Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor
2002; Springer Nature; Volume: 21; Issue: 18 Linguagem: Inglês
10.1093/emboj/cdf487
ISSN1460-2075
Autores Tópico(s)Pancreatic function and diabetes
ResumoArticle16 September 2002free access Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor Sanjay K. Mishra Sanjay K. Mishra Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Peter A. Keyel Peter A. Keyel Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Matthew J. Hawryluk Matthew J. Hawryluk Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Nicole R. Agostinelli Nicole R. Agostinelli Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Simon C. Watkins Simon C. Watkins Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Linton M. Traub Corresponding Author Linton M. Traub Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Sanjay K. Mishra Sanjay K. Mishra Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Peter A. Keyel Peter A. Keyel Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Matthew J. Hawryluk Matthew J. Hawryluk Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Nicole R. Agostinelli Nicole R. Agostinelli Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Simon C. Watkins Simon C. Watkins Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Linton M. Traub Corresponding Author Linton M. Traub Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA Search for more papers by this author Author Information Sanjay K. Mishra1, Peter A. Keyel1, Matthew J. Hawryluk1, Nicole R. Agostinelli1, Simon C. Watkins1 and Linton M. Traub 1 1Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, S325BST, Pittsburgh, PA 15261 USA ‡S.K.Mishra, P.A.Keyel, M.J.Hawryluk and N.R.Agostinelli contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4915-4926https://doi.org/10.1093/emboj/cdf487 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Clathrin-coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the μ2 subunit of the AP-2 adaptor complex, which recognizes tyrosine-based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled-2 (Dab2) recognizes the FXNPXY internalization motif in LDL-receptor family members via an N-terminal phosphotyrosine-binding (PTB) domain. Here, we show that in addition to binding AP-2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP-2 and clathrin-binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo-specific adaptor proteins, including β-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events. Introduction Clathrin-coated buds only assemble at the cell surface to convey select macromolecules into the cell interior. To gain entry into the clathrin coat, many transmembrane proteins utilize short endocytic sorting determinants located within the cytosolic domain. The best understood internalization signal employs the tyrosine-based YXXØ sequence, where Ø represents a residue with a bulky hydrophobic side chain (Bonifacino and Dell'Angelica, 1999). This particular class of internalization signals binds directly to the μ2 subunit of the AP-2 adaptor heterotetramer (Ohno et al., 1995). Structurally, the interaction occurs via an extended YXXØ sequence engaging the μ2 β-sandwich in a β-sheet augmentation, with specificity provided by two hydrophobic pockets that accommodate the key tyrosine and bulky hydrophobic side chains (Owen and Evans, 1998). As AP-2 also binds to clathrin directly, these findings explain nicely how the adaptors can efficiently coordinate clathrin lattice assembly with cargo selection. It is clear that not all cargo molecules cluster into clathrin-coated buds by direct engagement of the adaptor μ2 subunit. Heptahelical G protein-coupled receptors utilize an alternate system centered around β-arrestin (Miller and Lefkowitz, 2001). Normally soluble, β-arrestin translocates onto activated, and thus phosphorylated, heptahelical receptors while synchronously interacting with both clathrin and