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The Journal of Immunology, 2005, 175: 2123-2131.
Copyright © 2005 by The American Association of Immunologists

Transmembrane Sequences Are Determinants of Immunoreceptor Signaling 1

Julie A. Gosse, Alice Wagenknecht-Wiesner, David Holowka and Barbara Baird2

Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York, 14853


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To investigate structural features critical for signal initiation by Ag-stimulated immunoreceptors, we constructed a series of single-chain chimeric receptors that incorporate extracellular human Fc{epsilon}RI{alpha} for IgE binding, a variable transmembrane (TM) segment, and the ITAM-containing cytoplasmic tail of the TCR {zeta}-chain. We find that functional responses mediated by these receptors are strongly dependent on their TM sequences, and these responses are highly correlated to cross-link-dependent association with detergent-resistant lipid rafts. For one chimera designated {alpha}F{zeta}, mutation of a TM cysteine abolishes robust signaling and lipid raft association. In addition, TM disulfide-mediated oligomerization of another chimeric receptor, {alpha}{zeta}{zeta}, enhances signaling. These results demonstrate an important role for TM segments in immunoreceptor signaling and a strong correspondence between strength of signaling and cross-link-dependent partitioning into ordered membrane domains.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multichain immune recognition receptors are a structurally complex family whose members mediate hemopoietic cell activation in response to foreign Ags. Previous studies used chimeric single-chain transmembrane (TM) 3 analogues of these receptors to establish a critical role for ITAM-containing cytoplasmic (CT) segments in cross-link-dependent signaling (1, 2, 3). Other studies showed that tyrosine phosphorylation of ITAM sequences by Src-family kinases in response to receptor cross-linking is the initial biochemical event in multichain immune recognition receptor signaling that leads to recruitment and activation of the Syk/Zap70 tyrosine kinase family and cascades of downstream signaling (4, 5, 6).

In rat basophilic leukemia (RBL)-2H3 mast cells, a single-chain chimeric receptor consisting of the extracellular (EC) and TM regions of the IL-2 receptor {alpha}-chain, Tac, together with the ITAM-containing CT segment of Fc{epsilon}RI{gamma} or that of TCR {zeta}, was shown to be sufficient to mediate cross-link-dependent degranulation similar to endogenous Fc{epsilon}RI, the high affinity receptor for IgE (2). Subsequent studies showed that early signaling via these chimeric receptors is less robust than that of endogenous Fc{epsilon}RI (7, 8), probably because of the absence of the amplifying effects of the {beta} subunit of Fc{epsilon}RI (9, 10). Substitution of the IgE-binding EC domain of human Fc{epsilon}RI for EC Tac permitted IgE sensitization and anti-IgE triggering of these chimeric receptors, and the TM sequence of Fc{epsilon}RI{gamma} was found to substitute for the Tac sequence in these functional responses (11). In these various studies using chimeric single-chain receptors, a role for TM sequences in signal transduction was not systematically investigated.

Studies on the function of liquid-ordered regions of the plasma membrane, commonly called lipid rafts, have demonstrated that cross-link-dependent association of Fc{epsilon}RI with these membrane domains correlates strongly with the initiation of ITAM phosphorylation (12, 13), and other studies on Fc{epsilon}RI and the TM adaptor protein LAT pointed toward roles for TM segments in lipid raft association (14, 15). Field et al. (14) found that Fc{epsilon}RI{beta} is dispensable for cross-link-dependent lipid raft association, as human Fc{epsilon}RI{alpha}{gamma}2 showed similar association as rat Fc{epsilon}RI{alpha}{beta}{gamma}2. Fc{epsilon}RI{alpha} is not expressed at the cell surface unless it is associated with Fc{epsilon}RI{gamma}2 (human) or Fc{epsilon}RI{beta}{gamma}2 (rodent) (6). Roles for the TM segments of Fc{epsilon}RI{alpha} and/or -{gamma} were implicated in cross-link-dependent raft association by Field et al. (14), but the participation of these segments in Fc{epsilon}RI signaling were not critically evaluated.

To examine systematically the roles of TM segments in immunoreceptor signaling, we created a series of chimeric single-chain receptors in which the EC and CT segments remain constant, but the TM sequence is varied. We find that these receptors mediate cross-link-dependent signaling in RBL-2H3 mast cells with a wide range of efficiencies, and the magnitude of this signaling is highly correlated with cross-link-dependent association with lipid rafts. These results provide a critical test for the hypothesis that cross-link-driven immunoreceptor association with lipid rafts is an important feature for the initiation of signaling by this family of Ag-responsive multichain immune recognition receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Purified human IgE myeloma PS (huIgE) was a generous gift from C. Torigoe and H. Metzger (National Institutes of Health, Bethesda, MD) or was purchased from Cortex Biochem. Mouse monoclonal DNP-specific IgE (moIgE; Ref. 16) was purified as described previously (17). IgE was biotinylated with a 25-fold molar excess of 6-((6-((biotinoyl)amino) hexanoyl) amino)-hexanoic acid, succinimidyl ester (Molecular Probes) in borate buffered saline (pH 8.2) (200 mM boric acid, 33 mM NaOH, and 160 mM NaCl) for 1.75 h at 22°C, and then was dialyzed extensively at 4°C vs PBS with EDTA (PBS-EDTA: 0.15 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA (pH 7.4)). IgE was fluorescently labeled by incubating the contents of one vial from an Alexa Fluor 488 dye kit (Molecular Probes) with 1 mg of IgE in borate buffered saline (pH 8.2) for 2 h at 22°C with stirring, followed by extensive dialysis vs PBS-EDTA (pH 7.4) at 4°C. Mouse monoclonal anti-phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology. Affinity-purified goat polyclonal Ab to huIgE was obtained from MP Biomedicals, and Protein A-purified rabbit anti-moIgE was elicited as described previously (18). BSA conjugated with an average of 15 DNP groups (DNP-BSA, multivalent Ag) was prepared as described previously (19).

