to the Cell Surface - Cell Press

Molecular Cell, Vol. 3, 423–433, April, 1999, Copyright 1999 by Cell Press

A Role for a PDZ Protein in the Early Secretory Pathway for the Targeting of proTGF-a to the Cell Surface Juan Fernańdez-Larrea, Anna Merlos-Sua´rez, Jesu´s M. Urenã, Jose´ Baselga, and Joaquıń Arribas* Laboratori de Recerca Oncolo`gica Servei d’Oncologia Me`dica Hospital General Universitari Vall d’Hebron Psg. Vall d’Hebron 119-129 Barcelona 08035 Spain

Summary In general, plasma membrane integral proteins, such as the membrane-anchored growth factor proTGF-a, are assumed to be transported to the cell surface via a nonregulated, constitutive pathway. proTGF-a C-terminal mutants are retained in an early secretory compartment. Here, using a two-hybrid screen, we identify two TACIPs (proTGF-alpha cytoplasmic domain–interacting proteins) that contain PDZ domains and do not interact with proTGF-a C-terminal mutants. The binding specificity of one of them, TACIP18 (previously identified and named Syntenin or mda-9), coincides with that of the component that possibly mediates the normal trafficking of proTGF-a. TACIP18 colocalizes and interacts specifically with immature, intracellular forms of proTGF-a. Therefore, it appears that the interaction of TACIP18 with proTGF-a in the early secretory pathway is necessary for the targeting of the latter to the cell surface.

Introduction Through exhaustive genetic and biochemical analysis, it has become apparent that the subcellular location of numerous proteins along the secretory pathway largely relies on the recognition of specific signals by specialized sorting machineries. Much of the attention has been focused on the mechanisms that retain proteins in, and transport proteins between, the main secretory pathway compartments, the endoplasmic reticulum (ER), and the Golgi network. The protein components that constitute the ER contain specific signals, such as the -KDEL motif in soluble intraluminal proteins (Munro and Pelham, 1987) and the basic amino acid motif in transmembrane proteins (Letourneur et al., 1994). These ER resident proteins are either not included in transport vesicles or retrieved from the Golgi network through interaction with proteins that recognize their signals: the KDEL receptor or the COP I coatomer (reviewed in Teasdale and Jackson, 1996). Although the signals and mechanisms that maintain proteins in and transport proteins through the

* To whom correspondence should be addressed (e-mail: jarribas@ hg.vhebron.es).

Golgi network are known in less detail, signals and proteins for trans-Golgi localization are currently being elucidated (for example, see Wan et al., 1998). Thus, it is widely accepted that protein components of the ER or Golgi compartments need specific signals and mechanisms to be targeted to and maintained in their proper locations. In contrast with ER or Golgi proteins, it is frequently assumed that transmembrane proteins that reside at the cell surface are delivered to the plasma membrane via a nonregulated default pathway. However, several reports indicate the existence of specific mechanisms that transport several transmembrane proteins to the plasma membrane. For example, receptor activity modifying proteins (RAMPs) regulate not only the ligand specificity of calcitonin receptor–like receptors, but also the transport of these receptors to the plasma membrane (McLatchie et al., 1998). Also, Fas (CD95) and the neurotrophin receptor, TrkB, have been recently found to be rapidly transported to the cell surface from intracellular compartments after p53 activation and cAMP elevation, respectively (Bennett et al., 1998; Meyer-Franke et al., 1998). Therefore, at least some proteins that are usually resident at the plasma membrane, where they exert their function, need specific mechanisms to be transported to the cell surface. Commonly, mutations that prevent the transport of a transmembrane protein to the plasma membrane are interpreted to induce misfolding, leading to the retention and rapid degradation by “quality control mechanisms” of the ER (reviewed in Hammond and Helenius, 1995). Due to its biomedical importance, it has been particularly well established that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) interfere with normal folding of nascent polypeptides that are rapidly targeted for degradation by the ubiquitin– proteasome pathway (reviewed in Kopito, 1997). Mutations and truncations of the cytoplasmic domain of other transmembrane proteins that are normally located at the cell surface, such as a-1-proteinase inhibitor (Brodbeck and Brown, 1994), the tyrosine kinase receptor HER-2 (Scott et al., 1993), or the P-glycoprotein (Miura et al., 1994), have been also shown to induce ER retention, although in these and other cases it remains to be established whether these proteins are recognized as misfolded by the quality control mechanisms of the ER. Determinants found in the cytoplasmic tail of protransforming growth factor a (proTGF-a) have been also shown to control its subcellular distribution (Briley et al., 1997). TGF-a is a ligand for the epidermal growth factor receptor (EGFR) and is synthesized as part of a precursor, transmembrane, cell surface molecule: proTGF-a (Derynck et al., 1984; Lee et al., 1985). Although transmembrane proTGF-a is a functional EGFR ligand that transduces a mitogenic signal to adjacent cells (Wong et al., 1989), the ectodomain of proTGF-a can be shed from the cell surface, generating a diffusible mitogenic signal that acts at a distance from the TGF-a producing cell (Bringman et al., 1987). Through mutational analysis, the determinant necessary for the normal trafficking of

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Figure 1. Identification of Proteins that Interact with the Cytoplasmic Domain of WildType but Not with C-Terminal proTGF-a Mutants (A) Schematic of proTGF-a showing the signal sequence (black box), the TGF-a domain (shaded box), the transmembrane domain (hatched box), the site of insertion of HA or Myc epitopes, the sequence of the cytoplasmic domain, and the position of the mutation used to characterize the different cDNA clones isolated. (B) Yeasts cotransformed with a vector encoding the GAL4 AD fused to different cDNAs and an expression vector encoding the GAL4 BD alone (control) or fused to the cytoplasmic domain of wild-type or V159G proTGF-a were streaked in 2His/2Ade plates and scored for growth. (C) b-gal activity of the same yeasts was determined as described in the Experimental Procedures. The results shown are the averages 6 SD of triplicate determinations. (D) Radiolabeled alpha A1 Syntrophin and TACIP18 obtained by in vitro transcription and translation were incubated with equal amounts of wild-type proHA/TGF-a or proHA/ TGF-aV159G immunoprecipitated with antiHA antibodies from stably transfected CHO cells. Immunoprecipitates were washed and analyzed by SDS-PAGE.

