Sequence Requirements for Ligand Binding and Cell Surface ...

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Apr 5, 2018 - elements required for intracellular transport and li- gand binding by the human Tac interleukin-2 (IL-2) receptor, we prepared expression ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263,No. 10, Issue of April 5, pp. 4900-4906.1988 Printed in U.S.A.

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequence Requirements for Ligand Binding and Cell Surface Expression of the TacAntigen, a Human Interleukin-2 Receptor* (Received for publication, November 6, 1987)

Bryan R. Cullen*#,Frank J. Podlaski*, Nancy J. Peffern, Jane B. Hoskingn, and WarnerC. Greenell From the $Department of Molecular Genetics, Hoffmann-La Roche, Inc.,Nutley, New Jersey 07110 and the llHoward Hughes Medical Institute, Department of Medicine, Duke Uniuersity School of Medicine, Durham, North Carotinu27710

Discrete peptide domains withinthe primary se- as exposed sulfhydryl groups or regions of inappropriate hyquence of cell-surface receptor glycoproteins are be- drophobicity. lieved to regulatenot only their function but also their In this paper, we have used a similar strategy to examine targeting to the cell membrane. To identify sequence the sequence requirements for cellular transport and ligand elements required for intracellular transport and li- binding of the human Tac protein. The Tac protein is a gand binding by the human Tac interleukin-2 (IL-2) human interleukin-2 receptor (IL-2R)’ importantly involved receptor, we prepared expressionplasmids encoding a in the activation of T-cells by this lymphokine (7, 8). Treatseries of artificially mutated or naturally occurring ment of activated T-cells with anti-Tac antibody blocks invariants of the Tac cDNA. In particular, we sought to terleukin-2 (IL-2) binding and T-cell proliferation (9). The further delineate the functional role of the sequences Tac gene is composed of eight exons which together encode contributed by each of the eight exons that together the mature 55-kDa cell surface glycoprotein (10-12). The first encode the Tac protein.Deletion of exons 5 through 8 of the receptor had no detectable effect on IL-2 binding exon of this gene encodes a 21-amino acid signal peptide while or intracellular transport of the Tac protein, and re- exons 2-6 correspond to the219-residue extracellular domain sulted in secreted forms of this IL-2-binding protein. of the receptor (See Fig. 1).Exon 7 encodes the majority of Removal of sequences corresponding to all of exon 4 the membrane-spanning region while exon 8 contains most of the 13 residues that form the shortintracytoplasmic recepablated IL-2 binding activity yet still permitted transport to thecell surface. In contrast, partialdeletion of tor domain. In this paper, the terms Tac antigen and IL-2R exon 4 sequences resulted in proteins that not only are interchangeably used. lacked IL-2 binding activity but also were sequestered The critical amino acids that permit IL-2 binding by the within the endoplasmic reticulum. Removal of one or Tac antigen remain incompletely defined. Kuo et al. (13) have both of the N-linked glycosylation sites present in the demonstrated that lZ5I-IL-2bound and covalently cross-linked Tac proteindid not impair receptor transportor ligand to the Tac protein remains associated with a tryptic peptide binding. These results demonstrate that exon 4 of the corresponding to the NH2-terminal 83 amino acids. These Tac gene encodes amino acid residues that play an findings suggested that residues within exon 2, and perhaps important role in regulating both theintracellular exon 3, participatein the formation of the IL-2-binding transport andfunction of this IL-2 receptor. site(s). Virtually no information exists regarding the primary sequences required for the normal targeting and transport of the Tac protein to the plasma membrane. Once synthesized, the Tac protein is extensively modified by post-translational The display of an appropriate repertoire of cell-surface processing involving the early cleavage of the signal peptide, receptors and glycoproteins is essential for the correct func- the formation of intrachain disulfide bonds, and theaddition tion and interaction of individual cells within a multicellular of N-linked carbohydrate (12, 14). Following transfer from organism. Although many of the biochemical steps involved the endoplasmic reticulum to theGolgi complex,the receptor in protein transport and secretion have been elucidated ( l ) , is further modified by the addition of 0-linked carbohydrate the nature of the mechanisms and signals that ensure the and sialic acid prior to insertion into the cell membrane. proper targeting of proteins through the endoplasmic reticuTo investigate the functional role of different peptide dolum and Golgi apparatus to their final destination at the cell mains encoded by specific exons of the Tac gene, we have surface remains largely unknown. One experimental approach constructed expression plasmids containing a series of 3‘to this problem involves the expression of genes encoding truncated forms of the Tac cDNA. In addition a cDNA naturally occurring or artificially created variantsof proteins corresponding to anaturally occurring, but alternatively normally expressed at thecell membrane (2-6). These studies spliced, form of Tac mRNA lacking only exon 4 sequences have led to thehypothesis that appropriate intracellular pro- (15, 16) was analyzed. To investigate the potential role of N tein sorting and transport may require the presence of both glycosylation in IL-2 binding and receptor transport, sitepositive signals, such as a signal peptide and appropriate directed mutagenesis was used to alter, individually and in glycosylation, as well as theabsence of negative signals, such combination, each of the two consensus sequences for Nlinked carbohydrate addition. Each of these various forms of * The costs of publication of this article were defrayed in part by the Tac cDNA were transfected into COS cells and analyzed the payment of page charges. This article must therefore be hereby by precipitation with Tac specific heteroantibodies or IL-2 marked “aduertisement” in accordance with 18 U.S.C. Section 1734 immobilized on Sepharose bead supports. This experimental solely to indicate this fact. 8 To whom correspondence should be addressed Howard Hughes Medical Institute, Duke University Medical Center, Box 3025, Durham, NC 27710.

