©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Metabolism of the Amyloid Precursor-like Protein 2 in MDCK Cells
POLARIZED TRAFFICKING OCCURS INDEPENDENT OF THE CHONDROITIN SULFATE GLYCOSAMINOGLYCAN CHAIN (*)

Amy C. Y. Lo (1) (3), Gopal Thinakaran (3) (2), Hilda H. Slunt (3) (2), Sangram S. Sisodia (3) (2)(§)

From the (1) Departments of Neuroscience and (2) Pathology and the (3) Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Deposition of -amyloid peptide in senile plaques is a principal neuropathological hallmark of Alzheimer's disease. -Amyloid peptide is derived from larger amyloid precursor proteins. Amyloid precursor protein is a member of a family of integral membrane glycoproteins that includes amyloid precursor-like protein (APLP) 1 and 2. Alternatively spliced pre-mRNAs encode several APLP2 isoforms; the APLP2-751 isoform is a substrate for modifications by a chondroitin sulfate glycosaminoglycan (CS GAG) chain, whereas the APLP2-763 isoform does not undergo CS GAG modification. In this report, we have examined the sorting and metabolism of APLP2-751 and APLP2-763 in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that, despite differences in post-translational modifications, both the APLP2-751 proteoglycan and APLP2-763 isoform were targeted and secreted to the basolateral compartment of MDCK cells. We document that the kinetics of intracellular maturation of full-length forms and secretion of soluble derivatives generated from each isoform were indistinguishable. Our results are consistent with the view that, in MDCK cells, the CS GAG chain of APLP2 has little influence on intracellular trafficking and that the principal basolateral targeting determinants are likely to reside in the APLP2 core protein.


INTRODUCTION

Amyloid precursor protein (APP)() is a member of a family of integral membrane glycoproteins that includes amyloid precursor-like proteins (APLP) 1 (1) and 2 (2, 3, 4) . We have demonstrated that, in cultured cells of non-neuronal or neuronal origin, mouse APLP2, like APP, matures through the constitutive secretory pathway (4, 5) . We and others (2, 4, 6) have also shown that APLP2 is encoded by several alternatively spliced transcripts. One isoform, APLP2-751, is modified by the addition of a chondroitin sulfate (CS) glycosaminoglycan (GAG) chain at a single serine residue (Ser-614) in the ectodomain (5) . Interestingly, the APLP2-763 isoform contains an additional 12-amino acid sequence, presumably encoded by an alternatively spliced exon, inserted two amino acids N-terminal to Ser-614 of APLP2-751. Insertion of this 12-amino acid peptide abolishes CS GAG modification of the APLP2-763 isoform.()

In previous efforts, we and others (7, 8) have examined the maturation and sorting of human APP in Madin-Darby canine kidney (MDCK) cells, a prototypic polarized epithelial cell line, and demonstrated that full-length APP is sorted to the basolateral compartment. Furthermore, C-terminally truncated APP derivatives are secreted into the basolateral medium. Because APP and APLP2 mature through a similar secretory/cleavage pathway in non-polarized cells, we were prompted to investigate the sorting of APLP2-751, a CS proteoglycan (PG), and APLP2-763 in MDCK cells. We demonstrate that both APLP2-751 and APLP2-763 are directly sorted to the basolateral surface of MDCK cells, releasing 95% of the total soluble derivatives into the basolateral compartment. Furthermore, the kinetics of secretion of soluble derivatives generated from either isoform is indistinguishable. Thus, the CS GAG chain has little influence on trafficking of the core protein in MDCK cells, and we argue that the principal basolateral targeting information resides within the core protein itself.


MATERIALS AND METHODS

Antibodies

The following antibodies were used in the experiments. D2-I antiserum was raised against a mouse APLP2-751 polypeptide synthesized in Sf9 cells using a baculovirus expression system (5) . Myc-I antiserum was raised against a synthetic peptide, MEQKLISEEDLN, corresponding to a region of the c-Myc oncoprotein (9) .

