Contains Both Unique and Functionally Redundant Signal

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl.269, No. 20, Issue of May 20, pp. 14698-14704, 1994 Printed in U.S.A.

The Cytoplasmic Domain of the Interleukin-2 Receptor p Chain Contains Both Unique and Functionally Redundant Signal Transduction Elements* (Received for publication, February 7, 1994, and in revised form, March 10, 1994)

Mark A. GoldsmithSOn, Weiduan Xu$,M. Catherine AmaralS, Elizabeth S. KuczekS, and Warner C.GreeneSOII From the $Gladstone Institute of Virology and Immunology, Sun Francisco General Hospital, Departments of §Medicine and Imicrobiology and Immunology, University of California, Sun Francisco, California 94141-9100

Binding of interleukin-2 (IL-2) to theIL-2 receptor (IL- as well as heterotrimeric complexes containing all three sub2R) triggers a series of intracellular events culminating units, can bind IL-2 and initiate transmission of proliferative in lymphocyte proliferation and differentiation. A novel signals into the nucleus. transient assay of signal transduction leading to prolif- The biochemical mechanisms underlying growth signal transeration is now described which allows the rapid funcmission in response t o the binding of IL-2 remain poorly untional assessment of wild type and mutant receptors, derstood. By analogy to other members of the cytokine receptor including theIL-2R and other members of the cytokine superfamily, the bindingof ligand may facilitate association the receptor superfamily. This assay has been used to define of extracellular ligand-binding domainsof the IL-2R subunits, domains and specific residues within theIL-BRP intra- thereby properly aligning the intracellular domains of these cellular region that contribute to growth signal transreceptor chains (4, 5 ) . Although many traditional second mesduction. In these studies, internal deletion of either the conserved “Box1” or “BOX2” proximal cytokine receptor senger systems apparently are not induced by ligation of the IL-2R and attainment of this “activated configuration (6-8), homologysegmentssignificantlyimpairedreceptor certain downstream consequences have been recognized. function. Similarly, mutation of specific key residues Among the changes in intracellular physiology that have been within or between Box 1 and Box 2, or deletion of the described are inductionof certain proto-oncogenes (9-12), actiC-terminal 94 residues of the IL9RP chain, impaired phosphorylation of growth signaling.In contrast, either replacement of the vation of p2lTa8*GTPand Raf-l(13-15), and transmembrane domain with that of theCD4 molecule a variety of cellular substrates on serinehhreonine or tyrosine or internal deletionof the 119 amino acids immediately residues (16-21). Since tyrosine kinase activity has been detected in directassociation with the receptorcomplex (22, 23), downstream of Box 2 had no impact on growth signaling tyrosine phosphorylation of key substrates may constitute a competence. These studies thus further define the functional architectureof the intracellular region of IL-2RP, critical initiating event in this signal transduction pathway. and reveal specific receptor domains that are dispensOne necessary step in defining the protein-protein interacable, unique, or functionally redundant. tions that mediate signal transductionis a thorough molecular characterization of the receptor components themselves. Studies of both the IL-2R and of other cytokine receptors have Interleukin-2 (IL-2)’ and its cognate receptor (IL-2R) medi- depended largely on the stable expression of receptor variants ate diverse biologic events in the immune system, including in growthfactor-dependent cell lines, followed bymeasurement clonalexpansion of antigen-specific T lymphocytes (1).The of short-term proliferation in response to appropriate ligands fully competent high affinity receptor complex is now recog- (24). To facilitate detailed and rapid assignment of structure/ nized to comprise at least three transmembrane subunits (2) function relationships within thereceptor, we have established including: 1) the 55-kDa a (Tac) chain, a n inducible subunit a versatile and efficient transient assay of the receptor-medithat critically modulates receptor affinity for ligand; 2) the ated growth signaling process. This protocol, which appears to 70-kDa p chain, a member of the cytokine receptor superfamily be useful for multiple receptors types, permits the rapid assessthat is importantly involved in signal transduction; and3) the ment of receptor function with a biological readout that has newly recognized 64-kDa y chain, a second member of the cy- broad dynamic range and high sensitivity t o functional impairtokine receptor superfamily, which corresponds to the site of ment of the receptor. Such functional assessmentof IL-2RP demutation(s) inX-linked severe combined immunodeficiency (3). letion mutants and chimerasformed between CD4 and IL-2Rp Both heterodimeric complexes containing ILSRP andy chains, has permitted the identification of large dispensable regions, including its single transmembrane segment and the central * This work was supported in part by the J. David Gladstone Insti- one-third of its cytoplasmic tail. In contrast, both the proximal tutes. The costsof publication of this article were defrayed in part by the and distal intracellular portions of this subunit importantly payment of page charges. This article must therefore be hereby marked “uduertisernent”in accordance with 18 U.S.C.Section 1734 solely to contribute to receptor function. These findings represent sigindicate this fact. a detailed functional mapof nificant steps toward constructing ll Supported in part by National Institutes of HealthGrant AI the IL-2Rp subunit. A similar strategy should be possible with 07395-04 through the University of California, San Francisco AIDS Training Program.To whom correspondence should beaddressed:Glad- other members of the cytokine receptor superfamily. stone Institute of Virology and Immunology,P. 0. Box 419100, San MATERIALS AND METHODS Francisco, CA 94141-9100. Tel.: 415-695-3775; Fax: 415-826-1514. Cell Lines and Antibodies-The cell line BAiF3 is an IL-3-dependent The abbreviations used are: IL-2,interleukin-2;IL-2R, interleumurine pro-B cell line originally derived as described (25) and was kin-2receptor; IL-3, interleukin-3; PCR, polymerase chainreaction; EPO, erythropoietin;EPOR, EPO receptor; TM,transmembrane; CMV, kindly provided by Dr. Gordon Mills. These cells were maintained in RPMI 1640 supplementedwith 10% fetal calf serum, 10% WEHI-3 cytomegalovirus; PWT, wild type IL-2RP.