The abbreviations used are: IL-2R, interleukin-2 receptor; IL-2, interleukin-2.

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Sequence Requirements for Tac Antigen Function approachhaspermitted identification of defined regions within the Tac protein that are involved in ligand binding and intracellular transport. EXPERIMENTALPROCEDURES

Construction of Molecular Clones-All Tac expression constructions were based on the eukaryotic expression vector pBC12MI (17). The previously described pIL-2R3 and pIL-2FM plasmids (15) were used, respectively, as sources of the full length IL-2R andIL2RAexon4 cDNAs. In each case, the IL-2R cDNA insert was excised with NciI, which cleaves a t sites that closely flank the Tac proteincoding sequences. These “trimmed” IL-2R cDNA fragments were insertedin a sense orientation into the expression vector at the HindIII and BamHI sites immediately downstream from the Rous sarcoma virus long terminal repeat promoter. This ligation regeneratedthe unique 3’ BamHIsite within the vector. Subsequently, COOH-terminal deletions were generated by cleavage at thisBamHI site anda t internal sites, followed byligation of a universal terminator oligonucleotide. The terminator oligonucleotide used initially to generate pIL-2RANae and pIL-2RLUlst had thesequence 5”TTAAGTTAACTTAA-3’. For technical reasons, all subsequent deletions used the terminator sequence 5’-CTGAATGAATGA-3’.Depending on the reading frame, the insertion of theseterminator oligonucleotides resulted in the addition of sequences encoding up to three foreign amino acids a t the COOH terminus of the IL-2R deletion mutants (see legend to Fig. 1). The Tac antigen contains two consensus sequences for N-linked glycosylation a t amino acid positions 40 (Gl: Am-Ser-Ser) and 68 (G2: Asn-Thr-Thr) (15). Single amino acids were altered at these sites in the pIL-2RWstconstruction using an oligonucleotide mutagenesis procedure (18). This procedure employs double-stranded plasmid DNA as a starting substrate. The oligonucleotides used were two 29-mers that each introduced a contiguous 4-nucleotide mutation. In the case of pIL-2RAG1, this mutation generated a novel BspMII site (tct.agc + tcC.GGA) and changed the encoded amino acid sequence to Asn-Ser-Gly. In the case of pIL-2RAG2, the change generated a novel PuuI site (cgg. aac + cgA.TCG) and changed the encoded amino acid sequence to Ser-Thr-Thr. pIL-2RAG2 served both as a substrate for the synthesis of the double mutant pIL2RAG1+2 and for the synthesis of the deletion mutant pIL-2RUuu. Cell Culture and DNA Transfection-COS cells were maintained as previously described and were transfected with -500 ng of plasmid DNA/35-mm culture dish using DEAE-dextran and chloroquine (19). Immunoprecipitation and Immunofluorescence-Cellular proteins were labeled with [36S]methionine (specific activity 1000 Ci/mmol, Amersham Corp.) -48 h after transfection (19). The period of labeling with [36S]methioninevaried from 15 to 30 min for intracellular IL2R and up to -16 h for secreted IL-2R. In selected experiments, cells were pulse-labeled with [%]methionine for 15-30 min andthen chased with excess amounts of unlabeled methionine for various periods of time. Immunoprecipitations of labeled Tac proteins using Tac-specific heteroantibody were performed as previously described (15, 19). For analysis of IL-2 binding by the various Tac protein derivatives, IL-2 and insulin were covalently conjugated to Sepharose 4B beads with cyanogen bromide (14). Extracellularproteinsor Triton X-100 cellular extracts were incubated with either the IL-2Sepharose or insulin-Sepharose at 4 “C for 4-16 h. The beads were then washed, suspended in 1% sodium dodecyl sulfate, boiled, and diluted in buffer (1%Triton X-100, 1%deoxycholate, 0.15 M NaCl, 10 mM Tris-HC1, pH 7.4, 1 mM EDTA, and 0.25 mM phenylmethylsulfonyl fluoride) to achieve a final concentration of 0.1% sodium dodecyl sulfate (RIPAbuffer) and thenimmunoprecipitated with the Tac heteroantibody. Immunofluorescent staining of transfected COS cells was performed as previously described (19) a t 72 h after transfection using a 1:500 dilution of the rabbit Tac-specific heteroantibody followed by a 1:50 dilution of rhodamine-conjugated goat anti-rabbit IgG antibody (Boehringer Mannheim). RESULTS