Plasmid Construction

To distinguish transgene-encoded APLP2 from endogenous APLP2, sequences encoding a region of the human c-Myc oncoprotein (MEQKLISEEDLN) were engineered into the extreme C terminus of either mouse APLP2-751 or APLP2-763 cDNA. Sequences encoding Myc-tagged mouse APLP2-751 were excised from pSVAPLP2-751myc (5) and subcloned downstream of the cytomegalovirus promotor in the pCB6 vector containing a gene that encodes resistance to the neomycin analog G418 (a kind gift of Dr. Mike Stinski, University of Iowa, Iowa City, IA). To generate expression plasmid pCB6APLP2-763myc, sequences encoding a Myc-tagged mouse APLP2-763 were excised from pSVAPLP2-763myc with EcoRI and XbaI. The gel-purified insert was subcloned into pBSII KS+ (Stratagene, La Jolla, CA) previously digested with EcoRI and XbaI to generate pBSAPLP2-763myc. Plasmid pBSAPLP2-763myc was subsequently digested with KpnI and XbaI, and the 2.2-kilobase fragment was subcloned into pCB6 vector containing KpnI and XbaI ends to generate pCB6APLP2-763myc.

Cell Culture and Transfection

MDCK cells (type II, ATCC CCL 34) (Rockville, MD) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.), penicillin (100 units/ml), and streptomycin (100 µg/ml) (complete medium).

To generate stable cell lines that express either mouse APLP2-751 or mouse APLP2-763, MDCK cells were transfected with CsCl-purified plasmid DNA using a high efficiency calcium phosphate method (10) . After selection in medium containing 500 µg/ml G418 (Life Technologies, Inc.), stable transfectants were assayed for steady-state expression of the transgene by Western blot analysis of cell lysates using D2-I antibody (5) .

Cell Labeling

For polarity assays, MDCK cells (1.5 10 cells) were plated onto 24-mm clear Transwell filters (Costar Corp., Cambridge, MA) and grown for 5 days. Transgene expression was induced with 10 mM butyrate at least 18 h before each experiment. Prior to labeling, the integrity of the tight junctions in the monolayer was assessed using a [H]inulin test as described previously (8) . Filter-grown cells were labeled with 500 µCi of [S]methionine (DuPont NEN) for 2 h in methionine-free Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum. At the end of the labeling period, apical and basolateral media were collected and centrifuged at 14,000 rpm for 1 min to remove particulate materials. To prepare detergent-soluble lysates, the filters were excised from the Transwell units and washed with phosphate-buffered saline. Cells were lysed in immunoprecipitation buffer containing freshly added protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A) (11) .

For pulse-chase polarity assays, cells growing on Transwell units were starved for 30 min and labeled with 200 µCi of [S]methionine for 10 min. At the end of the labeling period, cells were rinsed once with medium containing 1 mML-methionine. One Transwell of cells was immediately harvested and lysed in immunoprecipitation buffer; and the remaining Transwell units were incubated for various time periods in methionine-free Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM butyrate, and 1 mML-methionine. After each chase period, apical and basolateral media were collected; cells were harvested and lysed as described above.

For immunoprecipitation analysis, aliquots of the media and detergent-soluble lysates were incubated with D2-I or Myc-I antiserum, respectively, in immunoprecipitation buffer. Immune complexes were collected with immobilized protein A-agarose beads (Pierce) and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Quantitation was performed using a Molecular Dynamics (Sunnyvale, CA) PhosphorImager.

Chondroitinase Digestions

Chondroitinase digestions were performed as described previously (5) . Briefly, biosynthetically labeled APLP2-751 or APLP2-763 was immunoprecipitated in duplicate with Myc-I antiserum. Immunoprecipitates were collected with protein A-agarose beads. One set of the resulting immunoprecipitates was then subjected to digestion with 0.1 unit of chondroitinase AC (Sigma) in 100 µl of 100 mM Tris-HCl (pH 7.0), 30 mM sodium acetate, and a protease inhibitor mixture (10 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride) at 37 °C for 1 h. The other set of immunoprecipitates was mock treated by incubation at 37 °C in digestion buffer lacking chondroitinase AC.

