Eur. J. Immunol. 1992. 22: 851-857
Patrice M. Dubois'.., Jan Stepinski'.*, Jacques Urbain. and Carol Hopkins Sibley'. Department of Genetics (SK-SO)'., University of Washington, Seattle and Departement de Biologie Moleculaire., UniversitC Libre de Bruxelles, Rhode-St-Genese
Membrane domains in B cell activation
Role of the transmembrane and cytoplasmic domains of surface IgM in endocytosis and signal transduction* The cross-linking of membrane IgM (mIgM) triggers the activation and differentiation of B lymphocytes. One very rapid result of the cross-linking is the activation of phospholipase C, the subsequent mobilization of free calcium from internal stores and the activation of protein kinase C. This is followed by a redistribution of the receptor-ligand complexes to a small cap on the B cell surface, the first step in endocytosis and antigen processing. Cross-linking of major histocompatibility complex (MHC) class I neither stimulates the release of intracellular calcium nor does it induce capping and endocytosis of the cell surface receptors. In this study, we sought to determine the role of the two carboxyterminal domains of the p heavy chain in signal transduction, capping and endocytosis of mIgM. We took advantage of the clear differences between MHC class I molecules and mIgM, replacing the transmembrane and cytoplasmic domains of p by their MHC class I equivalents. Our results show that the hybrid heavy chain could still associate with light chains and assemble into a tetramer on the cell surface. However, cross-linking of the hybrid cell receptor produced neither release of calcium from internal stores, nor capping and endocytosis. These observations demonstrate that the two carboxy-terminal domains of p are critical to both signal transduction and modulation of the mIgM-ligand complexes from the surface of B lymphocytes.
1 Introduction Mature B lymphocytes are arrested in the Go phase of the cell cycle, but cross-linking of their membrane immunoglobulin (mIgM) triggers their activation and differentiation. Cross-linking of mIgM activates phospholipase C (PLC) which hydrolyzes phosphatidylinositol4,S-bisphosphate to inositol 1,4,5 trisphosphate and diacylglycerol [l-31. Generation of these two secondary messengers triggers the mobilization of free calcium from internal stores and the activation of protein kinase C [4]. Elevated levels of cytoplasmic free calcium subsequently contribute to the uptake of calcium via plasma membrane channels [5]. The aggregation of surface Ig also increases protein tyrosine kinase activity [6, 71 and the importance of this pathway in lymphocyte activation is becoming increasingly clear. Cross-linking of mIgM is followed by a redistribution, or capping, of the receptor-ligand complexes to a small area on the cell surface [8]. Capping requires energy, is dependent on association of the mIg with various components of
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This work was supported by NIH grant GM 42508 to C.H.S. Departement de Biologic Molkculaire, Universite Libre de Bruxelles, 67, Rue des Chevaux, B-1640 Rhode-St.-Genkse, Belgium Department of Clinical Biochemistry, Medical Academy, Gdansk. Poland
Correspondence: Carol Sibley, Department of Genetics (SK-50), University of Washington, Seattle. WA 98195, USA Abbreviations: mIgM: Membrane IgM p,: Membrane form of IgM heavy chain ps:Secreted form of IgM heavy chain
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
the cytoskeleton 19-11] and is followed by endocytosis of the receptor-ligand complexes [8].Capping and endocytosis are specific responses to cross-linking ligands and are thought be important steps in antigen presentation by B cells [12, 131. mIgM is a tetramer composed of two membrane-bound p chains and two light chains. The various immunoglobulin heavy chain isotypes each contain a single transmembrane span followed by a cytoplasmic domain that can vary in size from 3 to 28 amino acids [14-161. The transmembrane domain and the first three cytoplasmic residues (LysVal-Lys) are highly conserved among different species and Ig classes [14-161. These three amino acids are the only cytoplasmic residues in mIgM. The MHC class I 01 chain is similar in structure to p chains and consists of multiple extracellular domains, a transmembrane part and a cytoplasmic tail [17]. It associates into a dimer with (32 microglobulin to form cell surface class I [18]. In B cells, class I molecules differ from surface Ig in that antibody cross-linking leads to the passive formation of a wide cap or patches; this does not require cytoskeletal interactions or generation of ATP [9].The rate of endocytosis of the MHC class I-antibody complexes is also much slower than that of cross-linked surface IgM [19]. Finally, under normal conditions, cross-linking of class I has no effect on the intracellular calcium concentration as seen in response to cross-linking of mIgM [20]. To determine the role of the two carboxyterminal domains of the p heavy chain in signal transduction as well as capping and endocytosis of mIgM, we have replaced the transmembrane and cytoplasmic domains of IgM by their mouse MHC class I equivalents. Our results show that the hybrid molecule could still associate with light chains, was assembled into a tetramer and was transported to the cell surface. 0014-2980/92/0303-0851$3.50+ .25/0
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P. M. Dubois, J. Stepinski, J. Urbain and C. Hopkins Sibley
However, cross-linking of this hybrid receptor did not trigger the release of calcium from internal stores, or capping and endocytosis. These observations demonstrate that the two carboxyterminal domains of p are critical for both signal transduction and modulation of the mIgMligand complexes from the cell surface.
Eur. J. Immunol. 1992. 22: 851-857
to 15 pg of RNA was separated on 1% agarose-formaldehyde gels [28], the RNA was blotted onto nitrocellulose (Schleicher and Schuell, Keene, NH) and baked at 80 "C for 1-2 h. Hybridization and washings were performed following the manufacturer's directions. Probes were nick-translated using [32P]deoxycytidinetriphosphate to a specific activity of >lo8 cpm/pg DNA.
2 Materials and methods 2.1 Construction of the Mum-2 hybrid gene Plasmid pV~167p[21] containing the genomic sequence of a rearranged mouse p heavy chain gene was a gift from Dr. U. Storb (University of Chicago, IL). The mouse H-2 class I genomic sequence pC23 (Fig. 1) was a gift from Dr. B. Arnold (German Cancer Research Center, Heidelberg). This gene includes the extracellular domains of H-2Kkand the transmembrane and cytoplasmic domains of H-2Kd [22]. The Mu/H-2 (Fig. 1) was constructed as follows. A Cla I(blunt)/Hind I11fragment of pC23 containing the transmembrane and cytoplasmic sequences of H-2 class I was subcloned into Bam HI(b1unt) and Hind I11sites of pUC19. The transmembrane and cytoplasmic domains of V ~ 1 6 7 pwere removed by a Kpn I/Sal I(b1unt) digest and replaced by a Kpn I/Hind III(b1unt) fragment containing the corresponding sequences of H-2Kd.
2.5 Cell staining and endocytosis experiments Cells were stained with an FITC-conjugated goat F(ab')2 anti-mouse IgM (Tago, Burlingame, CA). Analyses were performed on an Ortho Cytofluorograph (Ortho Diagnostic System, Westwood, MA). In endocytosis experiments, cells were washed once at room temperature and resuspended in 400 pl of RPMI containing goat anti-mouse IgM coupled to biotin (Tago). After 15 min at 37 "C, cells were washed at room temperature, resuspended in 400 yl of medium and incubated at 37°C in 5 YOCOz. The reaction was arrested by the addition of cold medium containing 0.1 YONaN3. Cells were then stained with avidin-FITC.
2.6 Analysis of intracellular calcium mobilization
This method has been described in detail [29]. Cells were washed twice and resuspended at a final concentration of 2 x 106/mlin RPMI, 10 mM Hepes, pH 7.0 containing the 2.2 Cell lines and culture conditions acetoxymethylester form of Indo-1 ([30]; Molecular Probes, Junction City, OR) at a concentration of 4 p ~ . Clone CH12 LX2B4 was a gift from Dr. G. Haughton, Loading was carried out for 45 min at 37°C. RPMI (Duke Medical Center, Durham, NC). It is an IgM- variant supplemented with 5 YOFCS was added to each sample and of the B lymphoma CH12 LX [23] that has lost the the incubation continued for another 15 min. After washrearranged allele of the immunoglobulin heavy chain gene. ing, cells were stored at room temperature in the dark. The M12.4.1.4.5 line was a gift from Dr. R. Asofsky (NIH, Analysis was performed with an Ortho Cytofluorograph Bethesda, MD). It is an IgM- variant of the BALB/c 50HH, the UV exitation (351 to 364 nm) was provided by B lymphoma M12 [24]. The A20 line is a B lymphoma an argon laser, blue emission was detected at 480 to 520 nm, expressing the membrane form of IgG2, [24]. Cells were and violet emission at 383 to 407 nm. Cells were stimulated grown in RPMI 1640 supplemented with 7 % FCS, with 5 pg/ml of a goat anti-mouse IgM or with a goat 0.3 mg/ml L-glutamhe, 1.0 mM sodium pyruvate, lC5M F(ab'):! anti-mouse IgM coupled to biotin. In experiments 2-mercaptoethanol, 100 units/ml penicillin and 0.01 YO where stimulation was performed with antibodies coupled streptomycin at 37°C in 5 YO C02. to biotin, avidin was added at a final concentration of 10 to 15 pg/ml. 2.3 Establishment of transfected lines Cells in the exponential phase of growth were cotransfected with 5 pg of circular pSV2Neo and 30 pg of the linearized nonselectable marker by electroporation [25]. Briefly, lo7 cells and the dialyzed plasmids were mixed in a final volume of 500 pl of RPMI 1640 at room temperature. The electroporation device reproduced the discharge profile of the shorted Iscove 494 power supply [26]. Cells were then grown for 24 h in fully supplemented growth medium containing 20 YOto 40 % culture supernatant from untransfected cells. After 24 h cells were plated at 1 x lo4 to 1 x 105/well with 1 mg/ml G 418. G418-resistant clones were stained with fluoresce-in-coupled antibodies and screened by fluorescence microscopy.