the β2 subunit of AP-2 (Goodman et al., 1996; Laporte et al., 1999, 2000). The multivalent attachments involving β-arrestin recruit activated receptors into assembling clathrin-coated regions for downregulation. Thus, β-arrestin meshes the receptor with the standard cellular endocytic machinery (Santini et al., 2002; Scott et al., 2002). A role akin to that of β-arrestin for alternate classes of cargo molecules is probably fulfilled by other proteins. Mutagenic inactivation of the μ2 subunit potently blocks transferrin receptor uptake (which uses a YTRF internalization motif), but fails to affect epidermal growth factor (EGF) receptor endocytosis (Nesterov et al., 1999). This indicates that the sorting of transferrin and EGF receptors into clathrin buds is mechanistically distinct. In fact, when one is overexpressed, transferrin, EGF and low density lipoprotein (LDL) receptors do not appear to compete with each other for incorporation into clathrin-coated vesicles, despite all containing tyrosine-based internalization signals (Warren et al., 1998). Dedicated connector proteins, functionally analogous to β-arrestin, were postulated to account for the independent selection of these proteins into clathrin coats at the cell surface (Warren et al., 1998). Evidence for one additional connector-type protein comes from genetic disruption of UNC-11, the Caeno rhabditis elegans ortholog of AP180 (Nonet et al., 1999). Remarkably, in unc-11 mutant worms, synaptobrevin/VAMP fails to be appropriately recycled together with the synaptic vesicle membrane following exocytosis. Instead, the protein diffuses widely over the presynaptic plasma membrane (Nonet et al., 1999). The size distribution of neuronal clathrin-coated vesicles is also aberrant in these worms, suggesting that UNC-11 could integrate cargo selection temporally with clathrin-coat assembly. The N-terminal ENTH domain of AP180 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). With the adjacent AP-2- and clathrin-binding segment of the protein, the ENTH domain facilitates the assembly of highly ordered, invaginated clathrin-coated buds in the presence of soluble AP-2 and clathrin (Ford et al., 2001). We have recently shown that two endocytic accessory proteins, epsin and huntingtin-interacting protein 1 (HIP1), each of which has similar overall domain organization to AP180, can also promote optimal clathrin-coat assembly upon liposome templates (Mishra et al., 2001). These proteins too could be cargo-selective endocytic connectors. The NPXY internalization motif within the LDL receptor might utilize an alternate connector-type protein because transferrin receptor overexpression does not interfere with LDL receptor internalization (Warren et al., 1998). One candidate connector is Disabled-2 (Dab2). Dab2 contains an N-terminal phosphotyrosine-binding (PTB) domain with a marked preference for non-phosphorylated NPXY motifs, as is found in the LDL receptor and amyloid precursor protein (Morris and Cooper, 2001). At steady state, the intracellular distribution of Dab2 co-localizes well with AP-2 and clathrin. Dab2 binds to AP-2 directly via a central portion that, like epsin, AP180 and HIP1, engages the independently folded appendage domain of the AP-2 α-subunit (Morris and Cooper, 2001). We show here that Dab2 also binds robustly to clathrin, independently of the AP-2 association, and, like other PTB domains, the PTB domain of Dab2 associates directly with phosphoinositides. With synthetic liposome templates, Dab2 drives clathrin-coat assembly in the presence of purified AP-2 and clathrin and, consequently, displays all the properties of a cargo-specific adaptor protein. Results Dab2 binds the AP-2 α-subunit appendage directly using the same interaction surface engaged by epsin, eps15, amphiphysin, HIP1 and AP180 (Morris and Cooper, 2001). Tandem DPF triplets, positioned at 293DPF RDDPF, are potential