDlg-binding region within the stalk domain of GAKIN. The traditional view of kinesin motors implies that their globular C-terminal tails usually serve as the cargo binding modules (45Klopfenstein D.R. Vale R.D. Rogers S.L. Cell. 2000; 103: 537-540Google Scholar). However, in the case of GAKIN, the MBS domain that binds to hDlg is located within the N-terminal half of GAKIN downstream of its N-terminal motor domain (Fig. 2). Thus, the C-terminal half of GAKIN with a single copy of CAP-Gly domain either serves a regulatory region for cargo binding and/or binds to distinct cargo molecules. It is noteworthy here other intracellular motors, such as the Rab6-binding kinesin Rab6-KIFL, have been speculated to harbor cargo-binding domains in their coiled-coil regions (46Hill E. Clarke M. Barr F.A. EMBO J. 2000; 19: 5711-5719Google Scholar). In any case, the presence of the MBS domain in a variety of motor and non-motor proteins suggests that this region might represent a novel cargo-binding motif with implications in linking soluble adaptors to motor proteins within the scaffolding complex. For example, the KIF13A motor transports cargo vesicles via its C-terminal tail that interacts with β1-adaptin and mannose-6-phosphate receptor (21Nakagawa T. Setou M. Seog D. Ogasawara K. Dohmae N. Takio K. Hirokawa N. Cell. 2000; 103: 569-581Google Scholar). Our identification of the MBS domain in KIF13A raises the possibility that these motors could potentially bind additional cargoes within their long stalk domains. Similarly, the presence of two MBS domains in RIM-BP1 also implicates recruitment of additional proteins in the assembly of RIM and Rab3-based trafficking machinery in the brain (38Wang Y. Sugita S. Sudhof T.C. J. Biol. Chem. 2000; 275: 20033-20044Google Scholar). In summary, our mapping data on the direct interaction between hDlg and GAKIN reveals a novel protein-binding domain that could mediate similar interactions with a large number of GUK domain proteins.The MBS domain of GAKIN does not share any sequence similarity with proteins that bind to the GUK domains of MAGUKs. Indeed, our results indicate that binding of GAKIN to the GUK domain of hDlg is not regulated by intramolecular interactions of the SH3 and GUK domains (Fig. 3). Based on our observation that GKAP competes with the MBS domain of GAKIN for binding to the GUK domain of hDlg (Fig. 4), we speculate on a model that offers an explanation for at least one function of the intramolecular interactions of MAGUKs. According to this model, the MBS domain of GAKIN interacts with a "folded" state of hDlg in the cytoplasm and transports it to specialized membrane sites. This folded and thus closed state of hDlg does not permit binding with other GUK domain binders such as GKAP. Once the hDlg cargo reaches the target membrane sites, other membrane and protein interactions "unfold" the closed SH3-GUK conformation thus permitting the transfer of hDlg to another GUK domain binder such as GKAP. The final assembly of the mature scaffolding complex occurs at this site by recruitment of additional binding partners to the "open" conformation of hDlg. Further experimental verification of this model would require identification of other cargo molecules of the GAKIN-hDlg complex and further investigation as to whether novel segments of GAKIN regulate protein-protein interactions of the scaffolding complex at or during the assembly process.The data presented in this manuscript suggest that human GAKIN is a biologically active kinesin motor, providing a molecular basis for the intracellular trafficking of hDlg in mammalian cells. Our results also suggest that direct binding of GAKIN to the GUK domain of hDlg could permit transport of a soluble multiprotein complex to specialized cell-cell contact sites. Alternatively, the GAKIN-hDlg interaction may also allow trafficking of the scaffolding complex attached to the intracellular vesicles. The hDlg-bearing vesicles are then delivered to the plus end of microtubules by GAKIN. The proposed model of GAKIN-dependent transport of intracellular vesicles is also consistent with the observed punctate and vesicular distribution of hDlg/SAP97 in neuronal, epithelial, and lymphoid cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar, 44Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.-P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-157Google Scholar, 47McLaughlin M. Hale R. Ellston D. Gaudet S. Lue R.A. Viel A. J. Biol. Chem. 2002; 277: 6406-6412Google Scholar). In addition to hDlg, the GAKIN-dependent trafficking could also play a pivotal role for the transport of PSD-95, based on the fact that GAKIN binds to the GUK domain of PSD-95 and is expressed abundantly in neuronal cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar).The proposed model adds a new example to the emerging general paradigm that scaffolding adaptor proteins act as molecular links between specific motor proteins and cargo vesicles. Recent examples supporting this paradigm include the mLin-10 adaptor that links KIF17 motor to vesicles via mLin-2, mLin-7, andN-methyl-d-aspartic acid receptor subunit complex (20Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Google Scholar), the role of JIPs in connecting the conventional kinesin to cargo vesicles via the Reelin receptor (22Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Google Scholar), and also the GRIP1 mediated linkage of kinesin heavy chains to vesicles via an AMPA receptor subunit (48Setou M. Seog D.H. Tanaka Y. Kanai Y. Takei Y. Kawagishi M. Hirokawa N. Nature. 