Chimeric receptor construction

For construction of plasmids pSXSR{alpha}{alpha}{zeta}{zeta} and pSXSR{alpha}/{alpha}T{zeta}, the EC Tac domain was removed from plasmids pSXSR{alpha}/T{zeta}{zeta} and pSXSR{alpha}/TT{zeta} (20) (a gift from L. Samelson, National Institutes of Health, Bethesda, MD) by EcoRI/BglII digestion and replaced by the EC portion of human Fc{epsilon}RI{alpha} (21) (cDNA provided by H. Metzger) using the oligos 5'-CCG GCC GAA TTC GAT GGC TCC TGC CAT GGA ATC CC-3' and 5'-GAG CAG ATC TAG CCA GTA CTT CTT CTC ACG C-3', respectively. The constructs {alpha}P{zeta}, {alpha}45{zeta}, and {alpha}F{zeta} were generated by simultaneously removing the TM and CT {zeta}-domains of the pSXSR{alpha}/{alpha}{zeta}{zeta} by BglII/EcoRV digestion and replacing them with P{zeta}-, 45{zeta}-, and F{zeta}-fragments that were derived by three-step PCRs using the appropriate cDNA templates and sets of oligonucleotide primers (PTP{alpha} cDNA was kindly provided by D. Shalloway, Cornell University; CD45 cDNA was provided by A. Weiss, University of California at San Francisco, San Francisco, CA; and transferrin receptor cDNA was provided by T. McGraw, Weill Medical College of Cornell University). The three primers used to amplify the TM fragments and to fuse them with the {zeta} CT segment were as follows: for P{zeta}, 5'-GCC GCA GAT CTC ATT ATT GCG GTG ATG GTG GC-3', 5'-CAC TCC TGC TGA ATT TTG CTC TTA ACA TGT ACA AAA CTA TGA TAA TAA AC-3', and 5'-CCG CGG GAT ATC TTA GCG AGG GGC CAG GGT CTG C-3' (ASZETAEcoRV); for 45{zeta}, 5'-GCC GCA GAT CTC GCA CTG ATA GCA TTT CTG GC-3', 5'-GCA CTC CTG CTG AAT TTT GCT CTG TAG AGA ACA ACA AGC AGG-3', and ASZETAEcoRV; and for F{zeta}, 5'-GCC GCA GAT CTC AGT ATC TGC TAT GGG ACT ATT GC-3', 5'-CAC TCC TGC TGA ATT TTG CTC TAT AGC CCA AGT AGC CAA TCA TA-3', and ASZETAEcoRV. To generate the chimeras {alpha}{zeta}{zeta}DA and {alpha}F{zeta}CA, we introduced point mutations into pSXSR{alpha}/{alpha}{zeta}{zeta} and pSXSR{alpha}/{alpha}F{zeta} by using the QuikChange Site-Directed Mutagenesis kit from Stratagene. All constructs were sequenced for confirmation.

Cells

Monolayer cultures of RBL-2H3 cells (22) were maintained with MEM containing 20% FBS (Atlanta Biologicals) and 10 µg/ml gentamicin sulfate. All cell culture materials were from Invitrogen Life Technologies, unless otherwise noted. Chimeric IgE receptor DNA was stably cotransfected into RBLs with pcDNA3 using GenePORTER transfection reagent (Gene Therapy Systems) in OptiMEM I Reduced Serum Media (Invitrogen Life Technologies). Cells were incubated with the DNA/GenePORTER complexes for 1 h at 37°C, then 0.1 µM phorbol 12,13-dibutyrate (Sigma-Aldrich) in OptiMEM I was added to the cells to promote fluid phase phagocytosis and DNA uptake. After 5–7 additional hours at 37°C, the cells were washed thoroughly to remove the phorbol 12,13-dibutyrate, and cultured for 1 day in fresh RBL culture media. Transfected cells were selected in medium containing neomycin (G418) at 380 µg/ml, and colonies stably expressing chimeric receptors were screened for binding of Alexa 488-huIgE. Cells were subjected to 1–2 rounds of subcloning by limiting dilution to ensure clonal populations.

For stimulation of functional responses, chimeric IgE receptors, sensitized with variable concentrations of biotinylated huIgE (bhuIgE) to achieve equivalent bhuIgE-chimeric receptor densities on all the clones as described below (see Flow cytometric analysis), were cross-linked with streptavidin. In separate samples, native Fc{epsilon}RI, together with any chimeric receptors present, were sensitized with saturating amounts of anti-DNP moIgE, then cross-linked with DNP-BSA. Stimulations were performed in buffered saline solution (BSS; 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 20 mM HEPES, and 1 mg/ml BSA (pH 7.4)) at 37°C. Because native Fc{epsilon}RI is present at substantially higher densities than the chimeric receptors (≥5-fold; see Results) in each of the clones, the responses mediated by moIgE are largely due to signaling via Fc{epsilon}RI, and are subsequently referred to as such. Dose response experiments showed that concentrations of 0.1 µg/ml streptavidin for bhuIgE and 1 µg/ml DNP-BSA for moIgE elicited maximal degranulation responses for receptor densities used in these experiments (data not shown), and these doses were used for all phosphorylation, calcium, and degranulation responses compared.