Biotinylation

MDCK cells plated on Transwell filters were metabolically labeled with 500 µCi of [S]methionine for 1 h. At the end of the labeling period, the filters were washed with ice-cold phosphate-buffered saline containing CaCl and MgCl. Cell surface molecules on duplicate filters were biotinylated on ice with 0.5 mg/ml sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate/biotin (Pierce) in 10 mM borate buffer (pH 9) as described (12) from either the apical or basolateral side. After the biotinylation, filters were rinsed three times with ice-cold complete medium, incubated in ice-cold complete medium for 5 min, and then excised. Cells were lysed and processed as described above. Cellular APLP2 molecules were collected by immunoprecipitation with Myc-I antiserum. One-twentieth of the resulting immunoprecipitates was analyzed directly, whereas the remaining fraction was reimmunoprecipitated with immobilized streptavidin-agarose beads (Pierce) to isolate cell surface forms of APLP2 as described previously (8) . Myc-I-immunoprecipitated APLP2 and streptavidin-bound APLP2 were fractionated by SDS-polyacrylamide gel electrophoresis. The levels of surface-bound APLP2 molecules were quantitated using a PhosphorImager.


RESULTS

Expression and Post-translational Modification of Mouse APLP2-751 and Mouse APLP2-763 in MDCK Cells-To examine the metabolism of APLP2-751 and APLP2-763, we generated MDCK cells stably transfected with cDNAs encoding APLP2-751 and APLP2-763. The transgenes were epitope-tagged at the extreme C terminus in order to distinguish the transgene-encoded polypeptides from endogenously synthesized APLP2-751 and/or APLP2-763. Three independent clones expressing each APLP2 isoform were examined by polarity assays. Cells were plated onto Transwell units, induced with butyrate, and labeled for 2 h with [S]methionine. Detergent-soluble cell lysates were immunoprecipitated with Myc-I antiserum (Fig. 1a). In all clones expressing APLP2-751, Myc-I antiserum immunoprecipitated a full-length APLP2-related 120-kDa protein and a heterogeneous set of polypeptides migrating between 130 and 160 kDa. This pattern is similar to that observed in Chinese hamster ovary cells expressing Myc-tagged mouse APLP2-751, wherein an 120-kDa immature form matured to a set of heterogeneous polypeptides migrating between 120 and 200 kDa (5) . Thus, it is likely that the polypeptide of 120 kDa in MDCK cells represents the immature form of APLP2-751 that is subject to post-translational modifications. We recently showed that APLP2-751 is modified by a CS GAG chain (5) . To address whether MDCK-synthesized APLP2-751 is also a substrate for CS GAG addition, we immunoprecipitated full-length APLP2-751 molecules with Myc-I antiserum in duplicate. One set of the immunoprecipitates was treated with chondroitinase AC, an enzyme that cleaves CS GAG chains from core proteins. As shown in Fig. 1b, chondroitinase AC treatment of the Myc-I immunoprecipitates from cell line B17 expressing APLP2-751 converted the heterogeneous population of 130-160-kDa molecules seen in the undigested sample (Fig. 1b, lane 1) to a single band migrating at 130 kDa (Fig. 1b, lane 2), reflecting the removal of variable-length CS GAG chains from the APLP2-751 core protein. PhosphorImager analysis revealed that the radioactivity contained within the 130-kDa band (Fig. 1b, lane 2) was essentially identical to that contained within the heterogeneous polypeptides of 130-160 kDa in Fig. 1b (lane 1). Therefore, in MDCK cells, CS GAG modification of APLP2-751 occurs in a fashion similar to that shown in non-polarized Chinese hamster ovary and COS-1 cells.