2.4 RNA analysis Total cellular RNA was isolated from log-phase cells using 4 M guanidinium isothiocyanate and CsCl[27]; thereafter 5
2.7 Immunoprecipitations and Western blot analysis
Cells were lysed in 20 mM Tris, pH 7.4, 100 mM NaCl, 1% NP40, 2 mM PMSF and 10 pg/ml leupeptin (Sigma, St. Louis, MO) for 20 min at 4 "C. Sepharose beads coupled to a polyclonal goat anti-mouse IgM or to a rat monoclonal anti-mouse IgM (Zymed Laboratories, San Francisco, CA) were used for immunoprecipitations. Beads were then washed, resuspended in 100 mM NaH2P04,pH 8.0,10 mM EDTA, 0.5 YO Triton X-100, 0.1 YO SDS, 1YO 2-mercaptoethanol and boiled. Supernatants were divided for treatment with or without glycopeptidase F (Boehringer Mannheim, Indianapolis, IN), used at 0.1 U/ml for 12 h at 37 "C. Separations were performed according to Laemmli [31] on a 10 YOSDS/acrylamide gel. Electroblotting onto Immobilon membranes (Millipore, Bedford, MA) was done in 25 mM Tris, pH 8.3, 192 mM glycine and 20 %O methanol at 0.6-0.9 A for 5-6 h. Membranes were saturated with 5 YoBSA in 20 mM Tris, pH 7.4, 100 mM NaCl for 1 h at
Eur. J. Immunol. 1992. 22: 851-857
Membrane domains in B cell activation
37”C,washed in the same buffer containing0.1 YOBSA, and incubated with a polyclonal rabbit anti-mouse IgM (Zymed) in 20 mM Tris, pH 7.4, 100 mM NaCl, 1YOBSA, 0.5 YO Tween-20 for 1 h at 37°C. Goat anti-rabbit IgG (Zymed) and recombinant protein G coupled t o alkaline phosphatase (Zymed) were used for detection. Staining was done with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
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pVH167 p
1Kb
L
3.1 Construction of the hybrid gene and transfection into B cell lines The starting plasmids, pV~167i.t and pC23, contained genomic copies of a productively rearranged p gene and a H-2K MHC class I gene (Fig. 1).The hybrid gene, pMu/H2, encodes the variable and constant regions of IgM and the transmembrane and cytoplasmic regions of H-2Kd (Fig. 1). The construction was verified by sequencing the junction. The linear p V ~ 1 6 7 pand pMu/H-2 plasmids were cotransfected into three mouse B lymphoma lines, CH12 LX2B4, M12.4.1.4.5 and A20. Clones resistant to G418 were screened by staining with FITC-coupled goat antimouse IgM antibodies. Several clones were chosen for detailed study; they are described in Table 1.
VDI
CH1 CHZ CH3
CH4
TM CyTo+3’untr.
pMuRI-2 Kpnl*/Sall* HindIII*l Sall’
Hidm’l salt’
L
3 Results
EcoRl
Kpnl
EcoRl
2.8 Iodinations Cells (2 x 107-3 x lo7) were surface labeled with 500 yCi 1251 using glass vials coated with 300 pg of IODO-gen (Pierce, Rockford, IL). The reaction was carried out in 750 yl of PBS for 15 min at room temperature. Membrane solubilization and immunoprecipitation were performed as described above except that formalin-fixed S. aureus were used for immunoprecipitations.
853
/I VDJ
CHI CH2 CH3
CH4 TM
CyTo+3’untr.
pC23 (H-2K) chl
Hind11
Hind111
Figure 1. Structure of the genes used in study. Plasmid pMu/H-2 was constructed as described in Sect. 2.1. Stars indicate bluntended restriction sites. Exons are shown as filled boxes and 3’ untranslated sequences as open boxes.
intron including the ys polyadenylation site, we expected pMu/H-2 to encode a primary transcript that could be spliced t o generate mRNA encoding both the usual secreted ps and a membrane-bound Mu/H-2 heavy chain [32]. The Northern blot hybridized with a fragment of the V ~ 1 6 7 yV region is shown in Fig. 2. Each of the lines transfected with the pMu/H-2 (CH12 MH60, CH12 MH85, M12 MH12.2 and A20 MHd4) expressed a ys message identical in size to the wild-type ys and a second band of about 3.4 kb which corresponds to the Mum-2 hybrid mRNA (lanes 3, 4, 6 and 8). Clone CH12 M6 processed the V ~ 1 6 7 ptranscript into both pm and ys mRNA. The untransfected lines showed no hybridization to t h e V ~ 1 6 7 y V region probe.
3.2.2 Protein analysis 3.2 Expression of the transfected genes To examine the protein product of each transfected gene. NP40 lysates were immunoprecipitated with antibodies directed against mouse y chains. Immunoprecipitates were In the CH12 and M12 cell lines, the endogenous p tran- size-separated by denaturing SDS-PAGE and electroblotscripts are processed into both ym and ys mRNA. Since the ted onto membranes. As expected, all clones produced a pMu/H-2 construct retained the 5‘ portion of the ys-ym protein of the appropriate size for ys (Fig. 3A). In addition,
3.2.1 RNA analysis
Table 1. Expression of transfected and wild-type IgM or Mu/H-2 molecules in B lymphoma linesa).
Clone
CH12 LX2B4 CH12 M6 CH12 MH60 CH12 MH85 M12 4.5 M12 M4 M12 M13 M12 MH12.2 A20 A20 MHd4
Plasmids
Membrane receptor
Fluorescence FITC-goat anti-mouse IgM
-
-
pSV2Neo + pVH167p pSV2Neo + pMum-2 pSV2Neo + pMu/H-2
IgM MUM-2 Mum-2
9.0 53.6 92.4 97.7 8.5 119.3 82.2 71.8 6.0 77.2
-
pSV2Neo pSV2Neo pSV2Neo + pMuM-2
-
IgM IgM Mum-2 IgG pSV2Neo + pMu/H-2 IgG + Mum-2
a) Cells were stained with an affinity-purified,
FITC-labeled goat F(ab’)z directed against mouse IgM heavy chains.The fluorescence represents the difference in mean channels between unstained and stained populations. Clones M12 M4 and M12 M13 spontaneouslyreverted to the expression of their wild-type rearranged IgM heavy chain gene.
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P. M. Dubois, J. Stepinski, J. Urbain and C. Hopkins Sibley
Eur. J. Immunol. 1992. 22: 851-857
8E
d
2
2 2 u X u
X
MU/H-2\
E
X
W
(MUM-2) K
-
2L
P2K2
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Figure 2. MRNA levels of wild type and hybrid genes in parental and transfected B lymphoma lines.Total RNA was denatured with fomaldehyde and size-separated by formaldehyde-agarose gel electrophoresis, transferred to a nitrocellulose membrane and probed with a 32P-labeled1.2-kbBam HI DNA fragment containing the VH167 variable region genomic sequence. CH12 LX2B4, M12 M13 and A20: untransfected cell lines; CH12 M6: transfected with pVH167p;CH12 MH60, CH12 MH85, M12 MH12.2 and A20 MHd4: cells transfected with pMu/H-2.
they synthesized higher molecular weight forms which correspond to either ~n, or the hybrid protein. Because the cytoplasmic domain of the H-2 is about 25 amino acids longer than that in pm, the hybrid protein was correspondingly larger than pm (compare lanes CH12M6 and CH12 MH60 in Fig. 3A). In glycosylated samples (Fig. 3A), the membrane form of the Mum-2 was represented by one band in CH12 transformants and by two bands in M12 and A20 clones. However, in samples treated with glycopeptidase F (Fig. 3B), only one band corresponding to the membrane form of Mum-2 was detectable in CH12 and M12 cells. A single Mu/H-2 band was also seen in a similar experimant done on A20 MHd4 cells (data not shown). This observation could be related to a difference between CH12 and the other cell lines in the glycosylation pattern of Mu/H-2.