Flow cytometric analysis

Cells were plated at 1 x 106 cells/well in 2 ml of medium per well in 6-well plates and were sensitized overnight with varying concentrations of Alexa488-hu or -moIgE. Cells were harvested with 135 mM NaCl, 5 mM KCl, 20 mM HEPES, and 2 mM EDTA, washed into BSS, then fixed in PBS-EDTA with 3.7% formaldehyde for 15 min at room temperature, and quenched with 10 mg/ml BSA in PBS-EDTA, and stored at 4°C until measured. Morphology and fluorescence gates were set with a negative control of untransfected RBL cells that had been incubated with Alexa488-huIgE, and flow cytometric analysis was performed on a FACScan cytometer (BD Biosciences) to assess clonality and receptor density of the stably transfected cell lines. Only clonal cell lines with huIgE-chimeric receptor densities that were equalized among clones (achieved by binding variable huIgE concentrations to the clones, predetermined by flow cytometry with Alexa 488-huIgE) were used in functional experiments.

Confocal microscopy

Cells were incubated overnight with 1 µg/ml Alexa488-IgE and then fixed with formaldehyde in PBS-EDTA as described above. Imaging was conducted using an MRC 1024 confocal system (Bio-Rad) in conjunction with an Olympus IX70 microscope (Olympus) with a 40x universal apochromat water objective (NA 1.15). The 488-nm line of a krypton-argon ion laser was used for excitation, together with a 515-nm long pass emission filter, and images were collected with LaserSharp (Bio-Rad) software.

Degranulation assay

Degranulation was determined using the standard {beta}-hexosaminidase assay as described previously (23). For 2–3 clones of each stably transfected cell type, one set of samples was used to measure the response via chimeric receptors and another via Fc{epsilon}RI for each clone on each day. Degranulation responses stimulated via Fc{epsilon}RI in these experiments were typically in the range of 40–80% {beta}-hexosaminidase released, and spontaneous release was typically <5% and always <10% in these experiments. For each experiment performed, the ratio of the degranulation response elicited via each chimeric receptor to that of endogenous Fc{epsilon}RI for each clone (to control for differences in overall functional capacity of the various clones) was normalized to the maximum value for that day before averaging data from multiple days.

Ca2+ measurements

Calcium responses were measured as described previously (24), except that fluo-4 (Molecular Probes) was used as the Ca2+ indicator instead of indo-1, and sulfinpyrazone was used at 0.6 mM to prevent leakage of fluo-4 from the cells. For each clone of stably transfected cells, one sample was used to measure the response via its chimeric receptor sensitized with huIgE, and a second via Fc{epsilon}RI sensitized with moIgE in each fluorometer run.

Phosphorylation assays and immunoblotting

For whole cell lysate phosphorylation assays, attached cells were stimulated or not for 5 min at 37°C and then boiled in SDS sample buffer with 2% 2-ME, and 8000 cell equivalents were loaded per lane onto 12% SDS-PAGE gels.

For immunoprecipitation of chimeric IgE receptors before and after stimulation, three 60-mm dishes for each sample were plated with 5 x 106 cells/dish in 3 ml of medium with 3 µg bhuIgE and incubated overnight. Attached cells were stimulated for 5 min and then lysed (450 µl/dish) in radioimmunoprecipitation assay lysis buffer (25) with 2 mM N-ethylmaleimide to block free sulfhydryl groups. Receptors were immunoprecipitated from these detergent extracts (~1.5 ml) by incubating each sample with 4 µg of goat anti-huIgE for 1 h at 4°C, then rotating each sample for 1 h with 30 µl of ImmunoPure Immobilized Protein G (Pierce) at 4°C. In preliminary experiments, we verified that these conditions result in consistently high recoveries of huIgE. Immunoprecipitates were washed twice with lysis buffer with detergent and once without detergent and then were boiled in SDS sample buffer with or without {beta}-ME. On 10% SDS-PAGE gels, entire samples were loaded into single lanes. Anti-phosphotyrosine immunoblotting was performed as described previously (25).

Sucrose gradient analysis of detergent-resistant membranes

Sucrose gradient analysis was performed as described previously (14), with a final concentration of 0.04% Triton X-100 (Pierce) during cell lysis and a 50% sucrose layer in place of the 60% layer described previously. Alexa488-IgE (hu or mo) was bound to receptors by incubation overnight at saturating concentrations and was cross-linked (or not) on washed, suspended cells with 10 µg/ml anti-huIgE or anti-moIgE for 5 min at 37°C before cell lysis at 4°C. Experiments with varying densities of Alexa488-IgE bound to Fc{epsilon}RI or to chimeric receptors did not alter the results obtained (data not shown). For sucrose gradient analysis of Thy-1 in unstimulated cells, RBL cells suspended at 5 x 106 cells/ml were incubated in BSS with 5 µg/ml Alexa 488-modified OX-7, an anti-rat CD90 (anti-Thy-1) mAb (BD Biosciences Pharmingen), for 30 min at room temperature, then the cells were washed twice in BSS and warmed for 5 min at 37°C before cell lysis at 4°C and analysis as for Alexa488-IgE-labeled cells. Fluorescence of isolated fractions was measured in an SLM 8000C fluorescence spectrophotometer (SLM Instruments), and background signal due to buffer alone was subtracted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Clonal expression of single-chain chimeric IgE receptors