proTGF-a to the cell surface has been mapped to the very C-terminal cytoplasmic amino acid, valine (Briley et al., 1997; Urenã et al., 1999). proTGF-a C-terminal mutants are retained in the ER but are not rapidly targeted for degradation by ER quality control mechanisms since their half-lives are comparable to, or even longer than, those of wild-type molecules (Briley et al., 1997; Urenã et al., 1999). C-terminal valines seem to be determinants of the subcellular distribution of diverse transmembrane proteins since mutations in the C-terminal valine also disrupt the normal trafficking of a molecule structurally and functionally unrelated to proTGF-a, the transmembrane, cell surface, membrane-type matrix metalloprotease I (MT1-MMP) (Urenã et al., 1999). One possible explanation for these results is the requirement of the C-terminal valine for correct folding, and therefore exiting of the ER, of proTGF-a, and MT1-MMP. Alternatively, mutations in the C-terminal valine could disrupt the interaction of proTGF-a with component(s) necessary for the normal trafficking of proTGF-a and MT1-MMP. Here, we present the results of a two-hybrid screen aimed to identify proTGF-a cytoplasmic domain–binding proteins, which we have named TACIPs (proTGF-alpha cytoplasmic domain–interacting proteins), involved in the trafficking of proTGF-a. Two TACIPs (referred to as 1 and 18) show a lack of interaction with a proTGF-a C-terminal mutant that does not reach the cell surface

and are identified as the PDZ proteins alpha A1 Syntrophin (Ahn et al., 1996) and Syntenin/mda-9 (Grootjans et al., 1997; Lin et al., 1998), respectively. PDZ domains are known to bind to the C terminus of a variety of transmembrane proteins. Accordingly, the PDZ domains of alpha A1 Syntrophin and TACIP18/Syntenin/mda-9 (hereafter referred to as TACIP18) are shown to be responsible for the interaction with the cytoplasmic domain of proTGF-a. Analysis of a panel of proTGF-a C-terminal mutants shows that mutations that prevent the binding to alpha A1 Syntrophin, but not to TACIP18, do not disrupt the transport of proTGF-a to the cell surface in vivo. Furthermore, the cytoplasmic domain of Syndecan-2, which is also known to bind TACIP18 (Grootjans et al., 1997), efficiently replaces that of proTGF-a, since proTGF-a/Syndecan chimeric molecules are transported to the cell surface with normal kinetics. Mutations that prevent the binding of TACIP18 to the cytoplasmic domain of Syndecans also interfere with the normal trafficking of proTGF-a/Syndecan chimeras, further supporting a role of TACIP18 in the trafficking of proTGF-a. Unlike the majority of PDZ proteins, which localize near the plasma membrane, TACIP18 localizes early in the secretory pathway. Furthermore, in vivo, TACIP18 specifically interacts with immature proTGF-a in a perinuclear area that colocalizes with ER markers. In summary, our data indicate that the binding to a PDZ protein, TACIP18, is necessary for the targeting of

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Figure 2. Domains of Alpha A1 Syntrophin and TACIP18 that Bind proTGF-a Yeasts cotransformed with an expression vector encoding GAL4 BD fused to the cytoplasmic domain of proTGF-a and an expression vector encoding the GAL4 AD/alpha A1 Syntrophin (A), GAL4 AD/TACIP18 (B), or the different deletion constructs represented were seeded in 2His/ 2Ade plates and scored for growth or assayed for b-gal activity. The results are the averages of duplicate determinations.

proTGF-a, and probably other transmembrane molecules, to the cell surface. Results Identification of Proteins that Interact with Wild-Type but Not with C-Terminal proTGF-a Mutants proTGF-a C-terminal mutants are retained in the ER of several cell lines, indicating that such mutants are misfolded or, alternatively, fail to interact with a factor necessary for the normal transport of proTGF-a to the cell surface (Briley et al., 1997). To identify possible TACIPs involved in the trafficking of proTGF-a, first we used the cytoplasmic domain of proTGF-a as a bait in a two-hybrid screen. Then, we analyzed the interaction of the TACIPs identified with C-terminal proTGF-a mutants that are retained in the ER, such as proTGF-a V159G (Figure 1A). Thus, yeasts expressing the cytoplasmic tail of proTGF-a fused to the GAL4 DNA-binding domain (GAL4 BD) were mated to yeasts transformed with a HeLa cDNA library fused to the GAL4 activation domain (GAL4 AD), and clones yielding positive interactions were selected. Whereas 76 clones produced positive phenotypes when coexpressed with the cytoplasmic tail of proTGF-a, five of them showed a lack of interaction with the cytoplasmic tail of proTGF-a containing the mutation V159G (Figures 1B and 1C; data not shown). The sequence of these clones revealed that two of them (TACIP1 and 33) contained identical inserts that corresponded to amino acids 48–507 of alpha A1 Syntrophin (Ahn et al., 1996). The remaining three clones (TACIP18, 19, and 27) contained different inserts, but all of them comprised the entire coding sequence of a protein known as Syntenin or mda-9, independently identified by two groups (Grootjans et al., 1997; Lin et al., 1998). The interactions detected were further confirmed using radiolabeled in vitro transcribed and translated alpha

A1 Syntrophin and TACIP18 and cold wild-type or mutant proTGF-a immunoprecipitated from stably transfected CHO cells. As shown in Figure 1D, the interaction between proTGF-a and both TACIPs was readily detected, indicating that the results of the two-hybrid screen were due to direct interactions. Also in agreement with the two-hybrid analysis, little or no interaction was detected between alpha A1 Syntrophin or TACIP18 and proTGF-aV159G. These results show the existence of proteins whose interaction with proTGF-a depends on the identity of the C-terminal amino acid of the latter. PDZ Domains Bind to the Cytoplasmic Tail of proTGF-a The only common feature between TACIP1/alpha A1 Syntrophin and TACIP18 is the presence of PDZ domains. PDZ domains are known to bind directly to the C terminus of certain type I transmembrane proteins (for a recent review, see Fanning and Anderson, 1998). The binding specificities of several PDZ domains have been extensively studied, and it has been shown that the C-terminal amino acid of the bound protein is a critical feature recognized by PDZ domains. Thus, we focused on the PDZ domains to make a series of deletion constructs to determine the minimal regions of alpha A1 Syntrophin and TACIP18 that interact with the cytoplasmic domain of proTGF-a. As expected, the single PDZ domain of alpha A1 Syntrophin is sufficient to bind the cytoplasmic domain of proTGF-a as judged by the Ade1/His1 phenotype and b-gal activity of yeasts expressing both proteins (Figure 2A). In contrast, both PDZs of TACIP18 are necessary for binding to proTGF-a, and only the N terminus of TACIP18 is dispensable (Figure 2B). Even small deletions in the regions that flank the PDZ domains profoundly affect the binding of TACIP18 to the cytoplasmic domain of proTGF-a (Figure 2B). This result is consistent