A Naturally Occurring Tac Variant Fails to Bind IL-2-To test whether the loss of exon 4 sequences affected the functional expression of the Tac protein, two expression vectors containing either the full length IL-2R or the IL-2RAexon4 cDNA clones were prepared (Fig. 1). These plasmids were

IL-2R

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5

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FIG. 1. Schematicstructure of IL-2RandIL-2RAexon4 cDNAs. Exon boundaries and extracellular, transmembrane, and cytoplasmic domains of the wild type (ZL-2R) and alternatively spliced (IL-2RAerod) Tac cDNAs are shown. N-Glycosylation and restrictionsites utilized for the construction of the truncated or mutated forms of these cDNAs are also indicated. All cDNAs were cloned in the sense orientation into the eukaryotic expression vector pBC12MI (17). The predicted amino acid ( M ) sizes of the mature forms of the different Tac proteins are as follows: pIL-2R, 251aa; ~IL-~RANN 223aa , (plus laa encoded by the terminator oligonucleotide); pIL-aRMst, 179aa (+laa); pIL-ZRAIVNde, 162aa (+3aa); pIL2RaEcoO1, 142aa (+laa); pIL-2RMga, 127aa (+3aa); pIL-fLRhPst, 102aa (+2aa); pIL-PRhPuu, 67aa; pIL-2RhRsa, 46aa (+2aa); pIL2RAexon4,179aa; pIL-2RAexon4AMst, 107aa.

introduced into COS cells byDNA transfection (19) and receptor expression analyzed by immunoprecipitation of [35S] methionine labeled IL-2R using eitherrabbit Tac-specific heteroantibody (Fig. 2.4) or IL-2 conjugated to Sepharose beads (Fig. 2B). Transfected COS cells were incubated in [35S] methionine for 30 min followed by a 3 h or overnight (16 h) chase with excess cold methionine. Intracellular and extracellular IL-2R production was examined. The full length IL2R gave rise to an initial -37-kDa polypeptide which chased to a -50-kDa form. Longer chases resulted in the loss of the larger cell-associated protein and the simultaneous appearance of an extracellular -40-kDa soluble form of the receptor. This pulse-chase behavior in the transfectedCOS cells recapitulated previous results obtained with endogenous receptor protein produced by mitogen-stimulated normal T-cells (14). The -37-kDa form represents a Tac polypeptide precursor which has been co-translationally modified by N-linked glycosylation. Further processing occurred with transport to the Golgi complex givingrise to the mature, more slowly migrating -50-kDa form. The smaller extracellular Tac protein appears to arise via “shedding” of the surface receptor (20) at least in part reflecting proteolytic cleavage at a site close to the cell membrane (21). Examination of the expression of the pIL-2RAexon4 variant of the Tac protein using Tac-specific heteroantibody (Fig. 2%) revealed a very similar pattern of synthesis and post-translationalprocessing, although this deleted IL-2R variant did appear to exit the endoplasmic reticulum somewhat more slowly than the full length protein. An initial -33-kDa form was observed to mature to anunexpectedly slow-migrating -48-kDaform. This formwas subsequently found to give rise to a smaller (-35 kDa) extracellular “shed” form of protein. Examination of theTac proteins produced during this labeling experiment using IL-2 Sepharose precipitation (Fig. 2B) revealed that all of the IL-2R forms encoded by the full length pIL-2R clone, including precursor and extracellular forms, were able to react with IL-2. In sharp contrast, none of the IL-PR moleculesencoded by the pIL-2RAexon4 cDNA bound IL-2. Thus, theIL-2RAexon4mRNA appears to encode a stable, smaller form of the Tac protein that is normally processed, transported, and shed by the expressiqg cell but lacks the capacity to bind IL-2. Other results supporting a

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for Tac Antigen Function

Sequence Requirements

FIG.2. Biosynthesis and a - 2 binding properties of the proteins encoded by the a - 2 R and IL2RAexon4cDNAs. COS cells were transfected with the indicated expression plasmids, and [35S]methionine pulse-chase labeling was performed 48 h later (19). Intracellular ( I ) and extracellular ( E ) proteins isolated at the indicated intervals were precipitated with the Tac heteroantibody ( p a n e l A ) or IL2 Sepharose ( p a n e l B ) as described under "Experimental Procedures" andanalyzed by electrophoresis through sodium dodecyl sulfate-10%-polyacrylamide gels. The migration of known molecular weight standards is shown on the left of each panel (30' = 30 min, 3' = 3 h). FIG. 3. Analysis of S'-truncated forms of the Tac cDNA. COS cells