interaction motifs for the α-appendage, yet amino acids 206–350 of murine Dab2 fused to glutathione S-transferase (GST) bind to soluble AP-2 relatively weakly (Morris and Cooper, 2001) (Figure 1B, lanes d). A GST fusion protein of Dab2 residues 206–492 appears to bind to AP-2 at least 4-fold more avidly (Morris and Cooper, 2001) (Figure 1B, lanes h), indicating that this distal region also contributes to the interaction of Dab2 with AP-2. We recently characterized an alternate α-appendage-binding sequence, the FXDXF motif (Brett et al., 2002), and Dab2 contains the sequence 480FLDLF within the distal segment. To explore the distal AP-2-binding determinant(s) further, we prepared a limited series of truncated Dab2 fusions (Figure 1A). Amino acids 206–368 is the minimal sequence required for optimal AP-2 binding in pull-down-type assays (Figure 1B, lanes f). Surprisingly, the removal of the FXDXF motif has no discernible effect on AP-2 binding, indicating that this sequence is not critical for the α-appendage association measured by these assays. The additional appendage-binding sequences in Dab2 remain to be characterized. Figure 1.Dab2 binds to both the AP-2 adaptor complex and clathrin. (A) Schematic illustration of the overall domain organization of Dab2 and the various GST–Dab2 constructs used. Phosphoinositide-, AP-2- and clathrin-binding properties of each fusion protein are indicated qualitatively. (B) Approximately 50 μg of either GST (lanes a and b), GST–Dab2(206–350) (lanes c and d), GST–Dab2(206–368) (lanes e and f) or GST–Dab2(206–492) (lanes g and h) immobilized on GSH–Sepharose were incubated with rat brain cytosol. After centrifugation, aliquots corresponding to 1/60 of each supernatant (S) and 1/5 of each washed pellet (P) were resolved by SDS–PAGE and either stained with Coomassie Blue or transferred to nitrocellulose. Portions of the blots were probed with the anti-AP-2 α-subunit mAb 100/2, anti-AP-2 μ2-subunit antiserum, the anti-clathrin heavy chain (HC) mAb TD.1 or the anti-clathrin light chain (LC) mAb Cl 57.3. The position of the molecular mass standards (in kDa) is indicated on the left and only the relevant portion of each blot is shown. Download figure Download PowerPoint Dab2 interacts directly with clathrin triskelia The central region of murine Dab2 also binds to a very prominent ∼180 kDa polypeptide in the pull-down assays (Figure 1B). Appropriate antibodies identify this protein as the clathrin heavy chain (Figure 1B). The smallest GST–Dab2 fusion tested, encoding only residues 206– 258, binds to clathrin (Figure 2, lanes d). Longer Dab2 segments fused to GST bind to soluble clathrin with noticeably greater affinity (Figure 2, lanes h) and remove trimers from cytosol more completely (lanes g compared with a and c). As little AP-2 binds to the GST–Dab2 (206–258) protein (lanes d), the interaction with clathrin is largely independent of the adaptor complex and may well be direct. Indeed, isolated GST–Dab2(206–258) binds to pre-assembled clathrin in vitro and, upon centrifugation, is recovered together with the polyhedral cages in the pellet (Figure 3, lane j). GST (Figure 3, lanes e and f) does not sediment appreciably with the cages (lane f) under the same conditions. In the absence of the clathrin, both GST (Figure 3, lane c) and the GST–Dab2 fusion (lane g) remain soluble and appear in the supernatant fraction, together with the carrier BSA. Similarly, a larger GST– Dab2 fusion (residues 206–350) binds to the assembled cages (Figure 3, lane n), but does not sediment significantly in the absence of clathrin (lane l). Figure 2.A clathrin-binding region within Dab2. Approximately 50 μg of either GST (lanes a and b) or GST–Dab2(206–258) (lanes c and d), GST–Dab2(206–258) (LVD→AAA) (lanes e and f), GST–Dab2(206–368) (lanes g and h), GST–Dab2(206–368) (LVD→AAA) (lanes i and j) or GST–Dab2(206–368) (LVD→AAA/W→A) (lanes k and l) immobilized