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Figure 3. Biosynthesis, Transport to the Cell Surface, and Subcellular Localization of Different proTGF-a Mutants (A, B, and C) CHO cells stably transfected with the different HA- or Myc-tagged constructs were pulsed for 20 min with 35S-Cys, chased for the indicated times, and lysed ([A and B] whole cell) or shifted to 48C and incubated with anti-HA antibodies to detect proHA/TGF-a at the cell surface ([A] cell surface) and lysed. Cell lysates were immunoprecipitated with anti-HA or anti-Myc antibodies, and immunoprecipitates were analyzed by SDS-PAGE and fluorography and quantified. The results shown in (C) are the averages 6 SD of triplicate determinations. (D) Cells transfected with the indicated Myctagged proTGF-a constructs were permeabilized and incubated with anti-Myc mouse monoclonal antibodies, FITC-labeled antimouse antibodies, rabbit anti-PDI polyclonal antibodies, and TRITC-labeled anti-rabbit antibodies. The images shown represent binary combined images with intermediate fluorescence corresponding to the colocalization areas of the proTGF-a mutants T157E or V159K with PDI.

with the fact that only TACIP18 clones containing the two PDZ domains were isolated in the two-hybrid screen. Recently, it has been shown that TACIP18/Syntenin binds to the cytoplasmic domain of Syndecans and that, as in the case of proTGF-a, both PDZ domains of TACIP18 are needed for this interaction (Grootjans et al., 1997). These results show that the PDZ domains of alpha A1 Syntrophin and TACIP18 are responsible for the interaction with the cytoplasmic domain of proTGF-a and that the PDZ domains of TACIP18 do not work in isolation. Biosynthesis and Transport of Different proTGF-a C-Terminal Mutants One of the mutations (V159G) that disrupts the trafficking of proTGF-a also disrupts the binding of the cytoplasmic domain of proTGF-a to the two PDZ proteins identified in the two-hybrid screen, alpha A1 Syntrophin and TACIP18, opening the possibility that the interaction with a PDZ protein is necessary for normal trafficking of proTGF-a. PDZ proteins have been classified according to the sequences they bind (see below). To identify the type of PDZ protein that possibly mediates the normal transport of proTGF-a to the cell surface, we analyzed the effect of mutations predicted to disrupt the binding of different types of PDZ domains to proTGF-a, on the trafficking of the latter.

The biosynthesis and maturation of proTGF-a have been extensively characterized by us and others (Arribas and Massague´, 1995; Briley et al., 1997; Urenã et al., 1999). proTGF-a is synthesized as a 18 kDa form that is N- and O-glycosilated to forms of 20–22 kDa during transport to the cell surface, where proteolytical removal of the glycosilated moiety takes place, leaving a cell surface 17 kDa form (Arribas and Massague´, 1995; Briley et al., 1997; Urenã et al., 1999). Consistently, pulse chase experiments performed with CHO cells stably transfected with wild-type proTGF-a allowed the detection of the expected 18, 20–22, and 17 kDa proTGF-a species located intracellularly or at the cell surface (Figure 3A). In agreement with previous reports (Urenã et al., 1999), proTGF-aV159G is not completely chased to the forms of 20–22 kDa, and little or no immunoreactive materials could be detected at the cell surface even after a 2 hr chase (Figure 4A; cell surface). The very C-terminal amino acid (position 0; V159 in proTGF-a) and the amino acid located two residues from the C terminus (position 22; T157 in proTGF-a) have been found critical for the binding of peptides or proteins to several PDZ domains (Songyang et al., 1997). All known PDZs bind proteins with hydrophobic amino acids in position 0. Therefore, PDZs have been classified according to the amino acid they bind in position 22. Type I PDZs bind to peptides with Ser or Thr in 22,


Figure 4. Analysis of the Binding of Different C-Terminal proTGF-a Mutants to Alpha A1 Syntrophin or TACIP18 Yeast coexpressing the different GAL4 BD/proTGF-a cytoplasmic domain mutants shown and alpha A1 Syntrophin or TACIP18 were seeded in 2His/2Ade plates and scored for growth or assayed for b-gal activity. The results shown in the lower panel are the averages of duplicate determinations.

while type II PDZs bind to peptides with hydrophobic amino acids in that position, preferentially Tyr or Phe (Songyang et al., 1997). To survey the transport to the cell surface of the different proTGF-a mutants in positions 0 and 22, we carried out simplified pulse chase experiments and examined the amount of 18 kDa proTGF-a intracellular precursor that is modified to the mature, cell surface, 20–22 and 17 kDa forms, after a 30 min chase. As expected, conservative mutations in position 0 had no effect (V159L), while mutations predicted to disrupt the interaction between proTGF-a and both types of PDZ domains, such as V159E and V159S (Urenã et al., 1999) or V159G, V159K, and V159M (Figures 3A and 3B) blocked the maturation of the 18 kDa proTGF-a intracellular precursor and caused the retention of these mutants in intracellular compartments that colocalize with ER markers (Figures 3B, 3C, and 3D; data not shown). However, proTGF-a mutants predicted to interact preferentially with type II PDZs (proTGF-a T157F and T157Y) showed kinetics of maturation very similar to those of wild-type or proTGF-a T157S (predicted to interact with type I PDZs) (Figure 3B; data not shown). Furthermore, mutations in position 22, such as T157A, T157E, T157L, T157M, and T157W, expected to disrupt the interaction of target molecules with any PDZ domain, did not affect the maturation and subcellular location of proTGF-a (Figures 3B and 3C), indicating that a very unusual PDZ domain with a very relaxed specificity in position 22 or, alternatively, some as yet unidentified type of molecule mediates the transport of proTGF-a.

Specificity of the PDZ Domains that Interact with the Cytoplasmic Domain of proTGF-a To determine the possible involvement of alpha A1 Syntrophin and/or TACIP18 in the trafficking of proTGF-a, we next analyzed the specificities of these PDZ proteins using a panel of proTGF-a cytoplasmic domains mutated in positions 0 and 22. As expected, the different proTGF-a C-terminal mutants in position 0 found to be retained in the ER showed a lack of interaction with alpha A1 Syntrophin or TACIP18 in two-hybrid assays (data not shown). In agreement with published results that analyzed the binding of Syntrophin to model peptides (Schultz et al., 1998), the introduction of any amino acid other than Ser or Thr in position 22 of the cytoplasmic tail of proTGF-a prevents its binding to alpha A1 Syntrophin (Figure 4). Thus, the PDZ of alpha A1 Syntrophin is classified as type I. The sequences bound by the individual PDZ domains of TACIP18 could not be determined since both PDZ domains are necessary to detect the interaction between proTGF-a and TACIP18 (Figure 4). Using the full-length molecule, we unexpectedly found that all proTGF-a mutants in position 22 except for those containing Pro or Lys showed detectable levels of interaction with TACIP18 (Figure 4), indicating that at least one of the PDZs of TACIP18 is an unusual one with a very relaxed specificity in position 22. These results indicate that the binding to alpha A1 Syntrophin is not likely necessary for the correct trafficking of proTGF-a since several proTGF-a mutants that are correctly transported to the cell surface do not bind to alpha A1 Syntrophin. In contrast, TACIP18 shows a very relaxed specificity, and its binding to proTGF-a in vitro correlates with the correct cell surface targeting of proTGF-a in vivo.