Figure 1: Expression and CS GAG modification of APLP2 in MDCK cells. a, filter-grown MDCK cells stably transfected with cDNA encoding either mouse APLP2-751 or APLP2-763 were labeled for 2 h with [S]methionine. Myc-tagged exogenous APLP2 molecules were immunoprecipitated from detergent-soluble cell lysates with Myc-I antiserum. Lanes 1-3 represent immunoprecipitates from three independent clones expressing APLP2-751 (751). Note the heterogeneity in the migration pattern of the mature forms marked by the bracket; lanes 4-6 represent Myc-I immunoprecipitates from three independent clones expressing APLP2-763 (763). b, MDCK cells expressing APLP2-751 and APLP2-763 were labeled for 2 h with [S]methionine. Exogenous APLP2 molecules were immunoprecipitated with Myc-I antiserum and collected with immobilized protein A-agarose beads. Immobilized immunoprecipitates were incubated without (-) or with (+) chondroitinase AC (ChAC) at 37 °C for 1 h. Lanes 1 and 2 represent immunoprecipitates from B17 cells expressing APLP2-751 (751); lanes 3 and 4 represent immunoprecipitates from A15 cells expressing APLP2-763 (763).



In transiently transfected COS-1 cells, APLP2-763 isoform is not modified by the addition of CS GAG. A similar result is obtained in MDCK cells expressing APLP2-763 (Fig. 1a, lanes 4-6); although an immature form of 120 kDa was detected, these forms appeared not to be modified to heterogeneous species (Fig. 1a, lanes 4-6). Instead, a single band of 130 kDa is now apparent. As expected, chondroitinase AC failed to alter the electrophoretic mobility of the 130-kDa species (Fig. 1b, lane 4); hence, we argue that insertion of the 12-amino-acid peptide inhibits CS GAG modification of the APLP2-763 core protein. Sorting of APLP2-751 and APLP2-763 to the Basolateral Compartment-Stimulated by the observation that APLP2-751 and APLP2-763 are differentially modified, we asked whether the CS GAG chain has any effect on the intracellular trafficking of the core proteins. To examine the delivery of each precursor to either the apical or basolateral surface, we selected cell lines B17 and A15, which express APLP2-751 and APLP2-763, respectively. Cells were grown on Transwell filters, labeled for 1 h, and then placed on ice. Surface molecules were biotinylated from either the apical or the basolateral compartment. Full-length APLP2 forms were first immunoprecipitated with Myc-I antibody. A fraction of the Myc-I immunoprecipitates was saved, and the remainder was subjected to a second round of binding with streptavidin. As expected, the total levels of labeled APLP2 forms immunoprecipitated with Myc-I were identical irrespective of whether biotinylation was performed from the apical or basolateral surface (Fig. 2a, lanes 1, 2, 5, and 6). On the other hand, streptavidin only detected modified, full-length forms of APLP2-751 and APLP2-763 when biotinylation was performed from the basolateral side (Fig. 2a, lanes 4 and 8, respectively). Thus, full-length forms of both APLP2-751 and APLP2-763 were preferentially delivered to the basolateral surface. As quantitated using a PhosphorImager, 95% of the surface-bound APLP2-751 and APLP2-763 molecules were found to reside on the basolateral side.


Figure 2: Polarized delivery and secretion of APLP2. A, apical; B, basolateral. a, filter-grown B17 and A15 cells were labeled with [S]methionine, and the cell surfaces were biotinylated as described under ``Materials and Methods.'' APLP2 molecules immunoprecipitated from cell lysates using Myc-I were subjected to a second round of binding with streptavidin. Lanes 1 and 2 and lanes 5 and 6 represent total Myc-I-immunoprecipitable (Myc) full-length forms from B17 and A15 cells, respectively. Lanes 3, 4, 7, and 8 represent cell surface biotinylated APLP2 molecules isolated with streptavidin (SAV). Note the preferential basolateral surface localization of both APLP2-751 and APLP2-763 molecules. b, B17 and A15 cells were grown on Transwell units and labeled for 2 h with [S]methionine. Equal aliquots of apical and basolateral media were immunoprecipitated with D2-I antiserum and analyzed by SDS-polyacrylamide gel electrophoresis. c, PhosphorImager analysis of APLP2 secretion from cells expressing APLP2-751 (751) and APLP2-763 (763) in 2-h labeling experiments. The values indicated represent average values obtained from three independent clones in two separate experiments. Solid columns, apical; shaded columns, basolateral.



