A Tyrosine-Based Signal Present in Ig α Mediates B Cell Receptor Constitutive Internalization

Chemicals

NHS-SS-biotin, NHS-LC-biotin, streptavidin coupled to horseradish peroxidase, and streptavidin-agarose beads were purchased from Pierce Chemcial Co. (Rockford, IL). Other chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO). RPMI 1640, FCS, PBS, penicillin, streptomycin, sodium pyruvate, nonessential amino acids, and l-glutamine were purchased from Life Technologies (Paisley, Scotland). RPMI depleted of methionine and ³⁵S Trans-label were obtained from ICN (Costa Mesa, CA). Protein A-Sepharose and cyanogen bromide-activated Sepharose 4B were purchased from Pharmacia (Uppsala, Sweden). Tf was coupled to FITC as previously described (22).

Plasmid construction and transfection

Ig α and Ig β chimeras were constructed by adding the cytoplasmic domains of Ig α and Ig β to the extracellular and transmembrane domains of cDNA encoding FcγRII as previously described (23). The tail minus FcγR (FcR/IC−) was described previously (16). The deletion in the cytoplasmic tail of Ig α or Ig β as well as the tyrosine mutants were constructed by recombinant PCR and then sequenced. The resulting amino acid sequences of the cytoplasmic tails of all the constructs are shown in Table II⇓. The cDNA encoding the chimeric proteins were inserted in SRα-driven expression vector that also contains the neomycin resistance gene. Vectors were linearized with ScaI enzyme before transfection into the B lymphoma cell line IIA1.6 as previously described (23). After selection in G418-containing medium, 50 to 100% of the selected cells expressed FcγR chimeras screened with the rat anti-mouse FcR mAb 2.4G2 (24). When necessary, FcγR-positive cells were selected by panning with 2.4G2.

Cell culture and Abs

The B lymphoma IIA1.6, a FcγR-defective variant of the A20 B cell, was grown in RPMI 1640 medium containing 50 mM 2-ME, 5 mM sodium pyruvate, and 10 mM glutamine and supplemented with 10% FCS. The J558Lμm mb1-transfected cells were provided by M. Reth and grown as previously described (20). Rabbit antiserum directed against the exoplasmic portion of the IgG FcR were provided by C. Sautes. 20.8.4s is an anti-K^d mAb. Texas Red- and FITC-labeled donkey IgGs directed against rabbit and rat IgGs were purchased from Immunotech (Marseille, France).

Constitutive endocytosis of cell surface biotinylated proteins

Cells (10⁷) were pulse labeled, then chased for 2 h at 37°C, and surface biotinylated with a solution containing 25 mg of NHS-SS-biotin in 3 ml of PBS at 4°C, with prior incubation at 37°C for indicated times. After chilling at 4°C, biotin remaining at the cell surface was or was not stripped with 1 ml of a solution containing 1.55 mg of glutathione, 150 mM NaCl, 10% FCS, and 5 ml of 50% NaOH or containing 20 mM 2-mercaptoethanesulfonic acid (MESNA), 50 mM Tris (pH 8.6), 100 mM NaCl, and 0.2% BSA. Cells were lysed in 500 μl of lysis buffer, and biotinylated proteins were sequentially precipitated first with the Ag NIP coupled to Sepharose 4B (for the IgM) or with the rabbit antiserum directed against the extracellular domain of FcγR coupled to protein A-Sepharose (for chimeras) and then with streptavidin-agarose for both (18). Biotinylated proteins were finally dissociated from streptavidin-agarose beads by boiling in Laemmli’s sample buffer under reducing conditions and were resolved by SDS-PAGE.

Immunofluorescence

Cells were first allowed to adhere on glass coverslips precoated with a 0.1% poly-l-lysine solution in water. Cells were fixed in 3% paraformaldehyde for 10 min at room temperature. After permeabilization with 0.05% saponin in PBS supplemented with 0.2% BSA, they were incubated with specific Abs and processed as previously described (18). When specified, adherent cells were incubated with 10 μg/ml of 2.4G2 mAb for 30 min at 4°C. They were then incubated for 30 min at 37°C and treated as described above for intracellular staining. Permeabilized cells were incubated with a rabbit serum directed against the mouse FcγR portion of the chimeric molecules (anti-FcγR) and finally stained with both FITC- and Texas Red-labeled donkey antisera directed against, respectively, rat and rabbit IgGs. FITC-transferrin endocytosis was performed as previously described (22).