on GSH–Sepharose were incubated with rat brain cytosol. After centrifugation, aliquots corresponding to 1/50 of each supernatant (S) and 1/5 of each washed pellet (P) were resolved by SDS–PAGE and either stained with Coomassie Blue or transferred to nitrocellulose. Portions of the blots were probed with the anti-AP-2 α-subunit mAb 100/2, anti-AP-2 μ2-subunit antiserum, the anti-clathrin HC mAb TD.1 or the anti-clathrin LC mAb Cl 57.3. Download figure Download PowerPoint Figure 3.Dab2 associates directly with assembled clathrin cages. Pre-assembled clathrin cages (∼0.5 μM), GST, GST–Dab2(206–258), GST–Dab2(206–350) (each ∼1 μM) or combinations thereof were incubated in MES–OH buffer on ice. After centrifugation, aliquots corresponding to 1/10 of each supernatant (S) or 1/8 of each pellet (P) were analyzed by SDS–PAGE and stained with Coomassie Blue. Download figure Download PowerPoint Sequence analysis reveals a putative type I clathrin-box sequence, 236LVDLN, located within the minimal segment of Dab2 (residues 206–258) that binds clathrin efficiently. To assess the contribution of this motif to clathrin binding, the first three residues of the sequence were mutated to Ala (LVDLN→AAALN). In the context of GST–Dab2(206– 258), the smallest clathrin-binding fragment, disruption of this type I sequence almost completely ablates clathrin association (Figure 2, lanes f compared with d). Introducing the LVDLN→AAALN substitution within a larger GST– Dab2(206–368) fusion has little effect on clathrin binding, however (Figure 2, lane j). The sequence 363PWPYP is similar to the type II clathrin-binding sequence 381PWDLW found in amphiphysin I and II (Ramjaun and McPherson, 1998; Slepnev et al., 2000). In amphiphysin, substitution of the proximal Trp with Ala completely inactivates the binding properties (Drake and Traub, 2001). Simultaneous mutation (LVD→AAA/W→A) of both the type I LVDLN and putative type II PWPYP sequences in GST–Dab2(206–368) reduces clathrin binding 4- to 5-fold in pull-down assays. The interaction of this Dab2 fusion with AP-2 is not similarly affected (Figure 2, lane l). These experiments clearly indicate that Dab2 contains multiple, tandemly arrayed clathrin-binding sequences. When Dab2 is immunoprecipitated from cytosolic extracts, clathrin trimers do not co-precipitate in significant amounts (not shown). However, we also fail to detect significant co-immunoprecipitation of clathrin with either epsin 1 (not shown) or AP-2 (Arneson et al., 1999), two proteins known to engage clathrin at coat assembly sites using clathrin-binding sequences related to those present in Dab2 (Drake et al., 2000; Dell'Angelica, 2001; Kalthoff et al., 2002). Previously, we have shown that tandemly arranged type I and type II sequences allow amphiphysin to bind to the terminal domain of the clathrin heavy chain and polymerize trimers into sedimentable assemblies (Drake and Traub, 2001). Similarly, if a GST–Dab2(1–368) fusion is mixed with soluble clathrin trimers, a portion of both proteins is recovered in a high-speed pellet after dialysis into either MES–OH buffer (pH 6.8; not shown) or a more physiological assay buffer (pH 7.2; Figure 4A, lane o). Substantially lower levels of the clathrin alone (Figure 4A, lane c) or the GST–Dab2(1–368) alone (lane l) sediment under the same conditions. Electron microscope (EM) examination of the Dab2 assembled population after negative staining reveals regular assemblies of polyhedral clathrin cages (Figure 4B). Despite binding to pre-assembled cages (Figure 3), the minimal clathrin-binding Dab2 fusion GST–Dab2(206–258) does not increase clathrin recovery in the high-speed pellet above background (lane i compared with c). GST is also unable to induce clathrin-coat assembly (data not shown). Compared with the GST–dimer form, thrombin-cleaved monomeric Dab2(206–350) shows a reduced capacity to assemble clathrin cages (not