To examine the secretion of soluble forms of APLP2-751 or APLP2-763, cells grown on Transwell filters were labeled for 2 h, after which both apical and basolateral media were collected and immunoprecipitated with D2-I antibody. As shown in Fig. 2b, cell line B17 expressing APLP2-751 secreted 94% of total soluble APLP2-751-related derivatives to the basolateral compartment (Fig. 2b, compare lanes 1 and 2, and Fig. 2c). These soluble derivatives migrated as a heterogeneous species on SDS-polyacrylamide gel electrophoresis reflecting the CS GAG modification of the secreted ectodomain. In addition, >95% of total soluble APLP2-763-related molecules were secreted into the basolateral medium in cells expressing APLP2-763 (Fig. 2b, lanes 3 and 4, and Fig. 2c). Thus, we conclude that, despite differences in post-translational modification of APLP2-751 and APLP2-763, both molecules appear to be targeted preferentially to the basolateral cell surface in polarized MDCK cells, and soluble APLP2-751 and APLP2-763 derivatives are secreted predominantly into the basolateral media. Kinetics of Secretion of APLP2-751 and APLP2-763 Are Indistinguishable-To examine the maturation and kinetics of secretion of APLP2-751 and APLP2-763 in MDCK cells, we performed pulse-chase experiments. Polarized MDCK cells grown on Transwell filters were pulse-labeled for 10 min with [S]methionine and chased for various periods of time in medium containing excess unlabeled methionine. Detergent-soluble lysates and conditioned media were subjected to immunoprecipitation with Myc-I and D2-I antisera, respectively. Fig. 3 (a and b, top panels) depicts the pattern of intracellular Myc-I-immunoprecipitated forms of APLP2-751 and APLP2-763, respectively, during the pulse-chase experiment. Maturation of each precursor to their respective modified forms occurred with similar kinetics and peaked at between 30 min and 1 h into the chase period. In addition, the levels of accumulated secreted APLP2-related derivatives in either the apical or the basolateral medium of both cell lines followed indistinguishable kinetics (Fig. 3, a and b, middle and bottompanels).


Figure 3: Kinetics of maturation and secretion of APLP2-751 (a) and APLP2-763 (b). Filter-grown cells were pulse-labeled with [S]methionine for 10 min and chased for the times indicated. At the end of each time point, cells and media were collected. Exogenous full-length APLP2 molecules were immunoprecipitated with Myc-I antiserum, whereas secreted APLP2 derivatives were immunoprecipitated with D2-I antiserum. Values indicated in the graphs (bottom panels) represent the average values obtained from two independent experiments. A, apical; B, basolateral.




DISCUSSION

APP, the precursor protein of A, is a member of a family of integral membrane glycoproteins that includes APLP1 and APLP2. We previously documented that APLP2, like APP, matures through the secretory pathway and that truncated soluble APLP2 derivatives are secreted (4) . We and others have also reported that APLP2 pre-mRNA is alternatively spliced to encode several APLP2 isoforms (2, 4, 6) . Here, we demonstrate that in stably transfected MDCK cells, a prototypic polarized epithelial cell line, the APLP2-751 isoform is modified by the addition of CS GAG chains, consistent with findings in non-polarized cells (5) . Furthermore, CS GAG modification is not apparent in the APLP2-763 isoform, which contains an additional 12-amino acid sequence, presumably encoded by an alternatively spliced exon that is inserted two amino acids upstream of the CS GAG addition site. In order to address whether the CS GAG chain plays a role in intracellular trafficking of the APLP2 core protein, we examined the polarized delivery and secretion of the precursor and its soluble derivatives, respectively, in stably transfected MDCK cells. In this report, we demonstrate that APLP2-751 is targeted to the basolateral plasma membrane and that truncated derivatives are released into the basolateral medium. The observation that a CS GAG modified form of APLP2 is targeted to the basolateral domain is consistent with earlier reports that demonstrate that several proteoglycans expressed endogenously in polarized epithelial cells are also delivered vectorially to the basolateral surface. One of these molecules, syndecan-1, a CS PG, is localized at the basolateral cell surface in mammary epithelial cells (13) . Moreover, a heparan sulfate PG of unknown core protein is released predominantly into the basolateral medium of MDCK cells (14) . Because both syndecan-1 and heparan sulfate PG are components of the extracellular matrix and are implicated to be involved in cell-matrix adhesion, the basolateral targeting and secretion of APLP2-751 may have functional implications for this molecule.