A series of single-chain chimeric IgE receptors were constructed and stably expressed in RBL-2H3 mast cells. All of these receptors contain the EC region of human Fc{epsilon}RI{alpha}, allowing selective sensitization with huIgE in the absence of sensitization of endogenous Fc{epsilon}RI (26), and they also contain the CT segment of the TCR {zeta} subunit, which has three ITAM sequences (2) (Fig. 1A). For the TM segment of our single-chain chimeric receptors, we used five different sequences, as well as several point mutations within these sequences to identify residues critical for signaling (Fig. 1B). Most of these chimeric receptors exhibit good expression at the plasma membrane, as shown in Fig. 2A, for selected clones sensitized with Alexa488-conjugated huIgE. One exception is {alpha}{zeta}{zeta} (with the TM region from TCR {zeta}), which exhibits a significant amount of internalized label when sensitized with monomeric Alexa488-huIgE. This constitutive internalization is absent in an aspartate to alanine point mutant of this construct, {alpha}{zeta}{zeta}DA (Fig. 2A).



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  		FIGURE 1. Structural features of chimeric IgE receptors. A, Constructs contain the EC segment of human Fc{epsilon}RI{alpha} for IgE binding ("{alpha}"), a variable TM region, and the ITAM-containing CT tail of the TCR {zeta} subunit ("{zeta}"). B, Chimeric receptors characterized are shown, including clone numbers, TM amino acid sequences, and TM protein of origin. Bolded, underlined amino acids are altered in site-directed mutagenesis constructs. Putative TM sequences, from end of EC domain to beginning of CT domain, were derived from sequence information for each parent protein from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/).

 


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  		FIGURE 2. Fluorescence characterization of stably transfected RBL-2H3 cell lines. A, Confocal fluorescence microscopy of Alexa488-moIgE bound to endogenous Fc{epsilon}RI and Alexa 488-huIgE bound to specified chimeric receptors. Images were acquired and displayed with identical settings, except that laser power was 10% of maximal for Fc{epsilon}RI and {alpha}T{zeta} and 30% for others shown. Scale bar, 10 µm. B, Clonality of stably transfected cell lines and the density of Alexa 488-huIgE-receptor complexes as evaluated by flow cytometry (shaded profiles). Cells were sensitized overnight with varying concentrations of Alexa488-huIgE as described in Materials and Methods. Mean fluorescence intensities are indicated in boxes, and these labeling conditions were used throughout in the functional assays to equalize densities of IgE-receptor complexes for the 14 clones evaluated. White profile curves represent background fluorescence due to incubation of untransfected RBL cells with Alexa488-huIgE.

 
All of the chimeric receptors exhibit uniform labeling at the plasma membrane with Alexa488-huIgE, consistent with expectations from labeled moIgE-Fc{epsilon}RI. We produced two or three stable clones expressing each of the seven chimeras listed in Fig. 1B for functional analysis. Because our chimeric receptors are typically expressed at levels that are ~5- to 20-fold lower than endogenous Fc{epsilon}RI in these stable cell clones, variable huIgE concentrations were used to sensitize chimeric receptors to compare functional responses with equal amounts of huIgE bound to chimeric receptors in the various clones (27). MoIgE was bound to endogenous Fc{epsilon}RI (and chimeric receptors) on each clone under saturating conditions. These equalized huIgE densities on stable cell clones are shown by flow cytometric analysis in Fig. 2B, together with the clonal distributions of huIgE bound to chimeric receptors.

Degranulation and calcium responses via chimeric receptors are highly dependent on TM sequence

Initial functional experiments compared the degranulation responses elicited by cross-linking bhuIgE bound to chimeric receptors using streptavidin, and we found that the chimera containing the TM sequence of the TCR {zeta}-chain, {alpha}{zeta}{zeta}, mediates a strong response that is 68 ± 7% of that elicited by an optimal dose of multivalent Ag, DNP-BSA, which cross-links moIgE bound to endogenous Fc{epsilon}RI, as well as the chimeric receptor on these clones (Fig. 3). By comparison, the chimeric receptor {alpha}T{zeta}, containing the TM sequence of the IL-2 receptor {alpha}-chain (Tac), which previously had been used to construct functional single-chain receptors (2, 8, 11), mediates a smaller response, 42 ± 5% of that to multivalent Ag.



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  		FIGURE 3. Degranulation via chimeric IgE receptors. Stably transfected clonal cell lines were incubated with bhuIgE to sensitize chimeric receptors alone or with anti-DNP moIgE to sensitize Fc{epsilon}RI together with chimeric receptors. Cells were stimulated with 0.1 µg/ml streptavidin for bhuIgE or 1 µg/ml DNP-BSA for moIgE, respectively, for 1 h at 37°C, and {beta}-hexosaminidase released into cell supernatants was measured. The ratio of the degranulation response via each chimeric IgE receptor to that via endogenous Fc{epsilon}RI for each clone was normalized as described in Materials and Methods. Error bars represent the SEM for data from six experiments with 2–3 clones for each chimeric receptor.