shown). However, monomeric Dab2(1–368) effectively recruits soluble clathrin trimers onto a membrane surface (see Figure 8D). Together, we believe our data show that in addition to AP-2 (Morris and Cooper, 2001), Dab2 binds to clathrin directly, interacts with pre-assembled clathrin cages and, by utilizing multiple binding sites, engages soluble clathrin trimers to productively assemble closed clathrin cages. Figure 4.Dab2 assembles soluble clathrin trimers into spherical polyhedral cages. (A) Clathrin (∼0.2 μM), GST–Dab2(206–258) (∼1 μM), GST–Dab2(1–368) (∼1 μM) or combinations thereof were mixed in 0.5 M Tris–HCl pH 7.0 and then dialyzed overnight against assay buffer. The samples were subject to differential centrifugation generating a low-speed pellet (PL) containing aggregated material, a high-speed supernatant (SH) containing soluble protein, and a high-speed pellet (PH) with the sedimentable assemblies. (B) Representative EM micrograph of a negatively stained aliquot of the high-speed pellet obtained from an incubation of clathrin with GST–Dab2(1–368) after resuspension in assay buffer on ice. Bar = 100 nm. Download figure Download PowerPoint Figure 5.Dab2 co-operates with AP-2 to drive the assembly of invaginated clathrin buds upon lipid membranes. Clathrin was added to 10% PtdIns(4,5)P2-containing lipid monolayers pre-incubated in the absence (A, inset) or presence (A and B) of GST–Dab2(1–368). An EM grid was used to remove each monolayer and then negatively stained. Bar = 50 nm. (C) Phosphoinositide-containing liposomes were first pre-incubated with AP-2 (lanes c, d, g–j and m, n), GST–Dab2(1–368) (lanes e–j) or GST–epsin 1(1–407) (lanes k–n) at 4°C for 60 min as indicated. After recovery by centrifugation, each liposome pellet was resuspended and then incubated at 4°C for 60 min with purified clathrin trimers in the presence of carrier BSA. After centrifugation, aliquots of 1/40 of each supernatant (S) and 1/4 of each pellet (P) were resolved by SDS–PAGE and stained with Coomassie Blue. Before centrifugation, Triton X-100 (1% final) was added in one reaction (lanes i and j). (D) PtdIns(4,5)P2-containing liposomes were first pre-incubated with (lanes a–j) or without (lanes k and l) thrombin-cleaved Dab2(1–368) at 4°C for 60 min. After recovery by centrifugation, each liposome pellet was resuspended and then incubated at 4°C for 60 min with increasing amounts of purified clathrin trimers in the presence of carrier BSA. After centrifugation, aliquots of 1/25 of each supernatant (S) and 1/5 of each pellet (P) were resolved by SDS–PAGE and stained with Coomassie Blue. Download figure Download PowerPoint Dab2 binds to phosphoinositides via the N-terminal PTB domain The capacity of the central region of Dab2 to interface with the core endocytic machinery is reminiscent of other so-called endocytic accessory proteins, including AP180, amphiphysin, epsin and HIP1. Indeed, in HeLa cells, epsin 1 and Dab2 show extensive co-localization at steady state (Figure 5), indicating that the two proteins are probably present within common clathrin-coated structures. AP180, epsin and HIP1 utilize an ENTH domain to interact with PtdIns(4,5)P2 and, tethered to a phosphoinositide-containing membrane surface, each protein can assist AP-2 in the assembly of clathrin-coated buds (Ford et al., 2001; Mishra et al., 2001). In Dab2, a PTB domain replaces an ENTH domain (Xu et al., 1995). PTB domains are structurally related to the pleckstrin homology (PH) domain, a known phosphoinositide-binding fold. In fact, the PTB domains of Shc (Ravichandran et al., 1997), IRS-1 (Takeuchi et al., 1998), Dab1 (Howell et al., 1999) and numb (Dho et al., 1999) all bind to phosphoinositides, leading us to test whether the Dab2 PTB domain similarly associates with phosphoinositides. In binding assays with mixed lipid liposomes, the Dab2 PTB domain (amino acids 1–205) fused to GST binds robustly