14698

Functional Architecture

of the IL-2Rp Chain

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fore developed a convenient assay scheme that relies upon transient transfection, bulk short-term cultures, and highly focused selection pressure. In thisprotocol, a n expression vector encoding the particular receptor subunit is transfected by electroporation into the IL3-dependent murinepro-B cell line BAA73 (day -1).After a 24-h culture inIL-3 to permitexpression of the transfectedgene, the cells are plated into dishes selection in medium containing the designated cytokine in theabsence of IL-3 (day 0). Cells acquiring a fully functional receptor by transfection proliferate continuously during this period, while untransfected parentalcells or cells acquiring a defective receptor cease to proliferate and subsequently die in theabsence of other growth factors. Viable cultures arereadily distinguishable from effete cultures by visual inspection after several days, and quantitative assessment is accomplished by measuring t3H1thymidine incorporation at selected time points as a marker of DNA synthesis. BAA73 cells express endogenous IL-2R-y chain, but notsufficient levels of IL-2Ra or P t o form IL-2-responsive heterodimeric or heterotrimeric receptor complexes. By day +6 following initiation of IL-2 selection, cultures ofBAA73 cells transfected with the IL-2Rp expression vector (PWT) demonstratedsubstantial L3H]thymidine incorporationcompared with a low background seen either in cultures transfected with vector alone or in cultures grown in the absence of IL-2 (Fig. lA). During the next several days such “positive” cultures reached peak incorporation of L3H1thymidine,with a 100-1000fold response relative t o the background incorporation in “vector only” transfectants. A representative assay with the mutant receptor PAA revealed full responsiveness as was seen previously in conventional assays with thecomparable “ A mutant (241, while the PS mutant (which is similar to the previously reported “SD” mutant) (24) demonstrated marked impairment of function (Fig. lA). Thus, this transient assay exhibits a wide dynamic range which faithfully reproduces the signaling phenotypes of these mutants as previously characterized using stable transfection methods. General Applicability to the Cytokine Receptor Superfamily-To test the general applicability of this method to other members of the cytokine receptor superfamily,we transfected a cDNA encoding the erythropoietin receptor (EPOR)into BA/F’3 cells and selected with erythropoietin (EPO) in theabsence of IL-3. The EPOR mediated similar largeproliferative responses in this setting (Fig. 1 B ) compared with the negative control, demonstrating the potential broad utility of this approach for studying other receptors. It should be noted that the EPOR is the constitutive mutant receptor usedintheseassays (cEPOR) containing a previously described substitution of cysteine for arginine 129 (361, which results in ligand-independent receptor homodimerization and signal transduction in stable transfectants (31, 36). However, in this transient system the cEPOR functions only in thepresence of the EPO ligand. This observation likely relates t o the observed requirement for a n “adaptation” period by host cells during which stable transfecRESULTS tants of this mutant receptor acquire ligand independence. As A Rapid Assay for Assessment of Receptor Functionindicated above, the transientmethod was designed in part to Lymphocyte proliferation represents a critical biologic end minimize such changes in cellular physiology that might acpoint that is stimulated by IL-2 binding to the IL-2R. One company long-term selection (see “Discussion”). conventional assay of receptor function has involved the stable One convenient method of comparing responses by various transfer of expression vectors encoding receptors into growth receptor variants is to display the maximal incorporation of factor-dependent hematopoietic lines, which consequently ac- L3Hlthymidine in response to saturating doses of ligand as a quire growth responsiveness to the cognate cytokine. The util- percentage of the response with the wild type receptor (Fig. ity of assays of proliferative potential thatrely upon such sta- 1C). Since expression of wild type IL-2RP using an alternate bly transfected cell lines is limited by the inconvenience of vector ( ~ 1 0 1 3supported ) comparable proliferation to that megenerating and maintaining long-term transfectants,by wide diated by the samereceptor expressedin theCMV-based vector clonal variability, and by the potential of unintended selection (Fig. lC), response in this assayis not vector-specific; the full pressures to altercellular physiology in subtleways. We there- responsiveness ofPAA and the severely impaired function of supernatant (as a source of IL-3), and 30 2-mercaptoethanol (complete medium). In our experience, parental BA/F3 cells maintained in long-term culture tend to develop independence from IL-3. For proliferation assays to evaluate receptor function it istherefore necessary to examine stock cultures regularly for the emergence of such growth factor-independent clones. The COS7 monkey kidney cell line (ATCC) was maintained as described (26). The monoclonal antibody DU-2 is specifically reactive with the IL-2RP chain (22). Recombinant human erythropoietin (Amgen Inc.) and recombinant human IL-2 (a gift of Chiron Corp.) were used as indicated under “Results.” Plasmids and Mutagenesis-The vectors pCMV4 and p1013 have been described previously (27, 28), and were provided by Drs. Mark Stinski and Roger Perlmutter, respectively. The vector pCMV4Neo, kindly provided by Dr. Mark Feinberg, was derived from pCMV4by insertion of an expression cassette containing the neomycin-resistance gene; the parental and modified vectors direct comparable levels of receptor expression. For the various functional analyses, the wild type IL-2RP cDNA (a gift from Dr. Tadatsugu Taniguchi) (29) was inserted into pCMV4, pCMV4Neo, or p1013. SM4 (provided by Dr. Taniguchi) (30) correspondsto a variantof IL-2RP containing a proline substitution for leucine 299, inserted into the p1013 vector. Truncation mutants of IL-2RP wereprepared by PCR amplification of the cytoplasmic domain of IL-2RP using 3’ primers containing the termination codon followed by a BanHI restriction site for cloning. Internal deletions were prepared by recombining cytoplasmic fragments obtained by PCR amplification using primers that contain convenient restriction sites adjacent to the deletions. For substitutions, PCR amplification was performed using primers that contain an adjacent restriction site as well as the appropriate mismatch. All amplified regions, including deletions and substitutions, were verifiedby sequencing using the Taq DyeDeoxy Terminator method and an AppliedBiosystems Automated Sequencer. The composition of each mutant is described in the relevant figure legend. The erythropoietin receptor cDNA (R129C) (31), a gift from Dr. Greg Longmore, was inserted into the pCMV4Neo vector forfunctional analysis. The human IL-2Ry cDNA,obtained by reverse transcriptase-PCR, was inserted into pCMV4 for binding studies with IL-2. ZYansient Functional Assay+DNA expression vectors were transfected into the IL-3-dependent mouse pro-B cell line BAD73 cells by electroporation. Briefly,20millioncellsgrowing exponentially were resuspended in 1ml of complete medium. Electroporation was carried out with 20 pg ofcDNA using a Gene Pulser (Bio-Rad) set at 960 microfarad and 300 mV. Transfectants then were maintained in 50 ml of complete medium for 24 h. After washing twice with phosphate-buffered saline, cells were cultured in medium containing either recombinant human IL-2 (10 m, Chiron Corp.), recombinant human erythropoietin (50 unitdml, Amgen, Inc.), 10%WEHI-3 supernatant (positive control), or nocytokine (negative control). On sequential days after plating, cells were aliquoted into 96-well plates (75,000 cellsin 0.2 ml/well) and then pulse-labeled with 1pCi of [3H]thymidine(DuPont NEN) for 4 h prior to harvest. L3H1Thymidine incorporation was measured using a Skatron Micro Cell Harvester and a Beckman liquid scintillation counter. Ligand Binding and Cross-linking-Equilibrium binding analysis was performed by co-transfecting IL-2Ry and IL-2RP expression plasmids into COS7 cells using the DEAE-dextran method (32). 1251-IL-2 (DuPont NEN) was used in these binding studies as described previously (33). Cross-linking studies were performedby co-transfecting IL2Ra and IL-2RP expression plasmids into COS7 cells followed by incubation with lz5I-IL-2,cross-linkingwith disuccinimidyl suberate (Pierce Chemical Co.), immunoprecipitation with DU-2, and analysis by SDSpolyacrylamide gel electrophoresis and autoradiography, as described (34, 35).