Confocal microscopy

Confocal laser scanning microscopy and double immunofluorescence analysis were performed using a TCS4D confocal microscope based on a DM microscope interfaced with a mixed gas argon/krypton laser (Leica Laser Technik, Heidelberg, Germany). Simultaneous double fluorescence acquisitions were performed using the 488 and 568 nm laser lines to excite FITC and Texas Red dyes with a ×100 oil immersion Neofluar objective (numerical aperture = 1.3). The fluorescence was selected with appropriate double fluorescence dichroic mirror and bandpass filters and measured with blue-green-sensitive and red-sensitive side 1 photomultipliers.

Electron microscopy

The c.Ig α and c.Ig β cells were cooled on ice for 15 min and then incubated with 2.4G2 mAb directly coupled to 12-nm gold particles for 1 h at 4°C. Abs were coupled to gold particles (25). Cells were then fixed as previously described (26). Briefly, an equal volume of cold 3% glutaraldehyde in 0.1 M Na cacodylate buffer containing 1% sucrose, pH 7.3, was added to cells for 1 h. After centrifugation, the cell pellet was resuspended in 1.5% glutaraldehyde in cacodylate buffer. Cells were then prepared for transmission electron microscopy as previously described (26). For quantitative analysis, cell surfaces were examined systematically, noting the location of every gold particle encountered.

BCR constitutive internalization is mediated by the Ig α cytoplasmic tail

We first evaluated the endocytic properties of the Ig α and Ig β BCR subunits in IgM-positive (IgM anti-NIP) J558Lμm cells expressing either wild-type (wt) or cytoplasmic tail-deleted Ig α (Ig α ΔIC) (20).

To follow the endocytosis of surface IgM, metabolically labeled cells were surface biotinylated with cleavable NHS-SS-biotin at 4°C and further incubated at 37°C for various times. To quantify the relative amounts of internalized IgM, each sample was divided into two equal aliquots; one of which was treated with MESNA at 4°C, a membrane-impermeant reducing agent. The residual cell-associated biotin corresponded to proteins protected from reduction by internalization. IgM were first precipitated with NIP-BSA coupled to Sepharose beads. The biotinylated IgM were then isolated by a second step of precipitation with streptavidin-agarose beads. Our results showed that the surface IgM were constitutively internalized in the wt cells (Fig. 1⇓a), as assessed by the detection of increasing amounts of protected biotinylated IgM. A plateau representing 35 to 40% of internalized IgM was reached within 10 to 20 min of incubation at 37°C (Fig. 1⇓b). In contrast, the Ig αΔIC cells showed no BCR constitutive internalization. The level of constitutive TfR internalization was equivalent in the two cell lines (data not shown). We concluded that the Ig α cytoplasmic domain is necessary to trigger IgM-based BCR constitutive endocytosis.

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FIGURE 1.

Constitutive internalization of surface IgM associated with Ig α/Ig β or with Ig αΔIC/Ig β. a, J558Lμm cells expressing either Ig α (wt) or intracytoplasmic deleted Ig α (Ig αΔIC) were metabolically labeled for 20 min and chased for 2 h. Plasma membrane proteins were then biotinylated at 4°C with NHS-S-S-biotin. After incubation at 37°C for the indicated times (0–60 min) cells were treated, or not, with MESNA at 4°C. After cell lysis, biotinylated heavy chains of IgM were sequentially immunoprecipitated with the Ag NIP coupled to Sepharose and then with streptavidin-agarose. b, The amount of internalized IgM protected from the external reduction was calculated as the percentage of biotinylated labeled IgM in samples untreated with MESNA. These ratios were deduced from scanning densitometric analysis of three experiments.

Ig α chimeras are constitutively internalized molecules

The constitutive internalization of the two BCR-associated subunits was further analyzed in B cells (IIA1.6) expressing chimeras where Ig α or Ig β intracellular domains were fused to the transmembrane and extracellular domains of the mouse FcγRII (c.Ig α and cIg β; Table I⇓). We therefore analyzed their rate of endocytosis essentially as described in Figure 1⇑. Chimera-expressing cells were metabolically labeled, and cell surface proteins were biotinylated with cleavable NHS-SS-biotin at 4°C before incubation at 37°C for various times. Only c.Ig α was efficiently internalized (Fig. 2⇓a), as assessed by the increasing amount of protected biotinylated c.Ig α after incubation at 37°C. In this set of experiments, equilibrium was reached between 10 and 20 min of incubation at 37°C. The size of the pool of internalized c.Ig α was 40 to 45% (Fig. 2⇓b). In contrast, lower amounts of c.Ig β were internalized, as the intracellular pool after 30 min at 37°C was 5% (Fig. 2⇓b). In c.Ig α-transfected cells, MHC class I H-2K^d molecules were sequentially immunoprecipitated, and no internalization was detected (Fig. 2⇓, b and c). The endocytosis of the mIgGs expressed on c.Ig β-transfected cells was examined. The amount of internalized mIgG reached a plateau at 25% (Fig. 2⇓, b and c) within 10 to 20 min at 37°C. The different sizes of the cIg α and cIg β intracellular pools suggested that the cytoplasmic tail of Ig α contains an internalization signal that mediates constitutive endocytosis of mIgs (7). However, we could not exclude that the both chimera were efficiently internalized, with cIg α addressed to degradative compartments and Ig β quickly recycling back to the cell surface.