to phosphoinositide-containing membranes in a dose-dependent fashion (Figure 6A, lanes d and f). The extent of the association with phosphoinositides is similar to an epsin 1 ENTH domain–GST fusion (Figure 6A, lane h) (Itoh et al., 2001; Mishra et al., 2001), while PTB-domain binding to liposomes devoid of phosphoinositides (lane b), or GST association with inositide-containing liposomes (lane j), is extremely poor. The monomeric PTB domain, cleaved from GST with thrombin, binds ∼25-fold better to PtdIns(4,5)P2 liposomes, rising from ∼2% without phosphoinositide to >50% bound (Figure 6B, lanes a, b compared with e, f). This suggests that the affinity of the Dab2 PTB domain for phosphoinositides is not massively increased by artificial GST-mediated dimerization. Protein–lipid overlays reveal that the Dab2 PTB domain displays a preference for PtdIns(4,5)P2, although there is binding to several phosphoinositides in this type of assay (Figure 6C). Figure 6.Dab2 co-localizes with epsin 1 at the plasma membrane. Saponin-permeabilized HeLa cells were fixed and probed with antibodies against Dab2 (A) and epsin 1 (B). The merged color images (C–E) show that areas of overlap are extensive (yellow), but there are clear regions containing each protein alone. Single 0.3 μm confocal sections of the ventral surface (D) and the medial region of the cell (E) through the nucleus (N) show Dab2 co-localized with epsin at the cell surface (arrows). The arrowhead indicates a pre/post-mitotic cell where the Dab2 staining pattern is principally nuclear, although some epsin is still visible at the cell surface; we note that the intracellular distribution of Dab2 appears to alter with the cell cycle. Importantly, the anti-Dab2 mAb used here detects all three splice isoforms of Dab2 (p67, p93 and p96), so we cannot conclude whether the different locations represent distinct Dab2 isoforms. Bar = 25 μm in (A–C) and 10 μm in (D) and (E). Download figure Download PowerPoint Figure 7.The PTB domain of Dab2 binds to polyphosphoinositides. (A) GST–Dab2(1–205) (lanes a–f), GST–epsin 1(1–163) (lanes g and h) or GST (lanes i and j), as indicated, were mixed with either control (lanes a and b) or phosphoinositide-containing (lanes c–j) liposomes and incubated together on ice for 60 min. After centrifugation, aliquots of 1/25 of each supernatant (S) or 1/4 of each pellet (P) were analyzed by SDS–PAGE and stained with Coomassie Blue. (B) Thrombin-cleaved Dab2(1–205) (lanes a–f) or GST (lanes g and h) was mixed with either control (lanes a and b) or phosphoinositide-containing (lanes c–j) liposomes and incubated on ice. After centrifugation, aliquots of 1/25 of each supernatant (S) or 1/4 of each pellet (P) were analyzed by SDS–PAGE and stained with Coomassie Blue or transferred to nitrocellulose. The blot was probed with an anti-Dab2 mAb. The asterisk denotes a stable ∼25 kDa thrombin degradation product that binds phosphoinositides and reacts with the anti-Dab2 mAb 52. (C) Nitrocellulose-immobilized phosphoinositides (100, 50, 25, 12.5, 6.2, 3.1, 1.5 pmol/spot) were incubated with GST–Dab2(1–205) PTB domain and then bound protein visualized with GSH-derivatized HRP. (D) The PTB domain of Dab2 binds to phosphoinositides and FXNPXF internalization motifs synchronously. Thrombin-cleaved Dab2(1–205) (PTB domain) was added to 3 μM, together with 0.4 mg/ml phosphoinositide-containing liposomes alone (lanes a and b) or liposomes plus either 15 μM NWRLKNINSIFDNPVYQKTT (lanes c and d) or NWRLKNINSIFDAPVAQKTT (lanes e and f) peptide and incubated on ice for 60 min. After centrifugation, aliquots of 1/25 of each supernatant (S) or 1/4 of each pellet (P) were analyzed by SDS–PAGE and stained with Coomassie Blue. The bound peptide migrates at the dye front and the asterisk denotes the stable ∼25 kDa Dab2 degradation product of the PTB domain. Download figure Download PowerPoint PTB domains are known to