Analysis of Transport and Targeting of Syndecan-1: Effect of Cytoplasmic Tail Deletions Heini M

Molecular Biology of the Cell Vol. 5, 1325-1339, December 1994 Analysis of Transport and Targeting of Syndecan-1: Effect of Cytoplasmic Tail Deletions Heini M. Miettinen,*t# Susan N. Edwards,* and Markku Jalkanen* *Centre for Biotechnology, FIN-20521 Turku, Finland; and tDepartment of Medical Biochemistry, University of Turku, FIN-20520 Turku, Finland Submitted October 6, 1994; Accepted October 26, 1994 Monitoring Editor: Richard Hynes Madin-Darby canine kidney (MDCK) cells and Chinese hamster ovary (CHO) cells were transfected with wild-type and cytoplasmic deletion mutants of mouse syndecan-1 to study the requirements for transport and polarized expression of this proteoglycan. Expression in MDCK cells revealed that wild-type syndecan-1 is directed to the basolateral surface via a brefeldin A-insensitive route. A deletion of the last 12 amino acids of the syndecan- 1 cytoplasmic tail (CT22) was sufficient to result in the appearance of mutant proteoglycans at both the basolateral and apical cell surfaces. Treatment with brefeldin A was able to prevent apical transport of the mutants. We thus propose that the C-terminal part of the cytoplasmic tail is required for steady-state basolateral distribution of syndecan-1. In CHO cells a deletion of the last 25 or 33 amino acids of the 34-residue cytoplasmic domain (CT9 and CT1, respectively) resulted in partial retention of the mutants in the endoplasmic reticulum (ER). A deletion mutant lacking the last 12 amino acids (CT22) was not retained. Interestingly, the unglycosylated core proteins of the CT9 and CT1 mutants showed a significantly lower apparent molecular weight when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than wild-type syndecan- 1. However, when CHO transfectants expressing the CT1 mutant were incubated with brefeldin A, causing fusion of the ER and Golgi, CT1 ran with an almost equally high apparent molecular weight as the wild-type molecule. This would suggest that syndecan-1 undergoes extensive post- translational modifications or forms an SDS-resistant dimer/complex after transit from the ER. INTRODUCTION (Kiefer et al., 1990; Rapraeger et al., 1991; Yayon et al., 1991). Heparin sulfate-bearing molecules at the cell surface To date, four members of the syndecan family (syn- are thought to act as coreceptors for both heparin-bind- decan-1, -2, -3, and -4) have been identified and named ing growth factors and extracellular matrix (ECM) pro- after the initial and best characterized member, syn- teins, thus participating in the regulation of cell behavior decan- 1. The extracellular domains show the greatest (for review see Rapraeger, 1993; Elenius and Jalkanen, divergence between members and, with the exception 1994). In vivo these functions may well be mediated of the sites of potential glycosaminolglycan-attachment, by the syndecans, a major family of transmembrane, are poorly conserved between species. In contrast, the and cell surface proteoglycans, which have been shown transmembrane and cytoplasmic domains of the syn- to bind a number of ECM molecules (Koda et al., 1985; decans are extremely conserved (Figure 1A) (for review Saunders and Bemfield, 1988; Sun et al., 1989; Salmi- see Bemfield et al., 1992; Jalkanen et al., 1993). In par- virta et al., 1991) and to function as coreceptors for the ticular, the cytoplasmic tail of syndecan-1 is identical binding of growth factors to tyrosine kinase receptors across the species so far examined, unlike its extracellular domain. This conservation suggests first that the cytoplasmic domain of syndecan-1 is crucial for its t Corresponding author and present address: Department of Mi- function, and second that it has a conserved role across crobiology, Montana State University, Bozeman, MT 59717. the syndecan family. To investigate the role of this do- © 1994 by The American Society for Cell Biology 1325 H.M. Miettinen et al. main, we have constructed a series of cytoplasmic dele- decan-1 in the ER and thus reduces normal processing tion mutants for murine syndecan-1 (Figure 1A) based of syndecan-1 in the Golgi. on the regions of greatest conservation between the We have also examined the effect of brefeldin A (BFA) syndecans. on trafficking of syndecan-1. BFA is a fungal metabolite It has previously been proposed that the cytoplasmic that has frequently been used as a tool to examine pro- tail of syndecan-1 binds to the intracellular cytoskeleton tein and membrane trafficking between different cellular and may thus act as an anchor between the extracellular compartments. In many cells BFA-treatment causes the matrix and cytoskeleton (Rapraeger et al., 1986). This Golgi to disperse and fuse with the endoplasmic retic- suggestion was based on results from Triton