Figure 4. Size of the molecules immunoprecipitated by anti-x antibodies from cell lines transfected with wild-type or hybrid gene. Cells were surface labeled with lZ5Iand NP40 lysates were immunoprecipitated with a goat anti-mouse x light chain. Immunoprecipitates were then separated on a 7 % SDS-acrylamide gel under nonreducing conditions. CH12 LX2B4: IgM- clone, CH12 M6 and M12 M4: mIgM+ clones, CH12 MH85 and M12 MH12.2: Mu/H-2+ clones, A20 MHd4: mIgG+/Mu/H-2+clone.
3.2.3 Structure of the hybrid receptor To determine that the hybrid heavy chain was expressed on the cell surface in the form of a (Mu/H-2)2K2 tetramer as expected for normal mIgM, intact cells were labeled with 1251, lysed in 1% NP40 and proteins precipitated with antibodies directed against x light chain. The isolated proteins were size-separated under non-reducing conditions on a 7 YOSDS-polyacrylamide gel.The autoradiogram shown in Fig. 4 demonstrates that the Mu/H-2 receptor is associated with x light chain and migrates, like mIgM, in the 200-300-kDa range. The additional band seen in the M12 M4 and A20 MHd4 lanes correspond to a p,x dimer and to surface IgG, respectively. Only one band corresponding to mIgG was observed in untransfected A20 cells (data not shown). Thus, the assembly of this hybrid molecule resembles the Ig pattern, forming a tetramer of heavy and light chains.
3.3 Endocytosis of cross-linked receptors B. Glycopeptidase treated
Figure 3. Western blot analysis of anti-IgM immunoprecipitatesin control and transfected B cell lines. Cells were lysed with NP40 and the membrane and cytoplasmic fractions were precipitated with a rabbit polyclonal (CH12 and M12 clones) or a rat monoclonal anti-mouse IgM (A20 clones) coupled to Sepharose beads. Immunoprecipitates were size-separatedunder reducing conditions on a 10 % SDS acrylamide gel and blotted onto Immobilon membranes. Staining was done as described in Sect. 2.7. A: Samples not treated with glycosidase F, and B: Samples treated with 0.1 unit of the glycopeptidase F at 37 "C for 12 h prior to analysis.
Our first step in evaluating the functional properties of the Mu/H-2 hybrid receptor was to study its capacity to cap and internalize rapidly in response to a cross-linking stimulus. In B cells, this process is characteristic of Ig receptors but not of MHC class I molecules [9]. Cells were incubated with biotin-coupled F ( a b ' ) ~anti-p antibodies, washed and incubated at 37 "C, 5 YOC02 for various times to allow capping and internalization to occur. The amount of mIgM remaining was then assayed by staining with FITC-coupled avidin. The top five panels of Fig. 5 show that both the parental line CH12 LX expressing surface IgM and clone CH12 M6 transfected with pVH167p internalized a large fraction of their mIgM during the 90-min incubation at 37°C. In contrast, in the two clones expressing the pMu/H-2 contrast (CH12 MH60 and CH12 MH85), a comparatively minor decrease in membrane fluorescence could be detected after
Membrane domains in B cell activation
Eur. J. Immunol. 1992. 22: 851-857
855
Mu/H-2 receptors formed small patches scattered evenly over the cell surface (results not shown).
3.4 Calcium studies In B cells, cross-linking of mIgM triggers the release of I* s)
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calcium from intracellular stores [2]. This is then a more immediate test of the function of the Mu/H-2 receptor. Cells were loaded with Indo-1 [30], and the effect of cross-linking their cell surface IgM or Mu/H-2 monitored on the cytofluorograph [29]. Fig. 6 (panels A and D) shows that cross-linking with F(ab')z anti-p antibodies induced a rapid intracellular calcium increase in CH12 and M12 cells expressing mIgM. However, in cells expressing Mu/H-2, no increase in the blue/violet ratio was observed upon crosslinking with anti-Ig antibodies (Fig. 6 B, C, E).
I
CH12 M6
3.0
Figure5. Analysis of surface IgM and NH-2 modulation in response t o an anti-IgM cross-linking stimulus. Cells (lo6) were incubated in goat F(ab')2 anti-mouse IgM coupled t o biotin, for 15 min at 37"C, in 0.2 ml of RF'MI 1640. Unbound antibody was removed by two washes in the same medium at room temperature. Cells were then resuspended in RF'MI and incubated at 37°C and the endocytosis reaction was arrested by the addition of NaN3 after 30 min (--------)and 90 min. (-.-.-.-.-).Samples incubated with antibodies in the presence of NaN3 (........), and unstained controls (-).
90min. This could reflect the normal turnover rate of Mu/H-2 proteins on the cell membrane. M12 cells expressing mIgM or the Mu/H-2 protein exhibit the same differences in their response t o cross-linking with anti-IgM antibodies (Fig. 5). A20 MHd4 cells also failed t o internalize the Mu/H-2 receptor in response t o cross-linking antibodies (data not shown). The same clones were tested t o determine the pattern of redistribution which followed cross-linking of their surface p or Mu/H-2. Clones which expressed mIgM redistributed their receptor-ligand complexes into tight caps, whereas the
Time (sec.)
Figure 6 . Measurement of Ca2+ mobilization in response to antiIgM stimulation. Relative cytoplasmic calcium concentrations expressed as the mean violet/blue indo fluorescence ratio. Cells were stimulated by the addition of 5 pgl6 x lo5 cells/ml of goat F(ab')2 anti-mouse IgM (A, B, D, F, G) or goat F(ab')z anti-mouse IgM coupled to biotin (C, E). In samples treated with biotincoupled antibodies, avidin was added at final concentrations ranging from 10 to 15 pglml. Antibodies directed against IgM triggered at least 80 % of the cells to increase their calcium levels above baseline in clones expressing the membrane form of p. An affinity-purified polyclonal goat anti-mouse IgG was used to activate the A20 B lymphoma through its IgG2, membrane receptor (F and G). A . CH12 M6: transfected with IgM ( p V ~ 1 6 7 ~B) ; and C: CH12 LX2B4 untransfected, CH12 MH60, CH12 MH85: transfected with pMuM-2; D. M12 M4 expressing IgM (wild type).E. M12 transfected with pMulH-2; F. A20, untransfected; G A20 transfected with pMulH-2.
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l? M. Dubois, J. Stepinski, J. Urbain and C. Hopkins Sibley
Since double cross-linking of class I has been shown to trigger Ca2+increases [20], cells expressing Mu/H-2 receptors were treated with a biotin-coupled goat F(ab')z anti-p followed by avidin. Panels C and E (Fig. 6) show that no variation in intracellular Ca2+ concentrations could be detected under these conditions. This inability to respond was not due to poor loading with indo-1, since a detectable increase in the violet/blue ratio followed treatment with ionomycin (Fig. 6, panels B, C and E). The failure of Mu/H-2+ cells to mobilize calcium could simply reflect an inability to respond to any cross-linking stimulus. To examine this possibility we compared A20 cells (mIgG+) with clone A20 MHd4 which expresses Mum-2. Panels F and G (Fig. 6) show that neither population increased intracellular calcium concentrations when stimulated with anti-p antibodies, while both responded to anti-IgG antibodies. This last experiment indicates that cross-linking of a Mum-2 receptor does not give rise to an increase in intracellular free calcium even in a cell that signals normally through another surface Ig.