2002; 417: 83-87Google Scholar). A more recent demonstration of SAP97 binding to the minus end-directed actin motor myosin VI also suggests a mechanism for the reversible translocation of MAGUKs in vivo (49Wu H. Nash J.E. Zamorano P. Garner C.C. J. Biol. Chem. 2002; 277: 30928-30934Google Scholar). In conclusion, the identity of various components of the hDlg and/or PSD-95 cargo complex transported by GAKIN could open an area of considerable interest for the assembly, transport, and regulation of this complex and more importantly could provide insights into the mechanism of dynamic regulation of the cell-cell contact structure in normal and disease states. Human Dlg protein and its rat orthologue SAP97 are localize at specialized membrane regions where cells form contacts such as the synaptic membrane in neuronal cells (10Muller B.M. Kistner U. Veh R.W. Cases-Langhoff C. Becker B. Gundelfinger E.D. Garner C.C. J. Neurosci. 1995; 15: 2354-2366Google Scholar), adherens junctions of epithelial cells (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 43Reuver S.M. Garner C.C. J. Cell Sci. 1998; 111: 1071-1080Google Scholar), and contact sites between T lymphocyte and antigen-presenting cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). In contrast, hDlg/SAP97 is present predominantly in the cytoplasm and appears to attach to intracellular membranes in cells that do not display cell-cell contacts (43Reuver S.M. Garner C.C. J. Cell Sci. 1998; 111: 1071-1080Google Scholar, 44Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.-P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-157Google Scholar). In addition to the PDZ domains that link hDlg to the cytoplasmic domains of transmembrane receptors (4Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Google Scholar), the primary structure of hDlg contains multiple protein-protein interaction domains that interact with cytoskeletal components and signaling molecules (9Lue R.A. Marfatia S.M. Branton D. Chishti A.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9818-9822Google Scholar, 19Hanada T. Lin L. Chandy K.G. Oh S.S. Chishti A.H. J. Biol. Chem. 1997; 272: 26899-26904Google Scholar). These multiple protein interactions presumably permit hDlg to function as a scaffolding protein by forming large protein complexes at the interface of the membrane-cytoskeleton (11Marfatia S.M. Morais Cabral J.H. Lin L. Hough C. Bryant P.J. Stolz L. Chishti A.H. J. Cell Biol. 1996; 135: 753-766Google Scholar). A major remaining issue of fundamental importance pertains to the mechanism of hDlg trafficking and its delivery to specialized sites. Our original identification of GAKIN was made by virtue of its association with the GUK domain of hDlg in the context of whole cell lysate (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar), therefore a possibility remained that the GAKIN-hDlg interaction might not be direct. In this manuscript, we demonstrate that GAKIN interacts directly with the GUK domain of hDlg (Fig. 1). A unique feature of this interaction is the unusual location of the hDlg-binding region within the stalk domain of GAKIN. The traditional view of kinesin motors implies that their globular C-terminal tails usually serve as the cargo binding modules (45Klopfenstein D.R. Vale R.D. Rogers S.L. Cell. 2000; 103: 537-540Google Scholar). However, in the case of GAKIN, the MBS domain that binds to hDlg is located within the N-terminal half of GAKIN downstream of its N-terminal motor domain (Fig. 2). Thus, the C-terminal half of GAKIN with a single copy of CAP-Gly domain either serves a regulatory region for cargo binding and/or binds to distinct cargo molecules. It is noteworthy here other intracellular motors, such as the Rab6-binding kinesin Rab6-KIFL, have been speculated to harbor cargo-binding domains in their coiled-coil regions (46Hill E. Clarke M. Barr F.A. EMBO J. 2000; 19: 5711-5719Google Scholar). In any case, the presence of the MBS domain in a variety of motor and non-motor proteins suggests that this region might represent a novel cargo-binding motif with implications in linking soluble adaptors to motor proteins within the scaffolding complex. For example, the KIF13A motor transports cargo vesicles via its C-terminal tail that interacts with β1-adaptin and mannose-6-phosphate receptor (21Nakagawa T. Setou M. Seog D. Ogasawara K. Dohmae N. Takio K. Hirokawa N. Cell. 2000; 103: 569-581Google Scholar). Our identification of the MBS domain in KIF13A raises the possibility that these motors could potentially bind additional cargoes within their long stalk domains. Similarly, the