The Cytoplasmic Tail of Syndecan-2 Functionally Replaces that of proTGF-a Syntenin, a PDZ protein identical to TACIP18, has been recently identified as a protein that binds the conserved motif Phe-Tyr-Ala, found at the C terminus of the cytoplasmic domain of Syndecans (Grootjans et al., 1997; see also Figures 5A and 5B). The results presented so far indicate that the interaction with TACIP18 could be necessary for the transport of proTGF-a to the cell surface. To strengthen this hypothesis, we tested whether the cytoplasmic tail of Syndecan is able to functionally replace that of proTGF-a by making a chimeric protein, composed of the extracellular and transmembrane domains of proTGF-a fused to the cytoplasmic domain of Syndecan-2 (M/Ta-Sd2) (Figure 5C), and analyzing the maturation of such chimeric molecule. As a control, we used a chimeric protein containing the cytoplasmic tail of Betaglycan (BG), a transmembrane protein whose transport to the cell surface is independent of the presence of its cytoplasmic tail (Urenã et al., 1999). As judged by pulse chase experiments, we found that while the maturation of M/Ta-BG is severely impaired, M/Ta-Sd2 is modified with approximately the same kinetics as wild-type proTGF-a (Figures 5D and 5E), indicating that the cytoplasmic domain of Syndecan-2 mediates the trafficking of proTGF-a/Syndecan-2 chimeras. To determine whether the cytoplasmic domain of Syndecan supports the transport of the chimeric molecule

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Figure 5. Biosynthesis and Maturation of proTGF-a/Syndecan-2 Chimeras (A) Schematic of Syndecan-2 showing sequence of the cytoplasmic domain and the position of the mutations introduced. (B) Yeasts coexpressing the GAL4 AD/ TACIP18 fusion protein and the GAL4 BD fused to the different Syndecan-2 constructs were assayed for b-gal activity as described in the Experimental Procedures. The results shown are the averages 6 SD of triplicate determinations. (C) Schematic of proTGF-a/Syndecan chimeric molecules. proTGF-a sequences are represented in white and hatched patterns, Syndecan sequences are shaded, and Betaglycan sequences are depicted as vertical bars. (D and E) CHO cells stably transfected with the different chimeric molecules were pulsed for 20 min with 35S-Cys, chased for the indicated times, and lysed. Cell lysates were immunoprecipitated with anti-Myc antibodies, and immunoprecipitates were analyzed by SDS-PAGE and fluorography and quantified. The results shown in (D) are the averages 6 SD of triplicate determinations.

M/Ta-Sd2 by means of its capability to bind TACIP18, we mutated the cytoplasmic domain of Syndecan in order to disrupt its interaction with TACIP18. We chose to introduce two mutations that, according to the data presented, would possibly destabilize the interaction between the cytoplasmic domain of Syndecan-2 and TACIP18: Gly and Pro in positions 0 and 22, respectively. Both mutations virtually abolish the interaction between Syndecan-2 and TACIP18 (Figure 5B). Consistent with a role for TACIP18 in the trafficking of M/TaSd2, the percentage of 17 kDa form that is chased to the 20–22 kDa forms dramatically decreased when the chimeric mutants M/Ta-Sd2A201G and M/Ta-Sd2F199P were analyzed (Figure 5D). These results further support a role for TACIP18 in the targeting of proTGF-a to the cell surface. TACIP18 Is Localized Early in the Secretory Pathway The majority of PDZ proteins described to date interact with the C-terminal region of transmembrane proteins and localize to submembranous sites close to the

plasma membrane (Fanning and Anderson, 1998). For example, alpha A1 Syntrophin localizes to the plasma membrane in skeletal muscle cells (Peters et al., 1997). However, the results presented so far indicate that the interaction between TACIP18 and proTGF-a takes place early in the secretory pathway, probably at or near the ER, since the disruption of this interaction leads to the retention of proTGF-a in a perinuclear area that colocalizes with ER markers (Briley et al., 1997; Urenã et al., 1999; see also Figure 3D). To determine the subcellular localization of TACIP18, its colocalization with the ER resident protein, PDI (protein disulfide isomerase) was analyzed using polyclonal anti-TACIP18 antibodies (see below) and anti-PDI. As shown in Figure 6 (upper panel), a perinuclear area that colocalizes with PDI is the primary location of TACIP18 in most cells; however, some variability was found between different cell preparations. In some cells, TACIP18 localizes to structures that resemble Golgi or intermediate compartment. In fact, in some preparations, TACIP18 colocalizes with gp74 (Figure 6), a protein of the cis-Golgi that cycles through


Figure 6. Subcellular Localization of TACIP18 Hela cells were permeabilized and incubated with FITC-labeled anti-TACIP18 polyclonal antibodies, anti-PDI or anti-gp74, and TRITC-labeled anti-rabbit antibodies. The right panels show binary combined images with intermediate fluorescence corresponding to the colocalization areas of TACIP18 and PDI or gp74, respectively.

the ER and intermediate compartment (Alcalde et al., 1994). These results suggest that TACIP18 locates to early secretory compartments, an unusual subcellular distribution for a PDZ protein. TACIP18 Interacts with proTGF-a Early in the Secretory Pathway Next, we analyzed the binding and colocalization of proTGF-a to TACIP18 in vivo using a polyclonal antibody made against recombinant, bacterially expressed TACIP18. To characterize such anti-TACIP18 polyclonal antibody, we performed Western blotting and immunoprecipitation experiments. Western blotting analysis showed that anti-TACIP18 specifically recognizes a unique band in total HeLa cell extracts that comigrates with in vitro transcribed and translated TACIP18 (Figure 7A, right panel). However, immunoprecipitation of lysates from metabolically labeled HeLa cells with antiTACIP18 revealed three specific proteins. One of these proteins was identified as TACIP18 because it comigrates with the unique band detected in Western blots (Figure 7A, left panel). The other two coimmunoprecipitating proteins, of apparent molecular mass 38 kDa and 45 kDa, are presumably not immunologically related to but form a complex with TACIP18 since they are not detected by Western blotting (Figure 7A). The binding of TACIP18 and proHA/TGF-a was analyzed by double immunoprecipitation of lysates from metabolically labeled HeLa cells expressing proHA/ TGF-a, prepared using low concentration of detergents, with anti-TACIP18 and anti-HA antibodies. Using antiHA antibodies, the typical array of proHA/TGF-a species plus bands that comigrate with TACIP18, P38, and P45 are specifically immunoprecipitated (Figure 7B). The identity of TACIP18 was further confirmed by subsequent immunoprecipitation with anti-TACIP18 of the materials primarily immunoprecipitated with anti-HA antibodies as described in the Experimental Procedures