4 Discussion The data presented in this study demonstrate that replacement of the transmembrane and cytoplasmic domains of p by the corresponding H-2 class I sequence still allowed normal expression of the heavy chain, assembly into (Mu/H-2)2~2tetramers and targeting of this receptor onto the cell surface. However, in clones expressing the Mu/H-2 receptor, cross-linking with anti-p antibodies failed to produce either signal transduction or internalization of the receptor-ligand complexes. The assembly of class I and mIgM are in many ways similar [33,34]. They both require the participation of two different polypeptides for membrane expression, the key interactions necessary for formation of the complex between the two proteins require extracellular domains, and their assembly occurs in the rough endoplasmic reticulum prior to expression on the surface. However, there is one important difference between the two systems: mIgM assembles into a disulfide-bonded tetramer, and the class I-Pz-microglobulin complex remains a dimer. The hybrid Mu/H-2 protein forms complexes of the size expected for tetramers, and thus resembles the immunoglobulin in this respect. This further supports the notion that the information controlling the assembly of mIgM is within the extracellular domains. Although expression of the VH167p protein has been shown in transgenic mice [21], it was expressed at low frequency (up to 1.8 %) compared to the Mum-2 protein (10 to 50 YO)on the surface of stable B cell transfectants. However, when a similar construct which could only produce p, was introduced into B cells, this frequency rose to 6.1 YO.Thus, processing of theVH167p transcript into pm or ps messages could be one element accounting for the difference between IgM (V~167p)and Mum-2 surface expression frequencies. Another important explanation for the difference in efficiency of mIgM and Mu/H-2 expression is that the p chains but not the Mu/H-2 protein require a number of auxiliary
Eur. J. Immunol. 1992. 22: 851-857
molecules such as IgM-a and Ig-P for cell surface expression [35-371. If expression of these molecules is frequently lost in IgM- lines, one would observe a corresponding decrement in the frequency of mIgM+ clones after transfection with pV~167p.This is in agreement with the observation that a chimeric receptor identical to Mum-2 can be targeted onto the surface of an IgH-plasmacytoma which is unable to express mIgM in the absence of IgM-a [37].Thus, a number of factors may combine to make the expression of wild-type IgM on the surface of these cells relatively rare. However, it is clear from our studies and those of Shaw et al. [38] that when a transfected p chain was expressed on the surface of a B lymphoma, its function was normal. This study complements two others which investigated the importance of the membrane and cytoplasmic domains of mIgM. In the first, Webb et al. replaced these two Ig domains with their MHC class I1 counterparts [39]. Although their study did not establish the structure of this surface receptor on the B lymphoma WEHI 231, the hybrid molecule did fail to signal growth inhibition after crosslinking [39]. The second is the work of Shaw et al. [38] in which mutant p chains were used to show that specific residues in the transmembrane domain and the presence of a cytoplasmic tail of similar length and charge were critical for antigen presentation and signalling. Thus, changes in either the membrane or cytoplasmic domain of p are sufficient to prevent proper receptor function. The failure of the Mu/H-2 receptor to transduce activation signals and internalize in response to cross-linkingcould be directly related to its expression on the cell surface in the absence of interactions with some, or all components of the B cell antigen receptor complex. Alternatively, the hybrid Mum-2 surface molecule could associate with this complex, but disruption of key interactions in the transmembrane and cytoplasmic regions would impair receptor function. Although further experiments are clearly required to distinguish between these models, two studies argue in favor of the latter. First, the extracellular domain of surface Ig directly adjacent to the plasma membrane has been shown to be a critical element in the association with the class specific form of Ig-a [40], and second, replacement of specific amino acids in the transmembrane or cytoplasmic region of IgM blocks calcium signaling and/or antigen presentation while still allowing the mutated IgM to be expressed on the cell surface [38]. It is thus likely that all three carboxy-terminal domains of the Ig receptor interact ~ of with members of the signaling complex. The C Hdomain IgM could be responsible for association with IgM-a (and possibly other components of the complex) whereas the transmembrane and cytoplasmic domains could affect the conformation of these accessory molecules, enabling them to transduce activation signals and target the receptorligand complexes to the appropriate intracellular compartement. Further studies of this kind will allow the identification of the exact interactions between auxiliary molecules and particular residues within the relevant domains of mIgM. They should also provide important information concerning the interactions between the antigen receptor complex and other molecules such as the lyn tyrosine kinase [41] thought to be involved in coupling the receptor to signal transduction.
Eur. J. Immunol. 1992. 22: 851-857 We would like to thank Drs. U. Storb and B. Arnold for plasrnids, G. Haughton and R. Asofsky for cell lines, K . Bomsztyk for help with calcium assays and E McConnell, I? Lane and R. Shapiro for helpful discussions and advice.
Received September 6, 1991; in revised form December 2, 1991.
5 References 1 Ransom, J. T., Harris, L. K. and Cambier, J. C., J. Immunol. 1986. 137: 708. 2 LaBaer, J., Tsien, R. Y., Fahey, K. A. and DeFranco, A. L., J. Immunol. 1986. 137: 1836. 3 Bijterbosch, M. K., Meade, C. J., Turner, G. A. and Klaus, G. G., Cell 1985. 41: 999. 4 Mizuguchi, J.,Tsang, W., Morrison, S. L., Beaven, M. A. and Paul, W. E., J. Immunol. 1986. 137: 2162. 5 Ransom, J. T., Chen, M., Sandoval, V M., Pasternak, J. A., Digiusto, D. and Cambier, J. C., J. Immunol. 1988. 140: 3150. 6 Campbell, M. A. and Sefton, B. M., Embo J. 1990. 9: 2125. 7 Gold, M. R., Law, D. A. and DeFranco, A. L., Nature 1990. 345: 810. 8 Taylor, R. B., Dufus, W. I? H., Raff, M. C. and de Petris, S., Nature 1971. 233: 225. 9 Braun, J., Fujiwara; K., Pollard, T. D. and Unanue, E. R., J. Cell Biol. 1978. 79: 409. 10 Rosenpire, A. L. and Choi, Y S., Mol. Immunol. 1982. 19: 1515. 11 Bourguignon, L. A. W. and Bourguignon, G. J., Int. Rev. Cytol. 1985. 87: 195. 12 Lanzavecchia, A . , Nature 1985. 314: 537. 13 Ron, Y and Sprent, J., J. tmmunol. 1987. 138: 2448. 14 Rabitts, T. H., Forster, A. and Milstein, C. F!,Nucl. Acids Res. 1981. 9: 4509. 15 Bernstein, K. E., Alexander, C. B., Reddy-Premkumar, E. and Mage, R. G., J. Imrnunol. 1984.132: 490. 16 Tyler, B. M., Cowman, A. F., Gerondakis, S. D., Adams, J. M. and Bernard, O., Proc. Natl. Acad. Sci. USA 1982. 79: 2008. 17 Steinmetz, M. and Hood, L., Science 1983. 222: 727.
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18 Nathenson, S., Geliebter, J., Pfaffenbach, G. M. and Zelf, R. A , , Annu. Rev. Immunol. 1986. 4: 471. 19 Pernis, B., tmmunol. Today 1985. 6: 45. 20 Gilliland, L. K., Noms, N. A., Grosmaire, L. S., Ferrone, S., Gladstone, P. and Ledbetter, J. A., Hum. Immunol. 1989. 4: 269. 21 Storb, U., Pinkert, C., Arp, B., Engler, P., Gollahon, K., Manz, J., Brady, W. and Brinster, R. F., J. Exp. Med. 1986. 164: 627. 21 Arnold, B. Burgert, H. G., Hamann, U., Hammerling, G., Kees, U. and Kvist, S., Cell 1984. 38: 79. 23 Haughton, G., Arnold, L. W., Bishop, G. A. and Mercolino, T. J., Immunol. Rev. 1986. 93: 35. 24 Kim, K. J., Kanellopoulos-Langevin, C., Mewin, R. M., Sachs, D. H. and Asofsky, R., J. Immunol. 1979. 122: 549. 25 Potter, H., Weir, L. and Leder, P., Proc. Nat. Acad. Sci USA 1984. 81: 7161. 26 Emery, D. W., Smith, S. and Hopkins-Sibley, C., Techniques 1989. 1: 153. 27 Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. R., Biochemistry 1979. 18: 5295. 28 Thomas, F! S., Proc. Natl. Acad. Sci. USA 1980. 77: 5201. 29 Rabinovitch, I? S., June, C. H., Grossman, A. and Ledbetter, J. A., J. Immunol. 1986. 137: 720. 30 Grynkiewicz, G., Poenie, M. and Tsien, R. Y., J. Biol. Chem. 1985. 260: 2719. 31 Laemmli, U. K., Nature 1970. 227: 680. 32 Early, F!, Rogers, J., Davis, M., Calame, K., Bond, M., Wall, R. and Hood, L., Cell 1980. 20: 313. 33 Hendershot, L., Bole, D. and Kearney, J. F., Immunol. Today 1987. 8: 111. 34 Klar, D. and Hammerberg, G., EMBO J. 1989. 8: 475. 35 Sakaguchi, N., Kashiwamura, S., Kimoto, M., Thalmann, F! and Melchers, F., EMBO J. 1988. 7: 3457. 36 Hombach, J., Tsubata, T., Leclercq, L., Stappert, H. and Reth, M., Nature 1990. 343: 760. 37 Hombach, J., Leclercq, L., Radbruch, A., Rajewsky, K. and Reth, M., EMBO J. 1988. 7: 3451. 38 Shaw, A. C., Mitchell, R. M., Weaver, Y. K., Campos-Tories, J., Abbas, A. and Leder, I?, Cell 1990. 63: 381. 39 Webb, C. E, Nakai, C. and Tucker, F! W., Proc. Natl. Acad. Sci. USA 1989. 86: 1977. 40 Wienands, J., Hombach, J., Radbruch, A., Riesterer, C. and Reth, M., EMBO J. 1990. 9: 449. 41 Yamanashi, I!, Kakiuchi, T., Mizuguchi, J.,Yamamoto, T. and Toyoshima, K., Science 1990. 251: 192.