 
Previous studies of TCR showed that mutation of a negatively charged aspartic acid residue in the TM sequence of TCR {zeta} to alanine converted it from a disulfide-bonded dimer to a monomer (28). We constructed an analogous point mutation in our {alpha}{zeta}{zeta} chimera ({alpha}{zeta}{zeta}DA) and found that it significantly reduces the degranulation response compared with {alpha}{zeta}{zeta}, making it more comparable to that of {alpha}T{zeta} (Fig. 3), despite increased surface expression of {alpha}{zeta}{zeta}DA compared with {alpha}{zeta}{zeta} at the plasma membrane (Fig. 2A). In contrast, directly changing the disulfide-forming cysteine in {alpha}{zeta}{zeta} to alanine ({alpha}{zeta}{zeta}CA) resulted in rapidly internalizing Alexa488-huIgE-receptor complexes, which appeared as punctate structures within the cell, with very few receptors remaining at the surface (data not shown). Because of this poor plasma membrane retention, we did not analyze the functional responses of this mutant.

We prepared another set of constructs to evaluate TM sequences that might exhibit different partitioning into lipid rafts. One of these is a chimeric receptor containing the inverted TM sequence of the transferrin receptor, which is a type II TM protein that does not usually fractionate with lipid rafts (29). We naively inserted this sequence (Fig. 1) into our Type I chimeric TM receptor, such that the orientation of the TM sequence of {alpha}F{zeta} in the plasma membrane is opposite to its normal orientation in the transferrin receptor itself. Cross-linking of this chimeric receptor ({alpha}F{zeta}) leads to a strong degranulation response similar to that for {alpha}{zeta}{zeta}. The significance of the sequence inversion is considered in Discussion. To test the possible role of a cysteine residue in the TM sequence of this chimera, we mutated it to alanine ({alpha}F{zeta}CA) and found a dramatic reduction in stimulated degranulation compared with that of the parent (Fig. 3; {alpha}F{zeta}CA vs {alpha}F{zeta}). Two other chimeric receptors, {alpha}P{zeta} and {alpha}45{zeta}, were constructed using the TM sequences of tyrosine phosphatases PTP{alpha} and CD45, respectively, that do not associate with lipid rafts (30, 31), and these were found to mediate relatively small degranulation responses, similar to that of {alpha}F{zeta}CA. The results summarized in Fig. 3 represent averages from six experiments using two or more clones for each construct. Table I shows the clonal variation in these experiments, which is modest for most of these constructs, with the largest difference seen for the {alpha}F{zeta} clones. In all functional experiments, samples of untransfected RBL cells incubated with huIgE showed no responses greater than unstimulated controls (data not shown).


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  		Table I. Average degranulation responses for separate clones to show extent of clonal variation

 
We also compared Ca2+ responses stimulated by cross-linking of these chimeric receptors, and representative time courses for these responses are shown in Fig. 4A. Reproducible differences in the kinetics and magnitudes of these responses are evident, and quantitative comparisons were made by integrating each response for 500 s following addition of an optimal dose of Ag or streptavidin. These results for multiple clones and experiments are summarized in Fig. 4B. More striking distinctions in Ca2+ responses among constructs were observed compared with degranulation responses, in part because stimulated degranulation requires only very small amounts of Ca2+ mobilization (32, 33). In the averaged Ca2+ responses, {alpha}{zeta}{zeta} and {alpha}F{zeta} are similar in magnitude as for degranulation, {alpha}{zeta}{zeta}DA is about half that of its parent, and {alpha}T{zeta} is even smaller than this, with an average value that is 14% of the response via endogenous Fc{epsilon}RI. Ca2+ responses for the other three chimeras, {alpha}P{zeta}, {alpha}45{zeta}, and {alpha}F{zeta}CA are substantially smaller than that for {alpha}T{zeta} and are undetectable in some experiments. It is clear from these results that functional responses mediated by these chimeric receptors are highly dependent on their TM segments.



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  		FIGURE 4. Ca2+ mobilization via chimeric IgE receptors. A, Representative time courses for Ca2+ responses stimulated with 0.1 µg/ml streptavidin for bhuIgE-sensitized chimeric receptors or with 1 µg/ml DNP-BSA for moIgE-sensitized Fc{epsilon}RI (and chimeric receptors). Additions of streptavidin or Ag are indicated by arrows. B, Average integrated calcium responses from the first 500 s of stimulation for chimeric IgE receptors relative to that of endogenous Fc{epsilon}RI in each clone were normalized to the maximum value in each set before averaging data from multiple data sets. Values are from 7 days of experiments with two or three clones for each chimeric receptor, and error bars represent the SEM.

 
Stimulated tyrosine phosphorylation

The earliest steps in signaling by these chimeric IgE receptors were evaluated by anti-phosphotyrosine Western blotting. As indicated by the whole cell lysate blot in Fig. 5A for {alpha}{zeta}{zeta} and {alpha}T{zeta}, stimulated tyrosine phosphorylation via chimeric receptors is detectable but weak compared with that via endogenous Fc{epsilon}RI, most likely because of the absence of Fc{epsilon}RI{beta}-mediated amplification of this response (9, 10) for these chimeric receptors. Stimulation via {alpha}{zeta}{zeta} and {alpha}T{zeta} elicits detectable stimulated tyrosine phosphorylation in two distinct bands, one at ~72 kDa, which is probably a substrate of activated Syk kinase (34), and the other at ~38 kDa, which is LAT (35). By comparison, Ag stimulates much stronger phosphorylation of these bands via endogenous Fc{epsilon}RI (in these same {alpha}T{zeta}/{alpha}{zeta}{zeta} stably transfected clones) and also stimulates phosphorylation of several other bands not detectable via chimeric receptor stimulation, including that of Fc{epsilon}RI{beta}. The apparent increase in Fc{epsilon}RI{beta} phosphorylation following stimulation of {alpha}{zeta}{zeta} in Fig. 5A, lane 2, is attributable to inefficient transfer for lane 1, as indicated by background signal in lane 5 and results from three additional experiments (data not shown).