(Figure 7B). P38 and P45 were not detected in this second immunoprecipitation, further supporting the possibility that they are not immunologically related to TACIP18. Using the reciprocal approach, cell lysates were first immunoprecipitated with anti-TACIP18. At the low concentrations of detergent used, the amount of nonspecific contaminants did not allow to specifically detect P45, P38, or proHA/TGF-a species (Figure 7B). However, subsequent immunoprecipitation with anti-HA revealed the presence of the 18 kDa intracellular proTGF-a (Figure 7B; see also Figure 4A). These results indicate that TACIP18 interacts specifically with the 18 kDa intracellular proTGF-a form and strongly support a role for TACIP18 in an early secretory compartment since little or no interaction with mature, cell surface proTGF-a was detected. Finally, we analyzed by immunofluorescence the colocalization of endogenous TACIP18 with wild-type proHA/TGF-a in HeLa cells. As previously reported (Shum et al., 1994; Urenã et al., 1999), proTGF-a is localized at the cell surface and is also detectable in a perinuclear area presumably in transit to the cell surface (Figure 7C). In agreement with the results shown in Figure 6, TACIP18 localizes primarily also in a perinuclear area where it colocalizes with proTGF-a (Figure 7C). Collectively, these results show that proTGF-a and TACIP18 colocalize and physically interact in a perinuclear area that probably coincides with the ER, further supporting the importance of the binding to TACIP18, in an early compartment of the secretory pathway, for the correct targeting of proTGF-a to the cell surface. Discussion In this report, we present evidences showing that the interaction with a PDZ protein, TACIP18, is necessary for the correct targeting of proTGF-a to the cell surface. In a screen designed to find TACIPs required for the

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Figure 7. Interaction and In Vivo Colocalization of proTGF-a with Endogenous TACIP18 (A) Characterization of the polyclonal anti-TACIP18 antibody. Total cell lysates from HeLa cells or in vitro transcribed and translated TACIP18 were analyzed by Western blotting with preimmune and immune sera as indicated. Lysates from HeLa cells metabolically labeled with 500 mCi of 35S-Translabel for 1 hr were immunoprecipitated with preimmune or immune sera and analyzed by SDS-PAGE and fluorography. (B) Metabolically labeled parental or HeLa cells expressing proHA/TGF-a were lysed in low detergent concentrations and immunoprecipitated with anti-HA, anti-TACIP18 antibodies, or pre-immune sera as indicated. Immunoprecipitates were analyzed by SDS-PAGE or treated with 1% SDS, boiled, diluted, immunoprecipitated with anti-TACIP18 or anti-HA as indicated, and analyzed by SDS-PAGE and fluorography. (C) HeLa cells expressing proHA/TGF-a were permeabilized and incubated with anti-HA mouse monoclonal antibodies, FITC-labeled antimouse antibodies, rabbit anti-TACIP18 polyclonal antibodies, and TRITC-labeled anti-rabbit antibodies. The right panel represents binary combined images with intermediate fluorescence corresponding to the colocalization areas of proTGF-a and TACIP18.

normal transport of proTGF-a to the cell surface, we found two candidate PDZ proteins that interact with a wild-type, but not with a C-terminal, proTGF-a point mutant that is not transported to the cell surface. Using a panel of point mutations, we determined that only one of these PDZ proteins, TACIP18, is most likely required for the correct targeting of proTGF-a to the cell surface. Furthermore, we show that the cytoplasmic domain of Syndecan-2 supports the transport to the cell surface of proTGF-a/Syndecan chimeras by virtue of its capability to bind TACIP18. Unlike the majority of PDZ proteins, such as alpha A1 Syntrophin, that are located near the cell surface, TACIP18 is located at or near the ER where it binds proTGF-a. These results indicate that the interaction with TACIP18 is necessary for the correct exit from the ER and, hence, transport of proTGF-a to the cell surface. Possible Functions of the Cytoplasmic Domain of proTGF-a The finding that TGF-a is synthesized as a transmembrane molecule (Bringman et al., 1987) lead to speculations on the possible role of the cytoplasmic domain of

proTGF-a. Previous reports indicated that the cytoplasmic domain determines the shedding of the ectodomain of proTGF-a, for proTGF-a C-terminal mutants showed a dramatically decreased rate of ectodomain shedding (Bosenberg et al., 1992). Recent publications show that a similar set of proTGF-a C-terminal mutants are retained in or near the ER (Briley et al., 1997; Urenã et al., 1999), indicating that such proTGF-a C-terminal mutants cannot be reached by TACE, the protease possibly responsible for proTGF-a ectodomain shedding (Peschon et al., 1998), that acts at the cell surface. Furthermore, the modest percentage of C-terminal mutants that reach the cell surface are shed with kinetics identical to those observed for wild-type molecules (Urenã et al., 1999), arguing that C-terminal mutations have no direct or indirect influence on the recognition of proTGF-a by TACE. Since there are no identifiable functional motifs in the cytoplasmic domain of proTGF-a, interacting proteins have been searched previously but, to date, only an unidentified protein, p86, has been reported to bind the cytoplasmic domain of proTGF-a at the cell surface (Shum et al., 1994). Another unidentified protein, p106,


forms a complex with p86 and proTGF-a but does not seem to directly bind to the cytoplasmic domain of proTGF-a (Shum et al., 1996). This complex is also associated to a kinase activity (Shum et al., 1994). The biological significance of this complex is unknown. p86 is not likely to be a PDZ protein since its binding to proTGF-a is dependent on a cysteine pair located six amino acids from the C terminus (Shum et al., 1996). On the other hand, the apparent molecular weights of the unidentified proteins, P38 and P45, found to coimmunoprecipitate with proTGF-a and TACIP18, indicate that they are different from p106 and p86. Thus, proTGF-a apparently interacts with different proteins in the ER (TACIP18) and at the plasma membrane (p86), probably forming functionally diverse complexes. Different PDZ Proteins Bind to the Cytoplasmic Domain of proTGF-a Several transmembrane proteins have been shown to interact with diverse PDZ proteins (Fanning and Anderson, 1998). In this report, we identified two PDZ proteins, alpha A1 Syntrophin and TACIP18, that interact with the cytoplasmic domain of proTGF-a. Alpha A1 Syntrophin does not seem involved in mediating the trafficking of proTGF-a, but it is a possible proTGF-a binding protein in vivo since the C terminus of proTGF-a perfectly matches the consensus sequence found to interact with the PDZ domain of alpha A1 Syntrophin (Glu-Ser/ThrXxx-Val) (Schultz et al., 1998) and some Syntrophin isoforms and proTGF-a are expressed in the same tissues (Derynck, 1992; Songyang et al., 1997). Syntrophins are a family of modular adapter proteins thought to recruit signaling proteins to the plasma membrane through interaction with Dystrophin and members of the Dystrophin family. Transmembrane proTGF-a and EGFR interact when expressed in adjacent cells. Conceivably, alpha A1 Syntrophin could play a role modulating the signal transduced by proTGF-a. The current availability of proTGF-a mutants that interact with TACIP18 and are efficiently transported to the cell surface, but do not bind Syntrophin, will be a helpful tool in determining the functional meaning of the interaction between proTGFa and alpha A1 Syntrophin. TACIP18 Contains Two PDZ Domains Frequently, several PDZ domains are contained within a single protein and bind different proteins. For example, the three PDZs present in PDS95 can bind the C terminus of NMDA receptors, potassium channels (reviewed in Gomperts, 1996), and neurolignins (Irie et al., 1997). PSD95, therefore, provides a scaffold for spatial colocalization of functionally related proteins. TACIP18 contains two PDZs and no other recognizable motifs, making it tempting to speculate that TACIP18 puts together proTGF-a and other protein(s) that could be involved in targeting proTGF-a to the cell surface. Supporting this possibility, TACIP18 coimmunoprecipitates with two proteins, P38 and P45. However, it remains to be established whether P38 or P45 interacts with the PDZ domains of TACIP18. Using peptide libraries to analyze their specificities, PDZ domains have been grouped into two main families: type I PDZs select targets with Ser or Thr in position