A. M, X

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Tac Hetero Ab plL-2R plLdRAExon 4 n

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Intracellular

were transfected with expression plas- A , X 10-3 M, X 103 mids containing either the full length Tac cDNA or cDNAs progressively trun60cated from the 3' end (see "Experimental 60Procedures"). Transfectants were bio43synthetically labelled with ["Slmethio43nine followedby immunoprecipitation of 25.7 25.7intracellular proteins ( p a n e l A , 15 min labeling) or extracellular proteins (panel 10.410.4B, 16 h labeling) with the Tac-specific heteroantibody. Panels C and D show results of [35S]methionine pulse-chase labeling of the indicated Tac cDNA deletion mutants immunoprecipitated with Tac heteroantibody. Lane I, intracellular proteins pulse-labeled with [?3]methionine for 15 min; lane 2, intracellular proteins following 1-h chase with cold D. IL-2RAMst IL-2RANde methionine; lune 3, intracellular pro" teins, 3-h chase; lune 4, extracellular proteins, 3-h chase. Immunoprecipitation of M, X 10-3 ( IGL I M, x 10-3 cells transfected with the expression vec6860tor lacking a cDNA insert is shown in 43the lastlane of panel D. Some of the IL432R deletion mutants yielded faster mi25.7 grating forms of Tac protein after short 25.7labeling periods (e.g. panel A ) . These 10.4molecules wereobserved to rapidly chase 10.4to a single, slower mobility form (panel D ) and may represent short-lived partially N-glycosylated precursors of the 1 2 3 4 1 2 3 4 Tac protein.

c*

-

role for exon4 on IL-2 binding by the Tacprotein have been recently described(22). Sequences Present in IL-2R Exon 4 Modulute Intracellulur Receptor Transport-To more fully dissect the role of sequences derived fromeach of the different Tac gene exons in receptor transport andligand binding, a nested set of COOHterminal IL-2R deletion mutants were constructed by introduction of in-frame termination codons at different restriction sites within the pIL-2R expression vector(Fig. 1). These mutations resulted in the progressive deletion of sequences corresponding to exons 7 and 8 (pIL-SRANue), exons5 and 6 (pII-ZRhMst), part or all of exon 4 (pIL-ZRANue, AEcoOl, M g a , and U s t ) , exon 3 (pIL-ZRA?'uu), and lastly part of exon 2 (pIL-2RARsa).We had previously shown that introduction of a translational terminator at the receptor NaeI site (pIL-2RANae)resulted in the production of a secreted form of the Tac protein that retained the ability to bind IL-2 (23, 24). Initially, the expression of these truncated Tac proteins was examined by immunoprecipitation of ["Slmethionine-

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labeled, transfected COS cells using the Tac heteroantibody (Fig. 3). The pIL-2RANae plasmidgave rise to an intracellular precursor of -36 kDa that migrated slightlymore rapidlythan the protein encoded by the full length pIL-2R vector (Fig. 3A).Similarly, the moreextensivelydeletedpIL-ZRAMst, ANde, AEcdl, and A?Iga clones each gave rise to high levels of receptor protein that, as expected, decreased in relative molecular size in a step-wise manner. The pIL-2RAPst mutant in contrast yielded somewhat reduced levels of an immunoprecipitated Tac protein which migrated more slowly than predicted by DNA sequence analysis (25). The extensively deleted pIL-2RAPuu and ARsa mutants, which, respectively, contain sequences derived from exons2 and 3 or from exon 2 alone, yielded no proteins that were detectable with the Tac heteroantibody. The reason for this finding has not been determined but could reflecteither an intrinsic instability of these IL-2R mRNAs or proteins, or alternatively, the deletion of critical sequences required for immunoprecipitation by the Tac heteroantibody.