Patrice M. Dubois'.., Jan Stepinski'.*, Jacques Urbain. and Carol Hopkins Sibley'. Department of Genetics (SK-SO)'., University of Washington, Seattle and Departement de Biologie Moleculaire., UniversitC Libre de Bruxelles, Rhode-St-Genese
Membrane domains in B cell activation
Role of the transmembrane and cytoplasmic domains of surface IgM in endocytosis and signal transduction* The cross-linking of membrane IgM (mIgM) triggers the activation and differentiation of B lymphocytes. One very rapid result of the cross-linking is the activation of phospholipase C, the subsequent mobilization of free calcium from internal stores and the activation of protein kinase C. This is followed by a redistribution of the receptor-ligand complexes to a small cap on the B cell surface, the first step in endocytosis and antigen processing. Cross-linking of major histocompatibility complex (MHC) class I neither stimulates the release of intracellular calcium nor does it induce capping and endocytosis of the cell surface receptors. In this study, we sought to determine the role of the two carboxyterminal domains of the p heavy chain in signal transduction, capping and endocytosis of mIgM. We took advantage of the clear differences between MHC class I molecules and mIgM, replacing the transmembrane and cytoplasmic domains of p by their MHC class I equivalents. Our results show that the hybrid heavy chain could still associate with light chains and assemble into a tetramer on the cell surface. However, cross-linking of the hybrid cell receptor produced neither release of calcium from internal stores, nor capping and endocytosis. These observations demonstrate that the two carboxy-terminal domains of p are critical to both signal transduction and modulation of the mIgM-ligand complexes from the surface of B lymphocytes.
1 Introduction Mature B lymphocytes are arrested in the Go phase of the cell cycle, but cross-linking of their membrane immunoglobulin (mIgM) triggers their activation and differentiation. Cross-linking of mIgM activates phospholipase C (PLC) which hydrolyzes phosphatidylinositol4,S-bisphosphate to inositol 1,4,5 trisphosphate and diacylglycerol [l-31. Generation of these two secondary messengers triggers the mobilization of free calcium from internal stores and the activation of protein kinase C [4]. Elevated levels of cytoplasmic free calcium subsequently contribute to the uptake of calcium via plasma membrane channels [5]. The aggregation of surface Ig also increases protein tyrosine kinase activity [6, 71 and the importance of this pathway in lymphocyte activation is becoming increasingly clear. Cross-linking of mIgM is followed by a redistribution, or capping, of the receptor-ligand complexes to a small area on the cell surface [8]. Capping requires energy, is dependent on association of the mIg with various components of
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*
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This work was supported by NIH grant GM 42508 to C.H.S. Departement de Biologic Molkculaire, Universite Libre de Bruxelles, 67, Rue des Chevaux, B-1640 Rhode-St.-Genkse, Belgium Department of Clinical Biochemistry, Medical Academy, Gdansk. Poland
Correspondence: Carol Sibley, Department of Genetics (SK-50), University of Washington, Seattle. WA 98195, USA Abbreviations: mIgM: Membrane IgM p,: Membrane form of IgM heavy chain ps:Secreted form of IgM heavy chain
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
the cytoskeleton 19-11] and is followed by endocytosis of the receptor-ligand complexes [8].Capping and endocytosis are specific responses to cross-linking ligands and are thought be important steps in antigen presentation by B cells [12, 131. mIgM is a tetramer composed of two membrane-bound p chains and two light chains. The various immunoglobulin heavy chain isotypes each contain a single transmembrane span followed by a cytoplasmic domain that can vary in size from 3 to 28 amino acids [14-161. The transmembrane domain and the first three cytoplasmic residues (LysVal-Lys) are highly conserved among different species and Ig classes [14-161. These three amino acids are the only cytoplasmic residues in mIgM. The MHC class I 01 chain is similar in structure to p chains and consists of multiple extracellular domains, a transmembrane part and a cytoplasmic tail [17]. It associates into a dimer with (32 microglobulin to form cell surface class I [18]. In B cells, class I molecules differ from surface Ig in that antibody cross-linking leads to the passive formation of a wide cap or patches; this does not require cytoskeletal interactions or generation of ATP [9].The rate of endocytosis of the MHC class I-antibody complexes is also much slower than that of cross-linked surface IgM [19]. Finally, under normal conditions, cross-linking of class I has no effect on the intracellular calcium concentration as seen in response to cross-linking of mIgM [20]. To determine the role of the two carboxyterminal domains of the p heavy chain in signal transduction as well as capping and endocytosis of mIgM, we have replaced the transmembrane and cytoplasmic domains of IgM by their mouse MHC class I equivalents. Our results show that the hybrid molecule could still associate with light chains, was assembled into a tetramer and was transported to the cell surface. 0014-2980/92/0303-0851$3.50+ .25/0
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P. M. Dubois, J. Stepinski, J. Urbain and C. Hopkins Sibley
However, cross-linking of this hybrid receptor did not trigger the release of calcium from internal stores, or capping and endocytosis. These observations demonstrate that the two carboxyterminal domains of p are critical for both signal transduction and modulation of the mIgMligand complexes from the cell surface.
Eur. J. Immunol. 1992. 22: 851-857
to 15 pg of RNA was separated on 1% agarose-formaldehyde gels [28], the RNA was blotted onto nitrocellulose (Schleicher and Schuell, Keene, NH) and baked at 80 "C for 1-2 h. Hybridization and washings were performed following the manufacturer's directions. Probes were nick-translated using [32P]deoxycytidinetriphosphate to a specific activity of >lo8 cpm/pg DNA.
2 Materials and methods 2.1 Construction of the Mum-2 hybrid gene Plasmid pV~167p[21] containing the genomic sequence of a rearranged mouse p heavy chain gene was a gift from Dr. U. Storb (University of Chicago, IL). The mouse H-2 class I genomic sequence pC23 (Fig. 1) was a gift from Dr. B. Arnold (German Cancer Research Center, Heidelberg). This gene includes the extracellular domains of H-2Kkand the transmembrane and cytoplasmic domains of H-2Kd [22]. The Mu/H-2 (Fig. 1) was constructed as follows. A Cla I(blunt)/Hind I11fragment of pC23 containing the transmembrane and cytoplasmic sequences of H-2 class I was subcloned into Bam HI(b1unt) and Hind I11sites of pUC19. The transmembrane and cytoplasmic domains of V ~ 1 6 7 pwere removed by a Kpn I/Sal I(b1unt) digest and replaced by a Kpn I/Hind III(b1unt) fragment containing the corresponding sequences of H-2Kd.
2.5 Cell staining and endocytosis experiments Cells were stained with an FITC-conjugated goat F(ab')2 anti-mouse IgM (Tago, Burlingame, CA). Analyses were performed on an Ortho Cytofluorograph (Ortho Diagnostic System, Westwood, MA). In endocytosis experiments, cells were washed once at room temperature and resuspended in 400 pl of RPMI containing goat anti-mouse IgM coupled to biotin (Tago). After 15 min at 37 "C, cells were washed at room temperature, resuspended in 400 yl of medium and incubated at 37°C in 5 YOCOz. The reaction was arrested by the addition of cold medium containing 0.1 YONaN3. Cells were then stained with avidin-FITC.
2.6 Analysis of intracellular calcium mobilization
This method has been described in detail [29]. Cells were washed twice and resuspended at a final concentration of 2 x 106/mlin RPMI, 10 mM Hepes, pH 7.0 containing the 2.2 Cell lines and culture conditions acetoxymethylester form of Indo-1 ([30]; Molecular Probes, Junction City, OR) at a concentration of 4 p ~ . Clone CH12 LX2B4 was a gift from Dr. G. Haughton, Loading was carried out for 45 min at 37°C. RPMI (Duke Medical Center, Durham, NC). It is an IgM- variant supplemented with 5 YOFCS was added to each sample and of the B lymphoma CH12 LX [23] that has lost the the incubation continued for another 15 min. After washrearranged allele of the immunoglobulin heavy chain gene. ing, cells were stored at room temperature in the dark. The M12.4.1.4.5 line was a gift from Dr. R. Asofsky (NIH, Analysis was performed with an Ortho Cytofluorograph Bethesda, MD). It is an IgM- variant of the BALB/c 50HH, the UV exitation (351 to 364 nm) was provided by B lymphoma M12 [24]. The A20 line is a B lymphoma an argon laser, blue emission was detected at 480 to 520 nm, expressing the membrane form of IgG2, [24]. Cells were and violet emission at 383 to 407 nm. Cells were stimulated grown in RPMI 1640 supplemented with 7 % FCS, with 5 pg/ml of a goat anti-mouse IgM or with a goat 0.3 mg/ml L-glutamhe, 1.0 mM sodium pyruvate, lC5M F(ab'):! anti-mouse IgM coupled to biotin. In experiments 2-mercaptoethanol, 100 units/ml penicillin and 0.01 YO where stimulation was performed with antibodies coupled streptomycin at 37°C in 5 YO C02. to biotin, avidin was added at a final concentration of 10 to 15 pg/ml. 2.3 Establishment of transfected lines Cells in the exponential phase of growth were cotransfected with 5 pg of circular pSV2Neo and 30 pg of the linearized nonselectable marker by electroporation [25]. Briefly, lo7 cells and the dialyzed plasmids were mixed in a final volume of 500 pl of RPMI 1640 at room temperature. The electroporation device reproduced the discharge profile of the shorted Iscove 494 power supply [26]. Cells were then grown for 24 h in fully supplemented growth medium containing 20 YOto 40 % culture supernatant from untransfected cells. After 24 h cells were plated at 1 x lo4 to 1 x 105/well with 1 mg/ml G 418. G418-resistant clones were stained with fluoresce-in-coupled antibodies and screened by fluorescence microscopy.