22, while type II PDZs select hydrophobic or aromatic amino acids in the same position (Songyang et al., 1997). The crystal structures of a type I PDZ (the third PDZ domain of PSD-95) (Doyle et al., 1996) and a type II PDZ (the PDZ of hCASK) (Daniels et al., 1998) show that the amino acid in position 22 of the target protein interacts with the first residue of the aB1 helix of PDZ domains, typically a basic residue in type I PDZs and a nonbasic residue in type II PDZs. Thus, the residue in position 1 of the aB1 helix is considered indicative of the specificity of a given PDZ. In the case of TACIP18, both PDZs contain a nonbasic amino acid in such position (Ser in the PDZ1 and Asp in the PDZ2) indicating that both are type II PDZs. However, as shown in this report, TACIP18 interacts with the C terminus of proTGF-a (Glu-Thr-ValVal-COOH), a typical target of type I PDZs, such as the PDZ of alpha A1 Syntrophin, indicating that at least one of the PDZs of TACIP18 is an unusual one. Furthermore, one or both PDZ domains of TACIP18 accomodate a variety of amino acids that are not usually found at the C termini of PDZ-binding proteins. Thus, the PDZs of TACIP18 have two unusual characteristics: they are not functional in isolation, and at least one of them shows a very relaxed specificity in position 22. Generality of the TACIP18-Mediated Cell Surface Targeting An important issue is the generality of the mechanism found necessary for the correct targeting of proTGF-a. Two lines of evidence indicate that a similar mechanism is necessary for the targeting of other cell surface molecules unrelated to proTGF-a. The cytoplasmic tail of Syndecans support the trafficking of proTGF-a to the cell surface (this report), opening the possibility that it is also necessary for the trafficking of Syndecan. Interestingly, deletion of the cytoplasmic tail has been found to cause retention of Syndecan-1 in the ER (Miettinen et al., 1994). On the other hand, a C-terminal valine is also necessary for the correct transport to the cell surface of membrane-type matrix metalloprotease 1 (MT1-MMP) (Urenã et al., 1999). MT1-MMP C-terminal mutants are also retained in an early compartment of the secretory pathway since they are not cleaved by Furin, a protease resident in the Golgi network that removes the propetide of MT1-MMP (Urenã et al., 1999). Thus, the results of this and other papers suggest the existence of a mechanism mediated by a PDZ protein, TACIP18, necessary for the correct targeting of certain transmembrane molecules to the cell surface. Identification of proteins that interact with TACIP18 will help to elucidate this mechanism. Experimental Procedures cDNA Constructs All cDNAs encoding proteins used as a bait in two-hybrid assays were fused in frame with the DNA-binding domain of GAL4 in the pAS2-1 vector (Clontech). cDNAs coding for the cytoplasmic domain of wild-type or various carboxy-terminal proTGF-a mutants were synthesized by polymerase chain reaction (PCR) amplification using rat proTGF-a as a template. The mutations were encoded in the 39 reverse complement oligonucleotide. cDNAs coding for wild-type or mutant cytoplasmic domain of human Syndecan-2 and the cytoplasmic domain of Betaglycan were obtained by PCR amplification using a HeLa cDNA library (Clontech) or rat Betaglycan as templates and the appropiate oligonucleotides. cDNA encoding proteins used

Molecular Cell 432

as a prey were cloned in frame with the GAL4 activation domain in the vector pGAD GH (Clontech). Deletion mutants of TACIP18 (see Figure 2) were also obtained by PCR. The cDNA encoding the PDZ domain of alpha A1 Syntrophin was obtained by deleting all the alpha A1 Syntrophin coding sequence downstream the unique AatII site. To generate cDNAs encoding the different full-length proTGF-a mutants and proTGF-a/Syndecan and proTGF-a/Betaglycan chimeric molecules, we used proTGF-a tagged in the ectodomain with the Myc epitope (Urenã et al., 1999; #584) containing an EcoRI site right after the transmembrane domain of rat proTGF-a (amino acid 136) introduced using PCR and standard techniques. The EcoRI/ BamHI cDNA fragments encoding for the various wild-type and mutant cytoplasmic domains of proTGF-a, Syndecan, or Betaglycan were introduced into the proMyc/TGF-a construct subcloned into the pcDNA 3.1 Zeo(1) (Invitrogene). All final constructs were confirmed by sequencing. Yeast Two-Hybrid Screen The yeast strain PJ69-2A (Clontech) transformed with cDNA encoding the cytoplasmic tail of proTGF-a subcloned in vector pAS2-1 was mated with the strain Y187 pretransformed with a HeLa cDNA library in vector pGAD GH (Clontech), following the instructions of the manufacturer. From 1.8 3 106 diploid cells obtained by mating, 78 colonies showing strong growth on quadruple drop-out medium (2Leu/2Trp/2His/2Ade) were selected, and plasmids encoding prey proteins were recovered as described (Ling et al., 1995). To confirm interactions, the diploid strain obtained by mating PJ69-2A and Y187 was cotransformed, using the lithium acetate method (Gietz et al., 1992), with positive prey clones and empty pAS2-1 vector or the same vector containing the wild-type or V159G proTGF-a cytoplasmic tail. To test growth, three to five independent Leu1/Trp1 colonies were separately grown to saturation in 2Leu/ 2Trp liquid medium; the saturated cultures were brought together, and 5 ml of this mixed culture was dropped on solid quadruple drop out medium. To determine b-gal activity, the saturated cultures were diluted 10-fold in fresh selective medium and collected separately after cells had undergone two divisions. b-gal activity was determined on permeabilized cells as described (Guarente and Mason, 1983). Cell Culture and Transfections CHO cells expressing proHA/TGF-a or proHA/TGF-aV159G constructs have been described elsewhere (Urenã et al., 1999). The rest of the stable cell lines expressing different Myc- or HA-tagged proTGF-a constructs was obtained using the calcium phosphate precipitate method as described (Arribas et al., 1995). Transient transfections were performed using the DEAE Dextran method as previously described (Carcamo et al., 1994). Production of Polyclonal Antibodies against TACIP18 cDNA encoding TACIP18 tagged at the C terminus with six histidines was produced using standard techniques, subcloned in the bacterial expression vector pET-21b (Novagen), and transformed into BL21(DE3) cells for expression of the recombinant protein. Histagged TACIP18 was purified by immobilized-Ni21 affinity chromatography using Ni-NTA agarose from Qiagen and instructions of the manufacturer. To obtain anti-TACIP18 antibodies, 100 mg of the purified protein emulsified with equal volume of complete Freund’s adjuvant were injected into New Zealand white rabbits. After three more subcutaneous injections (100 mg each) with incomplete Freund’s adjuvant at 1 week intervals, the titer of the rabbit sera was monitored every 2 weeks. Sera titers of 1/500 to 1/1000 were obtained 2 months after the last injection.