Sequence Requirements for Tac Antigen Function The level of extracellular IL-2R encoded by each of these mutants was next examined (Fig. 3B). As previously described (23,24), thepIL-2RANae mutant gave rise to high levelsof a secreted form of the Tacprotein. Similar results were observed with the next smaller deletion mutant, pIL-PRAMst, whichis deleted in exons 5 and 6. The wild type Tac protein was efficiently shed by expressing cells in a form intermediate in gel mobility between the 224-amino acid pIL-2RANae and the 180-amino acid pIL-2RAMst forms. This result is consistent with the published -192-amino acid size of the Tac protein shed by an HTLV-I transformed human T-cell line (21). Surprisingly, transfection of the pIL-2RANde mutant that encoded high levels of intracellular Tac protein did not result in the secretion of any detectable soluble receptor protein (Fig. 3B). The other two mutants exhibiting a partial deletion of exon 4-specific sequences, pIL-2RAEcoOl and pILBRAHga, also encoded proteins that were very inefficiently secreted by the transfected cells, although small amounts, especially in the case of pIL-PRAHga, were detected in the culture supernatants. In sharp contrast, thepII-2RAPst mutant, which lacks essentially all the sequences encoded by exon 4 of the Tac antigen, was efficiently secreted by the transfected cells. In order to examine further these apparent differences in intracellular receptor transport, we performed kinetic-labeling studies using COS cells transfected with either the pILZRAMst,pIL-SRANde, or pIL-2RAPst plasmids (Fig. 3, C and D). In cells transfected with pIL-2RAMst, the majority of the initial intracellular Tac precursor form was observed to convert to a secreted, more highly modified formwithin 3 h after labeling. Similarly, the majority of the Tac protein encoded by pIL-2RAPst also chased to an extracellular, more slowly migrating form within 3 h after labeling (Fig. 30). In contrast, the receptor encoded by pIL-2RANde showed no change in intracellular steady-state level or degree of posttranslational modification and was not detectably secreted by the transfected cells (Fig.3C). These results suggest that pIL2RANde encodes a receptor protein that is stable but that is not transported to thecell surface.The absence of an increase in apparent size during the chase suggests that the protein may fail to reach the Golgi apparatus where 0-linked sugar is added. To investigate more fullythe subcellular localization of the receptor protein encoded by pIL-2RANde, immunofluorescent staining of permeabilized transfected COS cells was performed (Fig.4). As expected, cellstransfected with either pIL2R or pIL-2RAexon4 gave similar fluorescence patterns consistent with the presence of receptor protein on the plasma membrane and within the Golgi complex (Fig. 4, A and B ) . Cells transfected with pIL-2RAMst surprisingly yielded no detectable fluorescence signal, suggesting that theintracellular steady-state level of the efficiently secreted IL-2AMst protein was below our level of detection with this technique. In marked contrast, cells transfected with pIL-2RANdeplasmid yielded a striking “filigree” pattern of fluorescence that is characteristic of protein accumulation within the endoplasmic reticulum. These results, together with the immunoprecipitation analysis presented in Fig. 3, suggest that the Tac mutants whichsuffered a partial deletion of exon4specific sequences encodedreceptor proteins which were unable to efficiently exit the cellular endoplasmic reticulum. In contrast, mutantsof the Tacprotein that retained all of exon 4 (e.g. pIL-2RAMst) or that had lost all of exon 4 as a result ofRNA splicing or by deletional mutagenesis (i.e. pIL2RAexon4, pIL-PRAPst), encoded proteins that were normally transported.

4903

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IL-2RAExon 4

IL-2R

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IL-2RAMst IL-2RANde FIG. 4. Subcellular localization of proteins encoded by the Tac cDNAs by immunofluorescent staining. The subcellular location of the indicated mutant Tac proteins was examined by treatment of fixed, transfected COS cells with rabbit anti-Tac heteroantibody followed by rhodamine-conjugated goat anti-rabbit antibody (19).Cells selected for photography were judged representative of the cultures. T w o cells were present in the IL-2RhMst field as detected by phase microscopy.

Binding of IL-2 Requires a Complete Exon 4 Domuin-Data shown in Fig. 2 demonstrated that both intracellular and extracellular forms of the full length IL-2R, but not the IL2RAexon4, proteins bound IL-2 conjugated to Sepharose beads. The IL-2 binding capacity of the Tacdeletion mutants was similarly analyzed (Fig. 5). Initially, mutants encoding secreted forms of the Tacproteins, i.e. pIL-ZRMst and pIL2RAPst as well as a similar IL-2R construction prepared by insertion of a termination codon at the MstII site in pIL2RAexon4 (pIL-2RAexon4AMst)were studied. As shown in Fig. 5A, each of these three vectors encodedhighlevels of secreted Tac protein detectable by immunoprecipitation with theTac heteroantibody. As predicted, thepIL2RAexon4AMst mutant, which isstructurally almost identical to pIL-2RAPst, shared the same secretion pattern and the unexpectedly slow migration on polyacrylamide gels previously noted forthe pIL-2RAPst mutant (Fig. 3). Precipitation with IL-2-Sepharose was found to be highly efficient with pIL-2RAMst but was not detected with the other two deletion mutants lacking all or most of exon 4. These results demonstrate that exon 4 but not exon 5 sequences are required for IL-2 binding. To more sharply delineate the region of exon 4 required for IL-2 binding,we also subjectedthe non-secreted deletion mutants to precipitation with IL-2-Sepharose. As shown in Fig. 5B, all of the deletions extending beyond the exon 4/exon 5 border abolished IL-2 binding, even though high intracellular levels of these truncated Tac proteins were detectable using the Tac heteroantibody (Fig. 5C). Thus, IL2 binding by the Tacprotein appeares to require a fully intact exon 4.