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  		FIGURE 5. Stimulated tyrosine phosphorylation via chimeric IgE receptors. A, Whole cell lysates are compared for stimulation via {alpha}T{zeta}, {alpha}{zeta}{zeta}, and Fc{epsilon}RI differentially sensitized in each clone. B–F, Anti-huIgE immunoprecipitates of {alpha}T{zeta}, {alpha}{zeta}{zeta}, {alpha}F{zeta}CA, {alpha}F{zeta}, {alpha}{zeta}{zeta}DA, and {alpha}P{zeta}, and anti-moIgE immunoprecipitate of Fc{epsilon}RI (F). Numbers after receptor names refer to minutes of stimulation (0 or 5) with 0.1 µg/ml streptavidin or 1 µg/ml DNP-BSA at 37°C. All blots shown are probed with anti-phosphotyrosine Ab 4G10. F, An overexposed blot to show the absence of detectable phosphorylated Fc{epsilon}RI{beta} in reduced {alpha}T{zeta} and {alpha}{zeta}{zeta} immunoprecipitates, whereas heavily phosphorylated Fc{epsilon}RI{beta} immunoprecipitated with anti-moIgE is seen in lane 2. Bands between 45 and 66 kDa in lanes 2, 4, 5, and 6 are nonspecific and appear in the absence of IgE.

 
Streptavidin-stimulated phosphorylation of the chimeric receptors themselves is generally only detectable following their immunoprecipitation with anti-huIgE, and relative extents are shown in Figs. 5, B–E. Although the polypeptide molecular mass of the monomeric chimeric receptors is ~38 kDa, they run on gels at ~70 kDa, likely due to glycoslylation of the Fc{epsilon}RI{alpha} EC domain (36, 37). Attempts to detect chimeric receptors by immunoblotting with Abs to the CT segment of TCR{zeta} or to the EC segment of Fc{epsilon}RI{alpha} were unsuccessful, but the size and stimulated phosphorylation profiles of these chimeric receptors immunoprecipitated with anti-huIgE could be evaluated using anti-phosphotyrosine mAb. As expected from the Ca2+ responses elicited, cross-linking of {alpha}{zeta}{zeta} (Fig. 5B) and {alpha}F{zeta} (Fig. 5C) stimulate the strongest phosphorylation responses, whereas those of {alpha}T{zeta} (Fig. 5B) and {alpha}{zeta}{zeta}DA (Fig. 5D) are weak, and those of {alpha}P{zeta} (Fig. 5E) and {alpha}45{zeta} (data not shown) are undetectable. Also undetectable with any of the chimeric receptors is coprecipitated, phosphorylated Fc{epsilon}RI{beta}, as indicated in the overexposed blot in Fig. 5F.

Fig. 5B shows that reduction of immunoprecipitated {alpha}{zeta}{zeta} causes a clear shift to a band with a lower apparent molecular weight (MW), and this difference was found to be 16 kDa (±6 kDa SD) in nine experiments. It is difficult to distinguish whether the unreduced band is a disulfide-bonded homodimer of {alpha}{zeta}{zeta} with an anomalously low MW, or a hetero-oligomer with some lower MW polypeptide. It is possible that a{zeta}{zeta} is disulfide-bonded to endogenous Fc{epsilon}RI{gamma}, as {zeta}{gamma} dimers have been demonstrated in T cells (38), but we have seen no evidence for phosphorylated {gamma} or other low MW bands in reduced immunoprecipitates of this chimera (data not shown). As shown in Fig. 5D, {alpha}{zeta}{zeta}DA shows a small shift in apparent MW due to reduction that is reproducibly less than that for {alpha}{zeta}{zeta}. As discussed below, it is possible that this shift is due to a lipid modification, such as palmitoylation, at the TM cysteine residue of this mutant. In contrast to these results, {alpha}T{zeta} and {alpha}F{zeta} show no detectable shift in apparent MW upon reduction (Fig. 5, B and C). This indicates that the different functional responses for {alpha}F{zeta} vs {alpha}F{zeta}CA are not likely to be the result of differences in their covalent oligomeric state.