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FIGURE 2.

Newly synthesized c.Ig α, but not c.Ig β, molecules are rapidly internalized from the cell surface. a, Cells were treated as described in Figure 1⇑, except that another reducing molecule was used (gluthatione). Chimeras were immunoprecipitated with an anti-FcR antiserum, and biotinylated material was subsequently recovered on streptavidin-agarose. Constitutively expressed H-2K^d and membrane IgGs heavy chains were used as, respectively, negative and positive internal controls for endocytosis. b, The amount of internalized proteins protected from the external reduction was calculated as the percentage of biotinylated labeled molecules in samples untreated with gluthatione for c.Ig α (open circles), c.Ig β (filled circles), heavy chains of H-2K^d (filled squares), and heavy chains of mIgGs (open triangles). These ratio were deduced from scanning densitometric analysis. c, To compare the turnover rates of chimeras, cells were metabolically labeled for 20 min and chased for various periods of time. At the indicated times, cells were cell surface biotinylated at 4°C and lysed, and lysates were immunoprecipitated using an anti-FcR antiserum. Immunoprecipitated material was dissociated from the immunoadsorbant by boiling in SDS, and 90% of the immunoprecipitated proteins was reincubated on streptavidin-agarose (Biot−). The remaining 10% (Total) illustrates the different intracellular forms of cIg α and cIg β detected over the time course of the experiment.

To investigate the turnover rate of both chimera, we measured the behavior of newly synthesized and cell surface Ig α and Ig β chimeras in pulse-chase experiments. The appearance of both molecules at the plasma membrane was followed by biotinylation of the cell surface at various chase times, ranging from 0 to 6 h. Within 30 min to 1 h, most of the newly synthesized cIg α or cIg β molecules was terminally glycosylated, as assessed by their reduced mobility in SDS-PAGE compared with that at the zero time point (Fig. 2⇑c). No variation in the signal intensities was observed within the next 6 h, showing that degradation did not occur. As shown in the right panel of Figure 2⇑c, both cIg α and cIg β reached the cell surface, where they were biotinylated within the first hour after their synthesis, and no significant variation in signal for biotinylated molecules was observed during the next 5 h. We therefore concluded that both chimera had similar stability, and that, probably, the differences in the intracellular pool sizes observed in Figure 2⇑b were due to a more efficient constitutive internalization of the Ig α chimera.

To further investigate this question, we next examined whether the cytoplasmic tail of Ig α and Ig β determined the differential recruitment of the chimera in clathrin-coated pits, which is the first step of receptor internalization. Cells were immunolabeled with gold-coupled 2.4G2 mAb directed against the FcR extracellular domain of the chimeras and prepared for examination by electron microscopy (Fig. 3⇓). Quantitative analysis of the distribution of c.Ig α chimeric molecules at the cell surface showed that 12% of all gold particles were located inside or in the vicinity of clathrin-coated pits (Table I⇑). In contrast, only 1.7% of gold particles coupled to the 2.4G2 mAb decorated such structures on the plasma membrane of c.Ig β-transfected cells. These data show that the Ig α cytoplasmic domain allows the chimeras to be enriched up to sevenfold in coated pits (Table II⇓). Therefore, only the cytoplasmic domain of the Ig α subunit of the Ig α/β sheath determined recruitment in coated pits and constitutive internalization of BCR. Since these chimera seemed stable in pulse-chase experiments (Fig. 2⇑c), these data suggested that after internalization, Ig α chimera were recycled back to the cell surface.

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FIGURE 3.

Distribution of gold-labeled c.Ig α on IIA1.6-transfected cells. Cells were incubated with the rat mAb against FcR (2.4G2) directly labeled to gold particles. The cells were treated for electron microscopy, and c.Ig α chimeras were visualized. Here are shown Ig α chimeras localized in coated pits.