Sequence Requirements Antigen for Tac

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FIG.5. IL-2binding properties of the truncated formsof the Tac cDNA. COS cells were transfected with the indicated Tac cDNA derivatives and 48 h later radiolabeled with [3sS]methionine for 16 h. Panel A, immunoprecipitation of extracellular proteins with IL-2 Sepharose (ZL-2), insulin-Sepharose (Insulin), Tac heteroantibody (Tac Hetero Ab), or normal rabbit serum ( N R S ) (prebleed). Panel B, intracellular radiolabeled proteins prepared from COS cells transfected with the indicated cDNAs were precipitated with IL-2 or insulin conjugated to Sepharose. Panel C,the production of truncated forms of the Tacprotein by the ANde, AEcoOl, and AHga mutants was confirmed by precipitation of the same samples shown in panel B with the Tacheteroantibody.

A.

Intracellular

B.

---Extracellular

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FIG. 6. Analysis of N-glycosylation sites in the Tac protein. COS

cells were transfected with A M s t variants of the Tac cDNA site specifically mutated a t amino acid position 42 (ZL2RAGl), position 68 (ZL-2RAG2), or positions 42 and 68 (ZL-2RAGl+2) to remove each of the N-glycosylation sites individually and in combination. Panel A, COS cells were transfected with the indicated cDNAs in the presence or absence of tunicamycin (TNC, 5 pg/ml) and intracellular [36S]methionine-labeled proteins were precipitated with the Tac-specific heteroantibody. Panel B, extracellular proteins secreted by the transfected COS cells were precipitated with insulin-Sepharose (insulin), IL-2 Sepharose (ZL-2).or Tac heteroantibody as indicated.

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Effect of N-Linked Glycosylatwn on IL-2R Transport and Ligand Binding-The previous studies demonstrated that sequences present in exon 4 of the full length IL-2R can affect the secretion behavior of soluble variants of the IL-2R. A second attribute of proteins that may play a role in the regulation of intracellular transport is the present of N-linked glycosylation (2,26). Inorder to testwhether the two consensus N-linked glycosylation sites present in the Tac antigen (Fig. 1) are both utilized and whether this glycosylation has any detectable function, we mutated each site individually and in combination in the pIL-2RaMst clone (see “Experi-

14-

mental Procedures” for details). Each construction was then transfected into COS cells and intracellular IL-2R examined by immunoprecipitation of [35S]methionine-labeledproteins. Removal of either the more amino-terminal (pIL-2RAG1) or more COOH-terminal (pIL-2RAG2) glycosylation site alone was found to result in intracellular Tac proteins which demonstrated equivalent mobility on acrylamide gels but which migrated significantly more rapidly than the parental pIL2RaMst protein (Fig. 6A). Deletion of both N-linked glycosylation sites (pIL-2RAG1+2) resulted in the synthesis of a protein which migrated with the same mobility as theparental