Sucrose gradient analysis of chimeric receptor partitioning into detergent-resistant membranes

To evaluate the potential role of lipid raft association in the functional responses elicited by these chimeric receptors, we compared their distributions in sucrose gradients following cell lysis in Triton X-100, both before and after cross-linking by anti-IgE. In most experiments, uncross-linked Fc{epsilon}RI or chimeric receptors show little or no migration at low densities characteristic of lipid raft association, and cross-linking of endogenous Fc{epsilon}RI causes its efficient association with lipid rafts by this criteria, as expected from previous results (12) (Fig. 6, top left panel). Under these conditions of lysis and gradient analysis, the GPI-linked protein Thy-1 labeled with fluorescent anti-Thy-1 mAb shows substantial fractionation with lipid rafts in unstimulated cells (Fig. 6, top left panel). For the chimeric receptors, cross-link-dependent flotation in sucrose gradients is always less than that observed with Fc{epsilon}RI, but it is reproducibly detected for some chimeras and not for others. Fig. 6 shows representative experiments that illustrate these findings: {alpha}F{zeta}, {alpha}T{zeta}, and {alpha}{zeta}{zeta}DA show significant amounts of flotation at the top of the sucrose gradients as a consequence of cross-linking, whereas {alpha}{zeta}{zeta} shows a smaller amount, which may be attributed to lower surface expression of this chimeric receptor caused by its steady-state internalization (see Fig. 2A). Consistent with this possibility, we also see very little {alpha}{zeta}{zeta} in a high-density peak, which represents cross-linked receptors that do not associate with detergent-resistant membranes (39), indicating that the {alpha}{zeta}{zeta} is less completely cross-linked than other chimeras. Cross-linking of the poorly signaling chimeric receptors, {alpha}P{zeta}, {alpha}45{zeta}, and {alpha}F{zeta}CA, does not cause any significant flotation in sucrose gradients, although efficient cross-linking is evident from the high-density peak in each of these gradient samples.



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  		FIGURE 6. Sucrose gradient analysis of Alexa488-huIgE bound to chimeric receptors from detergent-solubilized cells. Gradient distributions are compared for uncross-linked ({circ}) or anti-IgE cross-linked (•) IgE receptors. The top left panel also shows the gradient distribution of Thy-1 ({triangleup}) in unstimulated cells. Fractions ~2–8 contain detergent-resistant lipid rafts, fractions ~10–17 contain solubilized components, and fractions 18–20 contain nonraft-associated cross-linked receptors. Results are representative of 2–3 experiments for each of the chimeric receptors and Thy-1.

 
These results are summarized in Table II, together with comparisons of the functional responses for these chimeric receptors. They indicate a high correlation between the functional responses elicited by these various chimeric receptors and their capacity to associate with detergent-resistant lipid rafts. For {alpha}F{zeta} in particular, it is striking that a single point mutation in the TM region from cysteine to alanine converts this single-chain receptor from a strong functional responder to one that is very weak and, in so doing, converts it from a receptor that associates with lipid rafts in response to cross-linking to one that does not.


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  		Table II. Summary of results for chimeric IgE receptors

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Much has been learned about signal transduction via multichain immune recognition receptors, including the identities and roles of many proteins involved in both early and later steps in the signaling cascades elicited by these receptors. For most of these signaling steps, however, our understanding of the molecular mechanisms is incomplete, including the earliest events in receptor-mediated signaling. Our previous investigations suggested a role for ordered lipid raft domains in the earliest steps of Fc{epsilon}RI signaling, but the mechanism by which these domains might facilitate cross-link-dependent receptor phosphorylation was unclear (40). More recent studies have indicated that ordered lipid domains provide a protective environment for the Src family kinase Lyn, such that Lyn isolated from detergent-resistant lipid rafts has a substantially higher specific activity than Lyn from detergent-solubilized regions of the plasma membrane due to a greater amount of active site tyrosine phosphorylation (25). This constitutively active Lyn in lipid rafts is sequestered from uncross-linked receptors, as well as from TM phosphatases that dephosphorylate nonraft Lyn kinase to inactivate it (30). As cross-linking of Fc{epsilon}RI promotes its stable association with lipid rafts (12), this process juxtaposes Fc{epsilon}RI with active Lyn in these ordered lipid domains and thereby facilitates receptor phosphorylation by Lyn.

The results described in the present study support and extend this mechanism for cross-link-driven signal initiation via Fc{epsilon}RI. Because the calcium and degranulation responses due to chimeric receptors range from 0 to 90% of that due to native Fc{epsilon}RI in the same clones, we are able to distinguish responses of the chimeras even for relatively modest differences, as between putative monomers ({alpha}{zeta}{zeta}DA) and oligomers ({alpha}{zeta}{zeta}). The strong dependence of signaling on the TM segment of single-chain receptors we observe is highly correlated with the capacity of these receptors to mediate association with detergent-resistant lipid rafts. This correlation extends to a single point mutation in {alpha}F{zeta} that converts this receptor from the most robust signaling chimera to one that does not to stimulate detectable tyrosine phosphorylation or Ca2+ responses despite uncompromised surface expression. The sequence of the TM segment of this chimera is an inverted version of the human transferrin receptor, which is a type II membrane protein (41). Thus, the critical cysteine residue in the {alpha}F{zeta} TM sequence is located at the EC border of this protein’s TM region, instead of at the inner leaflet border, as found in the native transferrin receptor. Our blotting results indicate that this cysteine residue does not participate in covalent dimerization of this chimera, and it is possible that it is palmitoylated similar to the outer leaflet palmitoylation of the EC human signaling protein Sonic hedgehog (42). Preliminary results indicate that 2-bromopalmitate, an inhibitor of cellular palmitoylation (43), prevents cross-link-dependent association of {alpha}F{zeta} with detergent-resistant lipid rafts (J.A. Gosse, unpublished observations). Lipid raft association of influenza virus hemagglutinin requires five large hydrophobic TM amino acids, which are in contact with the exoplasmic leaflet of the membrane (44), and thus location of a palmitoylated cysteine residue at the exoplasmic leaflet may be particularly effective in conferring lipid raft association.