Cell surface immunoprecipitation of the different HA- or Myctagged proTGF-a constructs were performed as previously described (Urenã et al., 1999). Transcription and translation of full-length cDNAs encoding alpha A1 Syntrophin or TACIP18 were performed using the TNT T7 coupled reticulocyte lysate system (Promega) following the instructions of the manufacturer. To examine physical interactions, radiolabeled alpha A1 Syntrophin or TACIP18 was incubated in binding buffer (PBS plus 0.1% NP40 and 1 mM MgCl) with anti-HA immunoprecipitates from lysates of 5 3 107 CHO cells expressing wild type or proHA/TGF-aV159G. Then, immunoprecipitates were washed three times with binding buffer. All samples were analyzed by SDS-PAGE and fluorography. For coimmunoprecipitation experiments, parental HeLa cells stably transfected with proHA/TGF-a were metabolically labeled with 35 S-Translabel for 1 hr, lysed in lysis buffer II (50 mM Tris-ClH (pH 7.4), 100 mM NaCl, 0.2%, 1 mM MgCl2, and 0.2% NP40), and immunoprecipitated with anti-HA monoclonal antibodies or preimmune or anti-TACIP18 immune sera as indicated. Immune complexes were recovered with protein A, and immunoprecipitates were washed three times in lysis buffer II. Then, immunoprecipitates were analyzed by SDS-PAGE or treated with 1% SDS, boiled for 5 min, diluted with PBS containing 1% NP-40 and 5 mM EDTA (final detergent concentrations, 1% NP40, 0.1% SDS), and centrifuged at 15,000 rpm for 15 min to remove insoluble materials. Supernatants were immunoprecipitated with anti-HA or anti-TACIP18 antibodies as indicated, and immunoprecipitates were analyzed by SDS-PAGE and fluorography. Confocal Microscopy Cells grown in coverslips were fixed and permeabilized as described (Urenã et al., 1999). Permeabilized cells were incubated for 45 min with 10 mg/ml of mouse monoclonal anti-HA or anti-Myc antibodies and with a 1 to 100 dilution of rabbit polyclonal antibody against the ER marker protein disulphide isomerase (PDI) or against TACIP18. Then, cells were washed with PBS and incubated for 45 min with FITC-conjugated anti-mouse and TRITC-conjugated antirabbit antibodies (Vector Laboratories) at a 1 to 100 dilution. For colocalization of TACIP18 with PDI or gp74, permeabilized cells were incubated first with a anti-PDI or anti-gp74 antibodies, then with TRITC-labeled secondary antibodies and, after extensive washing, cells were incubated with FITC-labeled anti-TACIP18 polyclonal antibodies (Harlow and Lane, 1988). Coverslips were washed with PBS, mounted, and viewed on a Leica TCS 4D laser scanning confocal microscope (Leica Lasertechnik GmbH). Acknowledgments We thank Drs. Louis M. Kunkel and Yiumo Chan for providing the full-length cDNA encoding for alpha A1 Syntrophin, Dr. Jose´ G. Castanõ and Joaquıń Oliva for the anti-TACIP18 polyclonal antibody, Dr. Ignacio Sandoval for the anti gp74 antibody and advise, Dr. Maria Martell for help with sequencing, Anna Bosch and Merce Martı´ for help with confocal microscopy, Cristina Go´mez-Martıń for assistance with computers, and Maria Borrell for critical reading of the manuscript. This work was supported by grants from the Spanish Comisioń Interministerial de Ciencia y Tecnologıá (SAF97-0229), Fundacio´ La Marato´ de TV3 (036/97), and Fundacio´ “la Caixa” (98/ 056-01) to J. A., a predoctoral fellowship from the Spanish Ministry of Education to A. M.-S., and a postdoctoral fellowship from the Fundacio´ per a la Recerca i Doce`ncia dels Hospitals Vall d’Hebron to J. F.-L. Received November 10, 1998; revised February 3, 1999. References

Metabolic Labeling, In Vitro Transcription and Translation, and Immunoprecipitation Exponentially growing CHO or HeLa cells stably transfected with the different HA or Myc proTGF-a constructs were labeled and lysed, and cell lysates were immunoprecipitated with anti-HA or anti-Myc monoclonal antibodies as previously described (Urenã et al., 1999). Quantifications were done using a BAS-1800 instrument and software.

Ahn, A.H., Feener, C.A., Gussoni, E., Yoshida, M., Ozawa, E., and Kunkel, L.M. (1996). The three human syntrophin genes are expressed in diverse tissues, have distinct chromosomal locations, and each bind to dystrophin and it relatives. J. Biol. Chem. 271, 2724–2730. Alcalde, J., Egea, G., and Sandoval, I.V. (1994). gp74 a membrane


glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment. J. Cell Biol. 124, 649–665.

Lin, J.J., Jiang, H., and Fisher, P.B. (1998). Melanoma differentiation associated gene-9, mda-9, is a human gamma interferon responsive gene. Gene 207, 105–110.

Arribas, J., and Massague´, J. (1995). Transforming growth factor alpha and beta amyloid precursor protein share a secretory mechanism. J. Cell Biol. 128, 433–441.

Ling, M., Merante, F., and Robinson, B.H. (1995). A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction. Nucleic Acids Res. 23, 4924– 4925.

Bennett, M., Macdonald, K., Chan, S.W., Luzio, J.P., Simari, R., and Weissberg, P. (1998). Cell surface trafficking of fas: a rapid mechanism of p53-mediated apoptosis. Science 282, 290–293. Bosenberg, M.W., Pandiella, A., and Massague´, J. (1992). The cytoplasmic carboxy-terminal amino acid specifies cleavage of membrane TGF-a into soluble growth factor. Cell 71, 1157–1165.

McLatchie, L.M., Fraser, N.J., Main, M.J., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M.G., and Foord, S.M. (1998). RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393, 333–339.