Sequence Requirements Antigen Function Tac for

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certain mutations of the influenza hemagglutinin have been shown to interfere with the transport of this viral protein through the endoplasmic reticulum (3, 4). These mutations appear to prevent the requisite quaternary conformational changes in hemagglutinin structure involving the trimerization of this viral protein. The abnormal transport of the Tac protein mutants is also reminiscent of the effects produced by some naturally occurring mutations in the low density lipoprotein receptor. For example, a spontaneous deletion mutation within the cysteine-rich ligand-binding region ofthe low density lipoprotein receptor results in the synthesis of a protein that is blocked in transport to the cell surface (5). Similarly, a COOH-terminal deletion mutant of the low denDISCUSSION sity lipoprotein receptor was found to become trapped within In these studies, we have explored the primary sequence the endoplasmic reticulum of the expressing cell (6). These requirements for IL-2 binding and intracellular transport of results suggest that eukaryotic cells may havea general mechof inapprotheTac antigen. Ourexperimentalstrategy involved the anism that functions to prevent the transport expression in COS cells of a series of artificially mutated priately folded proteins to the cell surface (4). Although the forms of the Tac cDNA as well as a naturally occurring, details of this mechanism are unclear, it appears to involve cellular “gate-keeper’’ protein(s) that may recognize signals alternatively spliced form. Expression of thislatterTac of inappropriate folding such as unpaired sulfhydryl groups cDNA, which lacks exon 4 sequences but is otherwise identical or exterior regions of inappropriate hydrophobicity (4, 6). In to wild type, resulted in the production of a smaller protein that was transported to thecell surface and subsequently shed this context, it is of interest to note that the 17-amino acid deletion which distinguishes the normally secreted pILintothe culturesupernatant.This is very similar to the pattern of expression for the full length Tac protein reported 2RAh4st mutant from the abortively transported pIL-2RANcle mutant indeed includes a cysteine residue which is involved here for COS cells and previously in human T-cell lines (12, in intramolecular disulfide bond formation.2 14). However, this polypeptide lacked the capacity to bind ILOne gate-keeper protein, termed BiP, has been shown to 2. Our results, therefore, suggest that human T-cells, which associate with several proteins that are abortively transported express a low but significant level of the Aexon 4 form of the from the endoplasmic reticulum. For example, in the absence Tac mRNA (15, 16), probably also express a low level of a of light chain immunoglobulin expression, BiP associates with Tac-related molecule that is unable to bind IL-2. These results heavy chain immunoglobulin molecules and blocks their imply that the Aexon4 mRNA encodes eithera defective transfer to the Golgi apparatus (27-29). Similar results have protein or an alternate form of the Tacantigen whose function been reported with the influenza hemagglutinin mutants (4) presently remains unknown. and with a nuclear protein, SV40 T-antigen, which was inIn contrast to the naturalpIL-2RAexon4 protein, artificial appropriately directed into the endoplasmic reticulum by admutants lacking sequences from exon 5 to 8 exhibited no loss dition of an NH,-terminal signal sequence (30). Our results of ligand binding activity. Coupled with previous evidence are fully consistent with a similar mechanism occurring in indicating that residues within exon 2 are involved in IL-2 the case of the Tac mutants thatfail to exit the endoplasmic binding (13), these findings suggest that the 72 amino acids reticulum. A novel aspect of our data, however, is the obserencoded by exon 4 may interact with residues in exon 2 to vation that further deletion of the IL-2R molecule results in produce the IL-2-binding site(s). Other results consistent with the restoration of normal proteintransport.This finding a critical role for exon 4 in IL-2 binding have recently been suggests that the putative signal for the action of BiP or a described by Neeper and colleagues (22). These authors have related protein is deleted in pIL-2RhPst protein but present also detected disulfide bonding between the cysteine residues in pIL-2RaHgu protein. The nature of this signal is unclear, located at amino acid position 3 within exon 2 and position but inspection of this 25-amino-acid stretch does reveal a 147 within exon 4. It seems likely that the formation of this possibly unpaired cysteine residue which would be removed disulfide bridge plays an important role in the proper post- in the PstI deletion. translational folding of the Tac protein, perhaps serving to Lastly, we examined the potential role of N-glycosylation allow interactions between residues located within exons 2 in IL-2 binding and intracellular transport of the Tacprotein. and 4 and thereby contributing to theformation of the IL-2- N-Glycosylation of the vesicular stomatitis virus “G” glycobinding site(s). protein has been shown to be required for normal transport In addition to producing a loss of IL-2 binding, truncation of this protein to the cell surface (2). Site-directed mutagenof the Tac cDNA within exon 4 was found to interrupt the esis was employed to eliminate each of the two consensus normal transport of receptor protein to the cell surface and sequences for N-glycosylation in the Tac protein both singly instead resulted in the accumulation of these Tac proteins and in combination. Transfection of these mutant cDNAs within the endoplasmic reticulum. Interestingly, normal provided clear evidence that the Tac antigen is normally transport of the truncated receptor protein was re-established modified with carbohydrate at both sites. However, this sugar when virtually all of the exon 4 sequences were deleted. This addition was not required either for IL-2 binding or for normal restoration of transport is consistent with the normal trans- intracellular transport. Thus, N-glycosylation does not appear port phenotype of the Tac cDNA completely lacking exon 4 to play a critical role in the function of the Tacprotein. sequences. It seems possible that partial deletion of sequences within exon 4 produces areas oflocal denaturation which Acknowledgments-We thank Dr. Jarko Kochan for the gift of the interfere with receptor folding and in turnlead to sequestra- universal translational terminator and SharonGoodwinand Lisa tion of the receptor within the endoplasmic reticulum. Prec- Nieves for assistance in the preparation of this manuscript. edence for similar mutation-induced abnormalitiesin protein transport exists in other biological systems. For example, * Y. C. Pan and B. R. Cullen, manuscript in preparation. pI1-2RaMst protein labeled in the presence of 5 pg/ml of tunicamycin (TNC), an inhibitor of N-linked glycosylation (Fig. 6A). These findings demonstrate that both N-glycosylation sites are normally modified during post-translational processing of the Tac antigen. Next, we examined whether the partialor complete loss of N-linked glycosylation resulted in any change in the transport or IL-2 binding capacity of these Tac molecules (Fig. 6 B ) . These studies demonstrated that the Tac proteins lacking N-linked glycosylation were secreted with apparently normal kinetics and appeared fully able to bind IL-2, as determined by IL-2-Sepharose precipitation.