2.4 RNA analysis Total cellular RNA was isolated from log-phase cells using 4 M guanidinium isothiocyanate and CsCl[27]; thereafter 5
2.7 Immunoprecipitations and Western blot analysis
Cells were lysed in 20 mM Tris, pH 7.4, 100 mM NaCl, 1% NP40, 2 mM PMSF and 10 pg/ml leupeptin (Sigma, St. Louis, MO) for 20 min at 4 "C. Sepharose beads coupled to a polyclonal goat anti-mouse IgM or to a rat monoclonal anti-mouse IgM (Zymed Laboratories, San Francisco, CA) were used for immunoprecipitations. Beads were then washed, resuspended in 100 mM NaH2P04,pH 8.0,10 mM EDTA, 0.5 YO Triton X-100, 0.1 YO SDS, 1YO 2-mercaptoethanol and boiled. Supernatants were divided for treatment with or without glycopeptidase F (Boehringer Mannheim, Indianapolis, IN), used at 0.1 U/ml for 12 h at 37 "C. Separations were performed according to Laemmli [31] on a 10 YOSDS/acrylamide gel. Electroblotting onto Immobilon membranes (Millipore, Bedford, MA) was done in 25 mM Tris, pH 8.3, 192 mM glycine and 20 %O methanol at 0.6-0.9 A for 5-6 h. Membranes were saturated with 5 YoBSA in 20 mM Tris, pH 7.4, 100 mM NaCl for 1 h at
Eur. J. Immunol. 1992. 22: 851-857
Membrane domains in B cell activation
37”C,washed in the same buffer containing0.1 YOBSA, and incubated with a polyclonal rabbit anti-mouse IgM (Zymed) in 20 mM Tris, pH 7.4, 100 mM NaCl, 1YOBSA, 0.5 YO Tween-20 for 1 h at 37°C. Goat anti-rabbit IgG (Zymed) and recombinant protein G coupled t o alkaline phosphatase (Zymed) were used for detection. Staining was done with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
-
pVH167 p
1Kb
L
3.1 Construction of the hybrid gene and transfection into B cell lines The starting plasmids, pV~167i.t and pC23, contained genomic copies of a productively rearranged p gene and a H-2K MHC class I gene (Fig. 1).The hybrid gene, pMu/H2, encodes the variable and constant regions of IgM and the transmembrane and cytoplasmic regions of H-2Kd (Fig. 1). The construction was verified by sequencing the junction. The linear p V ~ 1 6 7 pand pMu/H-2 plasmids were cotransfected into three mouse B lymphoma lines, CH12 LX2B4, M12.4.1.4.5 and A20. Clones resistant to G418 were screened by staining with FITC-coupled goat antimouse IgM antibodies. Several clones were chosen for detailed study; they are described in Table 1.
VDI
CH1 CHZ CH3
CH4
TM CyTo+3’untr.
pMuRI-2 Kpnl*/Sall* HindIII*l Sall’
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3 Results
EcoRl
Kpnl
EcoRl
2.8 Iodinations Cells (2 x 107-3 x lo7) were surface labeled with 500 yCi 1251 using glass vials coated with 300 pg of IODO-gen (Pierce, Rockford, IL). The reaction was carried out in 750 yl of PBS for 15 min at room temperature. Membrane solubilization and immunoprecipitation were performed as described above except that formalin-fixed S. aureus were used for immunoprecipitations.
853
/I VDJ
CHI CH2 CH3
CH4 TM
CyTo+3’untr.
pC23 (H-2K) chl
Hind11
Hind111
Figure 1. Structure of the genes used in study. Plasmid pMu/H-2 was constructed as described in Sect. 2.1. Stars indicate bluntended restriction sites. Exons are shown as filled boxes and 3’ untranslated sequences as open boxes.
intron including the ys polyadenylation site, we expected pMu/H-2 to encode a primary transcript that could be spliced t o generate mRNA encoding both the usual secreted ps and a membrane-bound Mu/H-2 heavy chain [32]. The Northern blot hybridized with a fragment of the V ~ 1 6 7 yV region is shown in Fig. 2. Each of the lines transfected with the pMu/H-2 (CH12 MH60, CH12 MH85, M12 MH12.2 and A20 MHd4) expressed a ys message identical in size to the wild-type ys and a second band of about 3.4 kb which corresponds to the Mum-2 hybrid mRNA (lanes 3, 4, 6 and 8). Clone CH12 M6 processed the V ~ 1 6 7 ptranscript into both pm and ys mRNA. The untransfected lines showed no hybridization to t h e V ~ 1 6 7 y V region probe.
3.2.2 Protein analysis 3.2 Expression of the transfected genes To examine the protein product of each transfected gene. NP40 lysates were immunoprecipitated with antibodies directed against mouse y chains. Immunoprecipitates were In the CH12 and M12 cell lines, the endogenous p tran- size-separated by denaturing SDS-PAGE and electroblotscripts are processed into both ym and ys mRNA. Since the ted onto membranes. As expected, all clones produced a pMu/H-2 construct retained the 5‘ portion of the ys-ym protein of the appropriate size for ys (Fig. 3A). In addition,
3.2.1 RNA analysis
Table 1. Expression of transfected and wild-type IgM or Mu/H-2 molecules in B lymphoma linesa).
Clone
CH12 LX2B4 CH12 M6 CH12 MH60 CH12 MH85 M12 4.5 M12 M4 M12 M13 M12 MH12.2 A20 A20 MHd4
Plasmids
Membrane receptor
Fluorescence FITC-goat anti-mouse IgM
-
-
pSV2Neo + pVH167p pSV2Neo + pMum-2 pSV2Neo + pMu/H-2
IgM MUM-2 Mum-2
9.0 53.6 92.4 97.7 8.5 119.3 82.2 71.8 6.0 77.2
-
pSV2Neo pSV2Neo pSV2Neo + pMuM-2
-
IgM IgM Mum-2 IgG pSV2Neo + pMu/H-2 IgG + Mum-2
a) Cells were stained with an affinity-purified,
FITC-labeled goat F(ab’)z directed against mouse IgM heavy chains.The fluorescence represents the difference in mean channels between unstained and stained populations. Clones M12 M4 and M12 M13 spontaneouslyreverted to the expression of their wild-type rearranged IgM heavy chain gene.
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P. M. Dubois, J. Stepinski, J. Urbain and C. Hopkins Sibley
Eur. J. Immunol. 1992. 22: 851-857
8E
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X
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X
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-
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Figure 2. MRNA levels of wild type and hybrid genes in parental and transfected B lymphoma lines.Total RNA was denatured with fomaldehyde and size-separated by formaldehyde-agarose gel electrophoresis, transferred to a nitrocellulose membrane and probed with a 32P-labeled1.2-kbBam HI DNA fragment containing the VH167 variable region genomic sequence. CH12 LX2B4, M12 M13 and A20: untransfected cell lines; CH12 M6: transfected with pVH167p;CH12 MH60, CH12 MH85, M12 MH12.2 and A20 MHd4: cells transfected with pMu/H-2.
they synthesized higher molecular weight forms which correspond to either ~n, or the hybrid protein. Because the cytoplasmic domain of the H-2 is about 25 amino acids longer than that in pm, the hybrid protein was correspondingly larger than pm (compare lanes CH12M6 and CH12 MH60 in Fig. 3A). In glycosylated samples (Fig. 3A), the membrane form of the Mum-2 was represented by one band in CH12 transformants and by two bands in M12 and A20 clones. However, in samples treated with glycopeptidase F (Fig. 3B), only one band corresponding to the membrane form of Mum-2 was detectable in CH12 and M12 cells. A single Mu/H-2 band was also seen in a similar experimant done on A20 MHd4 cells (data not shown). This observation could be related to a difference between CH12 and the other cell lines in the glycosylation pattern of Mu/H-2.