Previous studies with mutated subunits of Fc{epsilon}RI showed that neither the disulfide bond in the TM segment of Fc{epsilon}RI{gamma} nor any single CT domain is required for assembly and transport of the receptor to the cell surface (45, 46), and no CT domain of Fc{epsilon}RI is uniquely necessary for lateral immobilization or detergent insolubility of Fc{epsilon}RI caused by cross-linking (46). Our results and previous studies using single-chain chimeric receptors show that these constructs can reveal structural roles for specific segments more directly than selective mutations in the multichain receptors themselves, which may have evolved additive and/or compensatory contributions from multiple distinct sequences (47). In contrast, disadvantages of chimeric receptors can include misfolding and mislocalization due to incorrect trafficking. For example, in initial efforts with GFP-tagged constructs, we assembled several different chimeric IgE receptors containing the CT segment of the Fc{epsilon}RI{gamma}-chain, which is highly homologous to {zeta}, but we found that these receptors were inefficiently expressed at the plasma membrane and exhibited substantial CT localization.

Our present results argue against a necessary role for the {beta} subunit for signal initiation via IgE cross-linking. No stimulated phosphorylation or association of Fc{epsilon}RI{beta} is detected with any of the chimeric receptors we have characterized, yet two of these mediate Ca2+ and degranulation responses that are comparable to that of endogenous Fc{epsilon}RI. Stimulated phosphorylation mediated by these chimeric receptors is much less than that of endogenous Fc{epsilon}RI, and this difference is attributable to the amplifying effect of Fc{epsilon}RI{beta} on this early signaling. Such amplification may well play an important role in normal mast cell physiology under conditions of sensitization by IgEs of multiple specificities and stimulation with threshold concentrations of allergens (6). However, it is clear from our results and the previous study of Repetto et al. (11) that Fc{epsilon}RI{beta} is not essential for IgE receptor-mediated mast cell degranulation via the chimeric receptors. Thus, the transphosphorylation mechanism, which involves a critical role for Lyn binding to Fc{epsilon}RI{beta} of uncross-linked receptors, cannot account for the results we describe. In contrast, our results are consistent with a mechanism in which cross-link-driven association of ITAM-containing receptors with ordered lipid rafts localizes these aggregates to a plasma membrane environment that is enriched in active Lyn and depleted of inactivating TM phosphatases (30).

A question that remains is the mechanism by which cross-linking promotes lipid raft association of TM proteins containing certain TM segments that do not stably associate with these domains in the absence of cross-linking. Furthermore, our results with {alpha}{zeta}{zeta} and {alpha}{zeta}{zeta}DA suggest that covalent oligomerization of receptors can enhance their signaling capacity, and, in so doing, may enhance or compensate for marginal lipid raft association. For {alpha}{zeta}{zeta}, homo-dimerization or hetero-disulfide bonding to Fc{epsilon}RI{gamma} may enhance interactions with other signaling proteins and thereby compensate for relatively poor surface expression. {alpha}{zeta}{zeta}DA may be palmitoylated at the cysteine residue involved in disulfide bonding in {alpha}{zeta}{zeta}, and this lipid modification could account for its enhanced cell surface expression and lipid raft association. In future studies, these issues can be explored more systematically using radiochemical labeling and well-defined bivalent and oligovalent ligands to cross-link these single-chain receptors and compare their relative signaling responses. A recent study of chimeric receptors containing EC and TM CD8, together with CT Fc{epsilon}RI{gamma}, showed that the disulfide linkages in the EC domain are critical in the degranulation response due to anti-CD8 cross-linking (48); these results support that the hypothesis that disulfide-linked oligomerization can play a significant role in immunoreceptor signaling.

In summary, we find that the TM sequence of single-chain receptors for IgE can greatly influence the signaling responses mediated by these receptors, and both disulfide-mediated receptor oligomerization and cross-link-dependent lipid raft association can contribute to the signaling capacities of these receptors. Furthermore, cross-linked receptors that do not detectably partition into detergent-resistant lipid rafts exhibit virtually undetectable early signaling responses. These results enable a clearer understanding of minimal structural requirements for the initiation of ITAM-mediated immunoreceptor signal transduction in hemopoietic cells.


   Acknowledgments
 
We thank Bettina Wagner (Baker Institute for Animal Health, Cornell University) for providing expert flow cytometry assistance, data analysis, and advice. We thank C. Torigoe and H. Metzger for huIgE and human Fc{epsilon}RI{alpha} cDNA; L.E. Samelson for TT{zeta} and T{zeta}{zeta} chimeric constructs; A. Weiss for PTP{alpha} cDNA; D. Shalloway for CD45 cDNA; and T. McGraw for transferrin receptor cDNA.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI18306 and AI22449. J.A.G. was supported by the W.M. Keck Foundation Program in Biophysics of Signal Transduction at Cornell University. Back

2 Address correspondence and reprint requests to Dr. Barbara Baird, Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853. E-mail address: bab13{at}cornell.edu Back

3 Abbreviations used in this paper: TM, transmembrane; CT, cytoplasmic; EC, extracellular; huIgE, human IgE; moIgE, mouse IgE; bhuIgE, biotinylated human IgE; BSS, buffered saline solution; MW, molecular weight. Back

Received for publication October 29, 2004. Accepted for publication May 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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