presence of two MBS domains in RIM-BP1 also implicates recruitment of additional proteins in the assembly of RIM and Rab3-based trafficking machinery in the brain (38Wang Y. Sugita S. Sudhof T.C. J. Biol. Chem. 2000; 275: 20033-20044Google Scholar). In summary, our mapping data on the direct interaction between hDlg and GAKIN reveals a novel protein-binding domain that could mediate similar interactions with a large number of GUK domain proteins. The MBS domain of GAKIN does not share any sequence similarity with proteins that bind to the GUK domains of MAGUKs. Indeed, our results indicate that binding of GAKIN to the GUK domain of hDlg is not regulated by intramolecular interactions of the SH3 and GUK domains (Fig. 3). Based on our observation that GKAP competes with the MBS domain of GAKIN for binding to the GUK domain of hDlg (Fig. 4), we speculate on a model that offers an explanation for at least one function of the intramolecular interactions of MAGUKs. According to this model, the MBS domain of GAKIN interacts with a "folded" state of hDlg in the cytoplasm and transports it to specialized membrane sites. This folded and thus closed state of hDlg does not permit binding with other GUK domain binders such as GKAP. Once the hDlg cargo reaches the target membrane sites, other membrane and protein interactions "unfold" the closed SH3-GUK conformation thus permitting the transfer of hDlg to another GUK domain binder such as GKAP. The final assembly of the mature scaffolding complex occurs at this site by recruitment of additional binding partners to the "open" conformation of hDlg. Further experimental verification of this model would require identification of other cargo molecules of the GAKIN-hDlg complex and further investigation as to whether novel segments of GAKIN regulate protein-protein interactions of the scaffolding complex at or during the assembly process. The data presented in this manuscript suggest that human GAKIN is a biologically active kinesin motor, providing a molecular basis for the intracellular trafficking of hDlg in mammalian cells. Our results also suggest that direct binding of GAKIN to the GUK domain of hDlg could permit transport of a soluble multiprotein complex to specialized cell-cell contact sites. Alternatively, the GAKIN-hDlg interaction may also allow trafficking of the scaffolding complex attached to the intracellular vesicles. The hDlg-bearing vesicles are then delivered to the plus end of microtubules by GAKIN. The proposed model of GAKIN-dependent transport of intracellular vesicles is also consistent with the observed punctate and vesicular distribution of hDlg/SAP97 in neuronal, epithelial, and lymphoid cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar, 44Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.-P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-157Google Scholar, 47McLaughlin M. Hale R. Ellston D. Gaudet S. Lue R.A. Viel A. J. Biol. Chem. 2002; 277: 6406-6412Google Scholar). In addition to hDlg, the GAKIN-dependent trafficking could also play a pivotal role for the transport of PSD-95, based on the fact that GAKIN binds to the GUK domain of PSD-95 and is expressed abundantly in neuronal cells (18Hanada T. Lin L. Tibaldi E.V. Reinherz E.L. Chishti A.H. J. Biol. Chem. 2000; 275: 28774-28784Google Scholar). The proposed model adds a new example to the emerging general paradigm that scaffolding adaptor proteins act as molecular links between specific motor proteins and cargo vesicles. Recent examples supporting this paradigm include the mLin-10 adaptor that links KIF17 motor to vesicles via mLin-2, mLin-7, andN-methyl-d-aspartic acid receptor subunit complex (20Setou M. Nakagawa T. Seog D.H. Hirokawa N. Science. 2000; 288: 1796-1802Google Scholar), the role of JIPs in connecting the conventional kinesin to cargo vesicles via the Reelin receptor (22Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. J. Cell Biol. 2001; 152: 959-970Google Scholar), and also the GRIP1 mediated linkage of kinesin heavy chains to vesicles via an AMPA receptor subunit (48Setou M. Seog D.H. Tanaka Y. Kanai Y. Takei Y. Kawagishi M. Hirokawa N. Nature. 2002; 417: 83-87Google Scholar). A more recent demonstration of SAP97 binding to the minus end-directed actin motor myosin VI also suggests a mechanism for the reversible translocation of MAGUKs in vivo (49Wu H. Nash J.E. Zamorano P. Garner C.C. J. Biol. Chem. 2002; 277: 30928-30934Google Scholar). In conclusion, the identity of various components of the hDlg and/or PSD-95 cargo complex transported by GAKIN could open an area of considerable interest for the assembly, transport, and regulation of this complex and more importantly could provide insights into the mechanism of dynamic regulation of the cell-cell contact structure in normal and disease states. We thank Drs. Craig Garner and Alan Fanning for sharing their GKAP construct and MDCK cells, respectively. We are grateful to Donna-Marie Mironchuk for assistance with the artwork, Caroline Walsh for excellent editorial assistance, and Dr. Steven Oh for valuable suggestions.