X-100 (TX- ulum (ER), resulting in accumulation of newly synthe- 100) extractions and coimmunofluorescence analysis. sized proteins in the ER (Misumi et al., 1986; Doms et Our recent data, generated with these cytoplasmic al., 1989; Lippincott-Schwartz et al., 1989). In MDCK deletion mutants, however, suggest that the insolubility cells, however, the Golgi stacks remain morphologically of syndecan-1 in TX-100 is not caused by its cytoplasmic intact during BFA exposure (Hunziker et al., 1991; tail but rather by an interaction of the glycosaminogly- Sandvig et al., 1991; Apodaca et al., 1993), whereas the can chains with detergent insoluble molecules (Mietti- trans-Golgi network forms tubular extensions that may nen and Jalkanen, 1994). fuse with the endosomes (Hunziker et al., 1991; Lip- In adult tissues syndecan-1 is principally expressed pincott-Schwartz et al., 1991; Wagner et al., 1994). BFA in epithelia, where it is localized to the basolateral sur- inhibits some apical and some basolateral sorting routes face. During development, however, high levels of syn- in MDCK cells (Low et al., 1992; Apodaca et al., 1993) decan-1 expression have also been demonstrated in and also influences basolateral to apical transcytosis mesenchymal tissues. The polarized distribution ob- (Hunziker et al., 1991; Prydz et al., 1992; Matter et al., served for syndecan-1 at the cell surface, both in culture 1993; Barosso and Sztul, 1994). Our experiments indi- (Rapraeger et al., 1986) and in tissues (Hayashi et al., cated that BFA-treatment of MDCK cells had no effect 1987), implies that the cytoplasmic domain of syndecan- on basolateral targeting of wild-type syndecan-1. In 1 may contain targeting signals. contrast, BFA treatment of MDCK transfectants ex- To examine whether precise requirements for the tar- pressing mutant syndecan-1 prevented the mistargeting geting and processing of syndecan-1 can account for to the apical surface and restored the wild-type baso- the extreme conservation of the cytoplasmic tail of syn- lateral localization. In CHO cells BFA led to fusion of decan-1, we have expressed wild-type and three cyto- the ER and Golgi, thus reversing some effects of the ER plasmic deletion mutants in Madin-Darby canine kidney retention of the CT9 and CT1 mutants. (MDCK) epithelial cells and Chinese hamster ovary (CHO) fibroblasts. MATERIALS AND METHODS MDCK cells were used as a model system to investigate targeting of syndecan-1 in polarized cells. In po- Expression Vectors and Transfections larized cells newly synthesized proteins are either 1) For stable expression in MDCK cells, inserts coding for wild-type or MDCK mutant mouse syndecan-1 were subcloned into the EcoRI site of pBGS, delivered directly to their final destination (e.g., a vector containing an SRa promoter (Takebe et al., 1988) and a neo- cells), 2) transported first to the basolateral domain from mycin-resistance cassette (a gift from Bruce Granger, Montana State which apical proteins are transcytosed to the apical side University, Bozeman, MT). The constructs were transfected into the (e.g., hepatocytes), or 3) transported to the target do- cells using commercial lipofectin (GIBCO/BRL, Grand Island, NY) mains both routes (e.g., CaCo-2 intestinal cells) (Felgner et al., 1987) or transfectace according to Rose et al. (1991). by (for Transfectants were selected with 0.5 mg/ml Geneticin (G418, Sigma, review see Mostov et al., 1992; Matter and Mellman, St. Louis, MO). Single colonies were isolated using cloning cylinders 1994). In this paper we have shown that a deletion of and tested for expression by immunofluorescence. the last 12 amino acids from the 34-residue cytoplasmic For stable expression in CHO cells, cDNAs encoding wild-type or tail of syndecan-1 resulted in increased mistargeting of mutant mouse syndecan-1 were subcloned into the EcoRI site of the domain. This altered amplifiable expression vector pFRSV (Simonsen and Levinson, 1983; the molecule to the apical polar- Horwich et al., 1985; Miettinen et al., 1989). Transfections were per- ization was not due to changes in shedding or ECM formed as above, and stably expressing cells were selected by growth binding. in 0.2 ,uM methotrexate. The vector was amplified with increasing Previous studies have shown that partial or complete concentrations of methotrexate (final concentration 20 MM). deletions of the cytoplasmic tail of some viral and en- dogenous eukaryotic proteins resulted in slower trans- Oligonucleotide-directed Mutagenesis port of the proteins from the ER to the Golgi (for ex- cDNAs encoding mouse syndecan-1 mutants lacking parts of the cy- amples see Rose and Bergman, 1983; Wills et al., 1984; toplasmic tail were created by oligonucleotide-directed mutagenesis Zuniga and Hood, 1986). Using the

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