Figure 4. Size of the molecules immunoprecipitated by anti-x antibodies from cell lines transfected with wild-type or hybrid gene. Cells were surface labeled with lZ5Iand NP40 lysates were immunoprecipitated with a goat anti-mouse x light chain. Immunoprecipitates were then separated on a 7 % SDS-acrylamide gel under nonreducing conditions. CH12 LX2B4: IgM- clone, CH12 M6 and M12 M4: mIgM+ clones, CH12 MH85 and M12 MH12.2: Mu/H-2+ clones, A20 MHd4: mIgG+/Mu/H-2+clone.
3.2.3 Structure of the hybrid receptor To determine that the hybrid heavy chain was expressed on the cell surface in the form of a (Mu/H-2)2K2 tetramer as expected for normal mIgM, intact cells were labeled with 1251, lysed in 1% NP40 and proteins precipitated with antibodies directed against x light chain. The isolated proteins were size-separated under non-reducing conditions on a 7 YOSDS-polyacrylamide gel.The autoradiogram shown in Fig. 4 demonstrates that the Mu/H-2 receptor is associated with x light chain and migrates, like mIgM, in the 200-300-kDa range. The additional band seen in the M12 M4 and A20 MHd4 lanes correspond to a p,x dimer and to surface IgG, respectively. Only one band corresponding to mIgG was observed in untransfected A20 cells (data not shown). Thus, the assembly of this hybrid molecule resembles the Ig pattern, forming a tetramer of heavy and light chains.
3.3 Endocytosis of cross-linked receptors B. Glycopeptidase treated
Figure 3. Western blot analysis of anti-IgM immunoprecipitatesin control and transfected B cell lines. Cells were lysed with NP40 and the membrane and cytoplasmic fractions were precipitated with a rabbit polyclonal (CH12 and M12 clones) or a rat monoclonal anti-mouse IgM (A20 clones) coupled to Sepharose beads. Immunoprecipitates were size-separatedunder reducing conditions on a 10 % SDS acrylamide gel and blotted onto Immobilon membranes. Staining was done as described in Sect. 2.7. A: Samples not treated with glycosidase F, and B: Samples treated with 0.1 unit of the glycopeptidase F at 37 "C for 12 h prior to analysis.
Our first step in evaluating the functional properties of the Mu/H-2 hybrid receptor was to study its capacity to cap and internalize rapidly in response to a cross-linking stimulus. In B cells, this process is characteristic of Ig receptors but not of MHC class I molecules [9]. Cells were incubated with biotin-coupled F ( a b ' ) ~anti-p antibodies, washed and incubated at 37 "C, 5 YOC02 for various times to allow capping and internalization to occur. The amount of mIgM remaining was then assayed by staining with FITC-coupled avidin. The top five panels of Fig. 5 show that both the parental line CH12 LX expressing surface IgM and clone CH12 M6 transfected with pVH167p internalized a large fraction of their mIgM during the 90-min incubation at 37°C. In contrast, in the two clones expressing the pMu/H-2 contrast (CH12 MH60 and CH12 MH85), a comparatively minor decrease in membrane fluorescence could be detected after
Membrane domains in B cell activation
Eur. J. Immunol. 1992. 22: 851-857
855
Mu/H-2 receptors formed small patches scattered evenly over the cell surface (results not shown).
3.4 Calcium studies In B cells, cross-linking of mIgM triggers the release of I* s)
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calcium from intracellular stores [2]. This is then a more immediate test of the function of the Mu/H-2 receptor. Cells were loaded with Indo-1 [30], and the effect of cross-linking their cell surface IgM or Mu/H-2 monitored on the cytofluorograph [29]. Fig. 6 (panels A and D) shows that cross-linking with F(ab')z anti-p antibodies induced a rapid intracellular calcium increase in CH12 and M12 cells expressing mIgM. However, in cells expressing Mu/H-2, no increase in the blue/violet ratio was observed upon crosslinking with anti-Ig antibodies (Fig. 6 B, C, E).
I
CH12 M6
3.0
Figure5. Analysis of surface IgM and NH-2 modulation in response t o an anti-IgM cross-linking stimulus. Cells (lo6) were incubated in goat F(ab')2 anti-mouse IgM coupled t o biotin, for 15 min at 37"C, in 0.2 ml of RF'MI 1640. Unbound antibody was removed by two washes in the same medium at room temperature. Cells were then resuspended in RF'MI and incubated at 37°C and the endocytosis reaction was arrested by the addition of NaN3 after 30 min (--------)and 90 min. (-.-.-.-.-).Samples incubated with antibodies in the presence of NaN3 (........), and unstained controls (-).
90min. This could reflect the normal turnover rate of Mu/H-2 proteins on the cell membrane. M12 cells expressing mIgM or the Mu/H-2 protein exhibit the same differences in their response t o cross-linking with anti-IgM antibodies (Fig. 5). A20 MHd4 cells also failed t o internalize the Mu/H-2 receptor in response t o cross-linking antibodies (data not shown). The same clones were tested t o determine the pattern of redistribution which followed cross-linking of their surface p or Mu/H-2. Clones which expressed mIgM redistributed their receptor-ligand complexes into tight caps, whereas the
Time (sec.)
Figure 6 . Measurement of Ca2+ mobilization in response to antiIgM stimulation. Relative cytoplasmic calcium concentrations expressed as the mean violet/blue indo fluorescence ratio. Cells were stimulated by the addition of 5 pgl6 x lo5 cells/ml of goat F(ab')2 anti-mouse IgM (A, B, D, F, G) or goat F(ab')z anti-mouse IgM coupled to biotin (C, E). In samples treated with biotincoupled antibodies, avidin was added at final concentrations ranging from 10 to 15 pglml. Antibodies directed against IgM triggered at least 80 % of the cells to increase their calcium levels above baseline in clones expressing the membrane form of p. An affinity-purified polyclonal goat anti-mouse IgG was used to activate the A20 B lymphoma through its IgG2, membrane receptor (F and G). A . CH12 M6: transfected with IgM ( p V ~ 1 6 7 ~B) ; and C: CH12 LX2B4 untransfected, CH12 MH60, CH12 MH85: transfected with pMuM-2; D. M12 M4 expressing IgM (wild type).E. M12 transfected with pMulH-2; F. A20, untransfected; G A20 transfected with pMulH-2.
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l? M. Dubois, J. Stepinski, J. Urbain and C. Hopkins Sibley
Since double cross-linking of class I has been shown to trigger Ca2+increases [20], cells expressing Mu/H-2 receptors were treated with a biotin-coupled goat F(ab')z anti-p followed by avidin. Panels C and E (Fig. 6) show that no variation in intracellular Ca2+ concentrations could be detected under these conditions. This inability to respond was not due to poor loading with indo-1, since a detectable increase in the violet/blue ratio followed treatment with ionomycin (Fig. 6, panels B, C and E). The failure of Mu/H-2+ cells to mobilize calcium could simply reflect an inability to respond to any cross-linking stimulus. To examine this possibility we compared A20 cells (mIgG+) with clone A20 MHd4 which expresses Mum-2. Panels F and G (Fig. 6) show that neither population increased intracellular calcium concentrations when stimulated with anti-p antibodies, while both responded to anti-IgG antibodies. This last experiment indicates that cross-linking of a Mum-2 receptor does not give rise to an increase in intracellular free calcium even in a cell that signals normally through another surface Ig.