Briley, G.P., Hissong, M.A., Chiu, M.L., and Lee, D.C. (1997). The carboxyl-terminal residues of proTGF-a are required for its efficient maturation and intracellular routing. Mol. Biol. Cell 8, 1619–1631.

Meyer-Franke, A., Wilkinson, G.A., Kruttgen, A., Minjie, H., Munro, E., Hanson, M.G., Reichardt, L.F., and Barres, B.A. (1998). Depolarization and cAMP elevation rapidly recruits TrkB to the plasma membrane of CNS Neurons. Neuron 21, 681–693.

Bringman, T.S., Lindquist, P.B., and Derynck, R. (1987). Different transforming growth factor-a species are derived from a glycosylated and palmitoylated transmembrane precursor. Cell 48, 429–440.

Miettinen, H.M., Edwards, S.N., and Jalkanen, M. (1994). Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions. Mol. Biol. Cell 5, 1325–1339.

Brodbeck, R.M., and Brown, J.L. (1994). Study of the roles of proline 391 and a highly conserved sequence in the carboxyl-terminal region of members of the serpin family in the secretion of alpha 1-proteinase inhibitor. J. Biol. Chem. 269, 17252–17256.

Miura, Y., Kambe, F., Yamamori, I., Mori, Y., Tani, Y., Murata, Y., Oiso, Y., and Seo, H. (1994). A truncated thyroxine-binding globulin due to a frameshift mutation is retained within the rough endoplasmic reticulum: a possible mechanism of complete thyroxine-binding globulin deficiency in Japanese. J. Clin. Endocrinol. Metab. 78, 283–287.

Carcamo, J., Weis, F.M., Ventura, F., Wieser, R., Wrana, J.L., Attisano, L., and Massague´, J. (1994). Type I receptors specify growthinhibitory and transcriptional responses to transforming growth factor beta and activin. Mol. Cell. Biol. 14, 3810–3821.

Munro, S., and Pelham, H.R. (1987). A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907.

Daniels, D.L., Cohen, A.R., Anderson, J.M., and Brugner, A.T. (1998). Crystal structure of the hCASK PDZ domain reveals the structural basis of class II PDZ domain target recognition. Nat. Struct. Biol. 7, 317–325.

Peschon, J., Slack, J., Reddy, P., Stocking, K., Sunnarborg, S., Lee, D., Rusell, W., Castner, R., Johnson, R., Fitzner, J. et al. (1998). An essential role for ectodomains shedding in mammalian development. Science 282, 1281–1284.

Derynck, R. (1992). The physiology of transforming growth factor-a. Adv. Cancer Res. 58, 27–52.

Peters, M.F., Adams, M.E., and Froehner, S.C. (1997). Differential association of syntrophin pairs with the dystrophin complex. J. Cell Biol. 138, 81–93.

Derynck, R., Roberts, A.B., Winkler, M.E., Chen, E.Y., and Goeddel, D.V. (1984). Human transforming growth factor-a: precursor structure and expression in E. coli. Cell 38, 287–297. Doyle, D.A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. (1996). Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 85, 1067–1976.

Schultz, J., Hoffmuller, U., Krause, G., Ashurst, J., Macias, M.J., Schmieder, P., Schneider-Mergener, J., and Oschkinat, H. (1998). Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels. Nat. Struct. Biol. 5, 19–24.

Fanning, A.S., and Anderson, J.M. (1998). PDZ domains and the formation of protein networks at the plasma membrane. Curr. Top. Microbiol. Immunol. 228, 209–233.

Scott, G.K., Robles, R., Park, J.W., Montgomery, P.A., Daniel, J., Holmes, W.E., Lee, J., Keller, G.A., Li, W.L., Fendly, B.M. et al. (1993). A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol. Cell. Biol. 13, 2247–2257.

Gietz, D., St Jean, A., Woods, R.A., and Schiestl, R.H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 25, 1425.

Shum, L., Reeves, S.A., Kuo, A.C., Fromer, E.S., and Derynck, R. (1994). Association of the transmembranes TGF-a precursor with a protein kinase complex. J. Cell Biol. 125, 903–916.

Gomperts, S.N. (1996). Clustering membrane proteins: it’s all coming together with the PSD-95/SAP90 protein family. Cell 84, 659–662.

Shum, L., Turck, C.W., and Derynck, R. (1996). Cysteines 153 and 154 of transmembrane transforming growth factor-alpha are palmitoylated and mediate cytoplasmic protein association. J. Biol. Chem. 271, 28502–28508.

Grootjans, J.J., Zimmermann, P., Reekmans, G., Smets, A., Degeest, G., Durr, J., and David, G. (1997). Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. Proc. Natl. Acad. Sci. USA 94, 13683–13688. Guarente, L., and Mason, T. (1983). Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell 32, 1279–1286. Hammond, C., and Helenius, A. (1995). Quality control in the secretory pathway. Curr. Opin. Cell Biol. 7, 523–529. Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual, (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory). Irie, M., Hata, Y., Takeuchi, M., Ichtchenko, K., Toyoda, A., Hirao, K., Takai, Y., Rosahl, T.W., and Su¨dhof, T.C. (1997). Binding of neurolignins to PSD-95. Science 277, 1511–1515. Kopito, R.R. (1997). ER Quality control: the cytoplasmic conection. Cell 88, 427–430. Lee, D.C., Rose, T.M., Webb, N.R., and Todaro, G.J. (1985). Cloning and sequence analysis of a cDNA for rat transforming growth factor-a. Nature 313, 489–491. Letourneur, F., Gaynor, E.C., Hennecke, S., Demolliere, C., Duden, R., Emr, S.D., Riezman, H., and Cosson, P. (1994). Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell 79, 1199–1207.

Songyang, Z., Fanning, A.S., Fu, C., Xu, J., Marfatia, S.M., Chisthi, A.H., Crompton, A., Chan, A.C., Anderson, J.M., and Cantley, L.C. (1997). Recognition of unique carboxy-terminal motifs by distinct PDZ domains. Science 275, 73–77. Teasdale, R.D., and Jackson, M.R. (1996). Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the golgi apparatus. Annu. Rev. Cell Dev. Biol. 12, 27–54. Urenã, J., Merlos-Sua´rez, A., Baselga, J., and Arribas, J. (1999). The cytoplasmic carboxy-terminal amino acid determines the subcellular localization of proTGF-a and membrane type matrix metalloprotease (MT1-MMP). J. Cell Sci. 112, 773–784. Wan, L., Molloy, S.S., Thomas, L., Liu, G., Xiang, Y., Rybak, S.L., and Thomas, G. (1998). PACS-1 defines a novel gene family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94, 205–216. Wong, S.T., Winchell, L.F., McCune, B.K., Earp, H.S., Teixido´, J., Massague´, J., Herman, B., and Lee, D.C. (1989). The TGF-a precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction. Cell 56, 495–506.