bind directly to the sequence NPXpY (Margolis, 1996), but for Dab1 and Dab2, the PTB designation is a misnomer as both bind to FXNPXY preferentially in the non-tyrosine-phosphorylated state (Howell et al., 1999; Morris and Cooper, 2001). Dab2 PTB association with phosphoinositide-containing mixed liposomes does not preclude NPXY sequence engagement. Addition of a 3- to 5-fold molar excess of a peptide corresponding to residues 2–22 of the LDL receptor cytosolic domain (814NWRLKNINSIFDNPVYQKTT) does not diminish the extent of monomeric PTB domain association with the liposomes (Figure 6D, lane d compared with b). Rather, the wild-type peptide is recovered in the liposome pellet, along with PTB domain (lane d). On standard SDS–PAGE, the bound peptide migrates at the dye front but Tris–Tricine gels confirm the identity of the 2.7 kDa peptide (not shown). The Dab1 PTB domain also binds lipid and NPXY peptides simultaneously (Howell et al., 1999) and, like Dab1, mutation of an invariant Phe (F166V) involved in FXNPXY engagement (Howell et al., 1999; Hocevar et al., 2001) does not prevent phosphoinositide binding (not shown). If an altered peptide, NWRLKNINSIFDAPVAQKTT, is included, negligible amounts of the NPXY-sequence-altered peptide sediment with the liposome/PTB domain pellet (Figure 6D, lane f). Because alteration of the Asn and Tyr side chains in the LDL receptor FXNPXY internalization sequence disrupts endocytic uptake (Chen et al., 1990), the sequence determinants necessary for the LDL receptor peptide to engage the membrane-bound Dab2 PTB domain are the same as those required for LDL receptor internalization. If the Dab2 PTB domain can engage the FXNPXY internalization sequence in vivo, overexpression of this segment of Dab2 might produce a selective block in LDL receptor internalization. Transient transfection of COS-7 cells with myc epitope-tagged Dab2(1–205) (PTB domain) had little effect on LDL receptor distribution (not shown). The phosphatidylinositol 3-phosphate-binding FYVE domain only targets efficiently to the appropriate intracellular site when expressed in tandem (Gillooly et al., 2000), so we also examined the effect of myc-Dab2 PTBx2 overexpression. Two populations of labeled cells are seen after a 15 min pulse with diI-LDL (Figure 7). The majority of the cells display bright perinuclear punctae, corresponding to endosomes (Figure 7A). A minority of cells display diffuse surface diI-LDL labeling with little or no evidence of accumulation within intracellular structures. These cells invariably express the myc-PTBx2 construct (Figure 7B–D), which also appears, at least in part, associated with the plasma membrane. Figure 8.Dab2 PTB domain overexpression selectively interferes with LDL uptake. COS-7 cells transiently transfected with myc-tagged Dab2 PTBx2 were pulsed for 15 min with 20 μg/ml diI-LDL alone (A–D), both diI-LDL and 25 μg/ml biotinylated transferrin (E) or transferrin alone (F–H) for 15 min prior to fixation. Transfected cells were identified using mAb 9E10 (B–D, G and H, in green in C, D and H) and transferrin with Alexa 488– (E) or Alexa 594–streptavidin (F and H). A single confocal section of triple-labeled cells is shown in (E) (myc epitope, blue; transferrin, green; LDL, red). Download figure Download PowerPoint Importantly, the PTB domain only affects LDL receptor uptake, and does not produce a global perturbation in endocytic activity of transfected cells. PTBx2-expressing cells, unable to internalize diI-LDL during a 15 min pulse, nevertheless take up transferrin normally compared with the surrounding, untransfected cells (Figure 7E–H). These results verify the capacity of the Dab2 PTB domain to engage the LDL receptor family FXNPXY internalization motif in vivo. Dab2 as an endocytic adaptor protein We interpret our findings to indicate that the N-terminal PTB domain
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