Our studies also demonstrate that APLP2-763, an isoform that is not modified by CS GAG chains, is trafficked to the basolateral compartment in a manner similar to APLP2-751. Hence, we conclude that intracellular trafficking and appearance of APLP2 on the basolateral cell surface are mediated by signals contained within the core protein, independent of the CS GAG chain. The specific sequences that mediate APLP2 sorting in MDCK cells have not yet been determined. However, recent studies indicate that specific tyrosine-containing signals contained within the APP cytoplasmic domain are essential for basolateral targeting of the parent molecule (15) . Considering the high degree of homology throughout the cytoplasmic domains of APP and APLP2, we speculate that APLP2 trafficking and targeting to the basolateral surface are mediated by signals contained within the cytoplasmic domain.

Our observation that the CS GAG chain has little, if any, influence on the targeting of the APLP2 core protein in polarized cells is unique. On the other hand, studies of the invariant chain (Ii), a nonpolymorphic glycoprotein that associates with the major histocompatibility complex class II molecules during antigen presentation, reveal that CS GAG chains are an important requirement for intracellular trafficking and cell surface expression of the core protein in non-polarized cells. The predominant localization of Ii is in endosomal vesicles, a localization specified by sequences within the cytoplasmic N terminus (16, 17) . However, a fraction (2-5%) of Ii is expressed at the cell surface (18) , and these cell surface Ii molecules are modified by the addition of CS GAG at a single serine residue (19, 20) . The presence of CS GAG-modified Ii on the cell surface indicates that the glycosaminoglycan moiety is a dominant signal that overrides the endosomal sorting signal.

The basolateral trafficking of APLP2 may also be related to one of the typical pathological features in Alzheimer's disease (i.e. the deposition of amyloid fibrils in cerebral microvessels). A, a major constituent of amyloid fibrils, is localized to the basement membrane of capillaries, including vessels in the meningeal space (21) . This preferential localization might be explained by the observation that A is delivered to the basolateral compartment of MDCK cells (7, 8) . Although APLP2 cannot generate A, the similarity in trafficking of A and APLP2 to the basolateral compartment may have some functional implications related to A deposition and amyloid fibril formation in cerebral vasculature. Indeed, the close association of proteoglycans with amyloid fibrils is well documented (for review, see Ref. 22). Moreover, both heparan sulfate and chondroitin sulfate proteoglycans have been localized within senile plaques (23, 24, 25, 26) . In addition, A binds with high affinity to glycosaminoglycans (27) , an interaction that results in the formation of fibrillar A-proteoglycan complexes that are resistant to both removal and/or degradation by cultured microglial cells (28, 29) . These latter findings are consistent with a postulated role for proteoglycans in amyloid accumulation and persistence. The role of CS PG in A aggregation and fibril formation and the persistence of amyloid deposits in vivo remain to be elucidated.


FOOTNOTES

*
This work was supported by United States Public Health Services Grants NIH AG 05146 and NS 20471, by funds from the American Health Assistance Foundation, and by a Zenith Award from the Alzheimer's Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Neuropathology Laboratory, The Johns Hopkins University School of Medicine, 558 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2196. Tel.: 410-955-5632; Fax: 410-955-9777.

The abbreviations used are: APP, amyloid precursor protein(s); A, -amyloid peptide; APLP, amyloid precursor-like protein; CS, chondroitin sulfate; GAG, glycosaminoglycan; PG, proteoglycan; MDCK, Madin-Darby canine kidney; Ii, invariant chain.

G. Thinakaran, H. H. Slunt, and S. S. Sisodia, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Philip Wong for providing Myc-I antiserum and Dr. Mike Stinski for providing the pCB6 vector. We also thank Drs. David Borchelt and Ora Weisz for helpful comments and discussions.


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