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Function Antigen Sequence Requirements Tac for REFERENCES

1. Sabatini, D.D, Kreibich, G., Morimoto, T., and Adesnik, M. (1982) J. Cell. Biol. 92, 1-22 2. Guan, J-L., Machamer, C. E., and Rose, J. K. (1985) Cell 4 2 , 489-496 3. Gething, M.-J., and Sambrook, J. (1982) Nature 300,598-603 4. Gething, M.-J., McCammon, K., and Sambrook, J. (1986) Cell 46,939-950 5. Yamamoto, T., Bishop, R.W., Brown, M. S., Goldstein, J. L., and Russel, D. W. (1986) Science 2 3 2 , 1230-1237 6. Lehrman, M.A., Schneider, W. J., Brown, M. S., Davis, C. G., Elhammer, A., Russel, D.W., and Goldstein, J. L. (1987) J. Biol. Chem. 262,401-410 7. Greene, W. C., Leonard, W. J., and Depper, J. M. (1986) Prog. Hematol. 14,283-302 8. Taniguchi, T., Matsui, H., Fujita, T., Hatakeyama, M., Kashima, N., Fuse, A., Hamuro, J., Nishi-Takaoka, C., and Yamada, G. (1986) Zmmunol. Rev. 9 2 , 121-133 9. Leonard, W. J., Depper, J. M., Uchiyama, T., Smith, K.A., Waldmann, T. A., and Greene, W. C. (1982) Nature 300,267269 10. Leonard, W. J., Depper, J. M., Kronke, M., Peffer, N. J., Svetlik, P. B., Kanehisa, M., Sullivan, M., and Greene, W.C. (1985) Science 230,633-639 11. Ishida, N., Kanamori, H., Noma, T., Nikaido, T., Sabe, H., Suzuki, N., Shimizu, A., and Honjo, T. (1985) Nucleic Acids Res. 13,7579-7589 12. Leonard, W. J., Depper, J. M., Robb, R. J., Waldmann, T. A., and Greene, W. C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6957-6961 13. Kuo, L-M., Rusk, C.M., and Robb, R. J. (1986) J. Zmmunol. 137,1544-1551 14. Leonard, W. J., Depper, J. M., Kronke, M., Robb, R. J., Wald-

15.

16. 17. 18. 19. 20. 21. 22. 23. 24.

25. 26. 27. 28. 29. 30.

mann, T. A., and Greene, W.C. (1985) J. Bid. Chem. 2 6 0 , 1872-1880 Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J., Robb, R. J., Kronke, M., Svetlik, P. B., Peffer, N. J., Waldmann, T., and Greene, W. C. (1984) Nature 3 1 1 , 626-631 Cosman, D., Cerretti, D. P., Larsen, A., Park, L., March, C., Dower, S., Gillis, S., and Urdal, D. (1984) Nature 3 1 2 , 768771 Cullen, B. R. (1986) Cell 4 6 , 973-982 Morinaga, Y., Franceshini, T., Inoue, S., and Inoue, M. (1984) Biotechnology 2,636-639 Cullen, B. R. (1987) Methods Enzymol. 152,684-704 Rubin, L. A., Kurman, C.C., Fritz, M. E., Biddison, W.E., Boutin, B., Yarchoan, R., and Nelson, D. L. (1985) J . ZmmunoL 135,3172-3177 Robb, R. J., and Kutny R. M. (1987) J. Zmmunol. 139,855-862 Neeper, M. P., Kuo, L-M., Kiefer, M. C., and Robb, R. J. (1987) J. Zmmunol. 138,3532-3538 Treiger, B. F., Leonard, W. J., Svetlik, P., Rubin, L. A., Nelson, D. L., and Greene, W. C. (1986) J. Immunol. 136,4099-4105 Hakimi, J., Seals, C., Anderson, L. E., Podlaski, F. J., Lin, P., Danho, W., Jenson, J. C., Perkins, A., Donadio, P. E., Familletti, P. C., Pan, Y.-C.E., Tsien, W.-H., Chizzonite, R.A., Casabo, L., Nelson, D.L., and Cullen, B. R. (1987) J. Biol. Chem. 262,17336-17341 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 7 4 , 5463-5467 Olden, K., Parent, J. B., and White, S. L. (1982) Biochem. Biophys. Acta 650,209-232 Morrison, S. L., and Scharff, M. D. (1975) J. Immunol. 1 1 4 , 655-659 Haas, I. G., and Wabl, M. (1983) Nature 306,387-389 Bole, D. G., Hendershot, L. M., and Kearney, J. F. (1986) J. Cell Biol. 102,1558-1566 Sharma, S., Rodgers, L., Brandsma, J., Gething, M-J., and Sambrook, J. (1985) EMBO J. 4,1479-1489