The c.Ig α molecules accumulated in Tf-containing endosomal compartments

We first analyzed the steady state distribution of Ig α and Ig β chimeras by immunofluorescence microscopy (Fig. 4⇓) after cell surface permeabilization. The chimera were labeled with rabbit anti-FcγR IgGs and Texas Red-coupled secondary Abs. The c.Ig α was clearly distributed between the plasma membrane and cytoplasmic structures (Fig. 4⇓a). In contrast, c.Ig β was essentially accumulated at the cell surface, as shown in a medial optical slice of labeled cells (Fig. 4⇓b). We then characterized the intracellular compartments where c.Ig α chimeric molecules accumulated using double immunofluorescence and confocal microscopy. As a sorted and recycling endosomal marker, FITC-coupled Tf (FITC-Tf) was continuously internalized for 30 min at 37°C before cell fixation (Fig. 4⇓c). The c.Ig α were labeled with rabbit anti-FcγR IgGs and Texas Red-coupled secondary Abs (Fig. 4⇓d). FITC-Tf was distributed between plasma membrane and vesicular intracellular structures that were also positive for anti-FcγR staining. Double labelings with later markers of the endosomal pathway such as cathepsin D and cation-independent mannose-6-phosphate receptor were also performed, but no clear colocalization was observed (not shown). Therefore, c.Ig α were constitutively endocytosed and were probably recycled to the surface.

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FIGURE 4.

Intracellular distribution of c.Ig α in IIA1.6 cells. IIA1.6 cells transfected with c.Ig α (a) or c.Ig β (b) were fixed in 3% paraformaldehyde before permeabilization with saponin and labeling with rabbit anti-FcR IgGs as described in Materials and Methods. Here are shown representative and medial optical slices of the specimen obtained from the confocal microscope. FITC-Tf (60 nM) was continuously internalized for 20 min at 37°C (c). Cells were then fixed, permeabilized, and labeled for hybrid molecules with rabbit anti-FcR IgGs and revealed by Texas Red donkey anti-rabbit IgGs (d). Space bar = 10 μm.

The constitutive internalization signal of Ig α is located in its ITAM and is dependent on tyrosine 23

To better characterize the internalization signal of Ig α, we analyzed the cell surface behavior of mutant molecules in which the cytoplasmic domain was essentially constituted by the ITAM (c.Ig αm; Table I⇑). The c.Ig αm was still efficiently internalized (Fig. 5⇓a), and its cellular distribution (Fig. 5⇓b) was identical with that observed for c.Ig α (Fig. 2⇑a). In contrast, chimeric molecules containing only the ITAM flanking sequences showed none of these characteristics (not shown). Together, these results showed that Ig α ITAM is sufficient to promote the constitutive internalization of the hybrid molecules and their intracellular distribution between plasma membrane and endosomal compartments.

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FIGURE 5.

Internalization of the c.Ig α mutants. a, c.Ig αm-, c.Ig αmA23-, and c.Ig αmA34-expressing cells were treated as described in Figure 1⇑. Confocal microscopic slices of cells expressing c.Ig αm (b), c.Ig αmA23 (c), and c.Ig αmA34 (d) were labeled with rabbit anti-FcR IgGs and revealed by Texas Red donkey anti-rabbit IgGs as described in Figure 4⇑.

Since the earlier work on the analysis of naturally occurring internalization-defective mutant low density lipoprotein receptors (27), the concept of tyrosine-containing internalization signals has been subsequently extended to other internalizing receptors (28, 29, 30). We then tested whether the two tyrosines present in Ig α ITAM were part of the endocytosis signal. These tyrosine residues were separately mutated into alanines in the c.Ig αm chimeras (c.Ig αmA23 and c.Ig αmA34; Table I⇑). We found that the mutation of the first tyrosine abolished the constitutive internalization of the chimeras (c.Ig αmA23), whereas the mutation of tyrosine 34 had no effect (c.Ig αmA34; Fig. 5⇑a). As expected from the above results, c.Ig αmA23 (Fig. 5⇑c) accumulated at the plasma membrane of IIA1.6-transfected cells, whereas c.Ig αmA34 (Fig. 5⇑d) distributed between plasma membrane and internal vesicles. In conclusion, the membrane-proximal tyrosine residue of Ig α ITAM involved in the B cell receptor activation pathway (20) is also part of a tyrosine-based signal for constitutive endocytosis.