4 Discussion The data presented in this study demonstrate that replacement of the transmembrane and cytoplasmic domains of p by the corresponding H-2 class I sequence still allowed normal expression of the heavy chain, assembly into (Mu/H-2)2~2tetramers and targeting of this receptor onto the cell surface. However, in clones expressing the Mu/H-2 receptor, cross-linking with anti-p antibodies failed to produce either signal transduction or internalization of the receptor-ligand complexes. The assembly of class I and mIgM are in many ways similar [33,34]. They both require the participation of two different polypeptides for membrane expression, the key interactions necessary for formation of the complex between the two proteins require extracellular domains, and their assembly occurs in the rough endoplasmic reticulum prior to expression on the surface. However, there is one important difference between the two systems: mIgM assembles into a disulfide-bonded tetramer, and the class I-Pz-microglobulin complex remains a dimer. The hybrid Mu/H-2 protein forms complexes of the size expected for tetramers, and thus resembles the immunoglobulin in this respect. This further supports the notion that the information controlling the assembly of mIgM is within the extracellular domains. Although expression of the VH167p protein has been shown in transgenic mice [21], it was expressed at low frequency (up to 1.8 %) compared to the Mum-2 protein (10 to 50 YO)on the surface of stable B cell transfectants. However, when a similar construct which could only produce p, was introduced into B cells, this frequency rose to 6.1 YO.Thus, processing of theVH167p transcript into pm or ps messages could be one element accounting for the difference between IgM (V~167p)and Mum-2 surface expression frequencies. Another important explanation for the difference in efficiency of mIgM and Mu/H-2 expression is that the p chains but not the Mu/H-2 protein require a number of auxiliary
Eur. J. Immunol. 1992. 22: 851-857
molecules such as IgM-a and Ig-P for cell surface expression [35-371. If expression of these molecules is frequently lost in IgM- lines, one would observe a corresponding decrement in the frequency of mIgM+ clones after transfection with pV~167p.This is in agreement with the observation that a chimeric receptor identical to Mum-2 can be targeted onto the surface of an IgH-plasmacytoma which is unable to express mIgM in the absence of IgM-a [37].Thus, a number of factors may combine to make the expression of wild-type IgM on the surface of these cells relatively rare. However, it is clear from our studies and those of Shaw et al. [38] that when a transfected p chain was expressed on the surface of a B lymphoma, its function was normal. This study complements two others which investigated the importance of the membrane and cytoplasmic domains of mIgM. In the first, Webb et al. replaced these two Ig domains with their MHC class I1 counterparts [39]. Although their study did not establish the structure of this surface receptor on the B lymphoma WEHI 231, the hybrid molecule did fail to signal growth inhibition after crosslinking [39]. The second is the work of Shaw et al. [38] in which mutant p chains were used to show that specific residues in the transmembrane domain and the presence of a cytoplasmic tail of similar length and charge were critical for antigen presentation and signalling. Thus, changes in either the membrane or cytoplasmic domain of p are sufficient to prevent proper receptor function. The failure of the Mu/H-2 receptor to transduce activation signals and internalize in response to cross-linkingcould be directly related to its expression on the cell surface in the absence of interactions with some, or all components of the B cell antigen receptor complex. Alternatively, the hybrid Mum-2 surface molecule could associate with this complex, but disruption of key interactions in the transmembrane and cytoplasmic regions would impair receptor function. Although further experiments are clearly required to distinguish between these models, two studies argue in favor of the latter. First, the extracellular domain of surface Ig directly adjacent to the plasma membrane has been shown to be a critical element in the association with the class specific form of Ig-a [40], and second, replacement of specific amino acids in the transmembrane or cytoplasmic region of IgM blocks calcium signaling and/or antigen presentation while still allowing the mutated IgM to be expressed on the cell surface [38]. It is thus likely that all three carboxy-terminal domains of the Ig receptor interact ~ of with members of the signaling complex. The C Hdomain IgM could be responsible for association with IgM-a (and possibly other components of the complex) whereas the transmembrane and cytoplasmic domains could affect the conformation of these accessory molecules, enabling them to transduce activation signals and target the receptorligand complexes to the appropriate intracellular compartement. Further studies of this kind will allow the identification of the exact interactions between auxiliary molecules and particular residues within the relevant domains of mIgM. They should also provide important information concerning the interactions between the antigen receptor complex and other molecules such as the lyn tyrosine kinase [41] thought to be involved in coupling the receptor to signal transduction.
Eur. J. Immunol. 1992. 22: 851-857 We would like to thank Drs. U. Storb and B. Arnold for plasrnids, G. Haughton and R. Asofsky for cell lines, K . Bomsztyk for help with calcium assays and E McConnell, I? Lane and R. Shapiro for helpful discussions and advice.
Received September 6, 1991; in revised form December 2, 1991.
5 References 1 Ransom, J. T., Harris, L. K. and Cambier, J. C., J. Immunol. 1986. 137: 708. 2 LaBaer, J., Tsien, R. Y., Fahey, K. A. and DeFranco, A. L., J. Immunol. 1986. 137: 1836. 3 Bijterbosch, M. K., Meade, C. J., Turner, G. A. and Klaus, G. G., Cell 1985. 41: 999. 4 Mizuguchi, J.,Tsang, W., Morrison, S. L., Beaven, M. A. and Paul, W. E., J. Immunol. 1986. 137: 2162. 5 Ransom, J. T., Chen, M., Sandoval, V M., Pasternak, J. A., Digiusto, D. and Cambier, J. C., J. Immunol. 1988. 140: 3150. 6 Campbell, M. A. and Sefton, B. M., Embo J. 1990. 9: 2125. 7 Gold, M. R., Law, D. A. and DeFranco, A. L., Nature 1990. 345: 810. 8 Taylor, R. B., Dufus, W. I? H., Raff, M. C. and de Petris, S., Nature 1971. 233: 225. 9 Braun, J., Fujiwara; K., Pollard, T. D. and Unanue, E. R., J. Cell Biol. 1978. 79: 409. 10 Rosenpire, A. L. and Choi, Y S., Mol. Immunol. 1982. 19: 1515. 11 Bourguignon, L. A. W. and Bourguignon, G. J., Int. Rev. Cytol. 1985. 87: 195. 12 Lanzavecchia, A . , Nature 1985. 314: 537. 13 Ron, Y and Sprent, J., J. tmmunol. 1987. 138: 2448. 14 Rabitts, T. H., Forster, A. and Milstein, C. F!,Nucl. Acids Res. 1981. 9: 4509. 15 Bernstein, K. E., Alexander, C. B., Reddy-Premkumar, E. and Mage, R. G., J. Imrnunol. 1984.132: 490. 16 Tyler, B. M., Cowman, A. F., Gerondakis, S. D., Adams, J. M. and Bernard, O., Proc. Natl. Acad. Sci. USA 1982. 79: 2008. 17 Steinmetz, M. and Hood, L., Science 1983. 222: 727.
Membrane domains in B cell activation
857
18 Nathenson, S., Geliebter, J., Pfaffenbach, G. M. and Zelf, R. A , , Annu. Rev. Immunol. 1986. 4: 471. 19 Pernis, B., tmmunol. Today 1985. 6: 45. 20 Gilliland, L. K., Noms, N. A., Grosmaire, L. S., Ferrone, S., Gladstone, P. and Ledbetter, J. A., Hum. Immunol. 1989. 4: 269. 21 Storb, U., Pinkert, C., Arp, B., Engler, P., Gollahon, K., Manz, J., Brady, W. and Brinster, R. F., J. Exp. Med. 1986. 164: 627. 21 Arnold, B. Burgert, H. G., Hamann, U., Hammerling, G., Kees, U. and Kvist, S., Cell 1984. 38: 79. 23 Haughton, G., Arnold, L. W., Bishop, G. A. and Mercolino, T. J., Immunol. Rev. 1986. 93: 35. 24 Kim, K. J., Kanellopoulos-Langevin, C., Mewin, R. M., Sachs, D. H. and Asofsky, R., J. Immunol. 1979. 122: 549. 25 Potter, H., Weir, L. and Leder, P., Proc. Nat. Acad. Sci USA 1984. 81: 7161. 26 Emery, D. W., Smith, S. and Hopkins-Sibley, C., Techniques 1989. 1: 153. 27 Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. R., Biochemistry 1979. 18: 5295. 28 Thomas, F! S., Proc. Natl. Acad. Sci. USA 1980. 77: 5201. 29 Rabinovitch, I? S., June, C. H., Grossman, A. and Ledbetter, J. A., J. Immunol. 1986. 137: 720. 30 Grynkiewicz, G., Poenie, M. and Tsien, R. Y., J. Biol. Chem. 1985. 260: 2719. 31 Laemmli, U. K., Nature 1970. 227: 680. 32 Early, F!, Rogers, J., Davis, M., Calame, K., Bond, M., Wall, R. and Hood, L., Cell 1980. 20: 313. 33 Hendershot, L., Bole, D. and Kearney, J. F., Immunol. Today 1987. 8: 111. 34 Klar, D. and Hammerberg, G., EMBO J. 1989. 8: 475. 35 Sakaguchi, N., Kashiwamura, S., Kimoto, M., Thalmann, F! and Melchers, F., EMBO J. 1988. 7: 3457. 36 Hombach, J., Tsubata, T., Leclercq, L., Stappert, H. and Reth, M., Nature 1990. 343: 760. 37 Hombach, J., Leclercq, L., Radbruch, A., Rajewsky, K. and Reth, M., EMBO J. 1988. 7: 3451. 38 Shaw, A. C., Mitchell, R. M., Weaver, Y. K., Campos-Tories, J., Abbas, A. and Leder, I?, Cell 1990. 63: 381. 39 Webb, C. E, Nakai, C. and Tucker, F! W., Proc. Natl. Acad. Sci. USA 1989. 86: 1977. 40 Wienands, J., Hombach, J., Radbruch, A., Riesterer, C. and Reth, M., EMBO J. 1990. 9: 449. 41 Yamanashi, I!, Kakiuchi, T., Mizuguchi, J.,Yamamoto, T. and Toyoshima, K., Science 1990. 251: 192.