Functional Architecture of the IL-2Rp Chain

14700

A-

TM

20ww

BOX 1

215

IPWLGHLLVGLSGAFGFIILVYLLINCFWTGF~WLKKVLK

254

CNT~~P@CFFSQLSSEHGGDVQK~LSSPFPSSSFSPGGL

293

APE~SPLEVLER~K_VTQ~.LL@DKVPEPASLSSNHSLTS

BOX

2

332 CFTNQG$FFFHLPDALEIEACQV?FT;DP$SEEDPDEGV 371 AGAPTGSSPQPLQ~~ZEDDA&TFPSRDDLLLFSPSLL B

C

410 GGPSPPSTAPGGSGAGEERMP%LQERVPRDWDPQPLGP

.B

449

PTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPW

40% SRPPGQGEFRALNARLPLNTDA$LSLQELQGQDPTHLV

FIG.2. Sequence and architecture of the IL-2RP intracellular region. Data were retrieved from GenBamMBL (accession number M26062). The TM, Box 1, and Box 2 domains, and the borders of the designated A, B, and C domains are indicated. Conserved amino acid residues are underlined. Cytoplasmic tyrosine residues are identified with a closed circle. The one-letteramino acidcode is used, and numbers to the left indicateamino acid positions inthe mature receptor protein.

PSD are also readily evident in this display. Additionally, the previously described P mutant SM4 (containing L299P substitution) demonstrated markedlyreduced function in this assay, as has been shown elsewhere in a conventional assay using stable transfectants (30).

Functional Architecture of the IL-2Rp Intracellular RegionSeveral intrinsic featuresof the IL-2Rp chain served as convenient landmarks to begin our mutagenesis analysis (Fig. 2). First, the 25-residue putative transmembrane domain (TM) 100000shares only its hydrophobic character with other cytokine receptors, and is followed by a relatively polar stretch of 7 amino acids. Next appear two 14-amino acid elements, termed Box 1 and Box 2 (37), which are partially conserved among many cytokine receptors and are separatedby a relatively divergent 5000035-residue “spacer” sequence (Fig. 2). Mutational analysis has revealed the functional importance of these domains in other cytokine receptors (37, 38). In addition, the impaired mutant PSD lacks Box 2 as well as 30 additional upstream and 13 0” downstream residues. For convenience we have divided the 5 6 7 8 9 10 remainder of the cytoplasmic tail into three additional segments. Segment A (73 aminoacid residues), spanning the previously described “acidic region” (also known as A region) of the receptor, includes the first4 of 6 cytoplasmic tyrosine residues that serve as potential phosphate acceptor sites duringreceptor C. Percent of control activation. The adjacent segment B (46 amino acid residues) (k S.E.M.) contains the fifth tyrosine residue, and the C-terminal segment Vector only