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J. Biol. Chem., Vol. 279, Issue 32, 33875-33881, August 6, 2004

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Modulation of FcRI (CD64) Ligand Binding by Blocking Peptides of Periplakin^*

Jeffrey M. Beekman, Jantine E. Bakema, Joke van der Linden, Bastiaan Tops, Marja Hinten, Martine van Vugt¶, Jan G. J. van de Winkel¶, and Jeanette H. W. Leusen||

From the Immunotherapy Laboratory, Department of Immunology, University Medical Center Utrecht, Lundlaan 6, 3584 EA, The Netherlands, Medarex Europe, Lundlaan 6, 3584 EA, Utrecht, The Netherlands, and ^¶Genmab, Yalelaan 60, 3584 CM, Utrecht, The Netherlands

Received for publication, January 29, 2004 , and in revised form, May 20, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
FcRI requires both the intracellular domain of the -chain and associated leukocyte Fc receptor (FcR) -chains for its biological function. We recently found the C terminus of periplakin to selectively interact with the cytoplasmic domain of the FcRI -chain. It thereby enhances the capacity of FcRI to bind, internalize, and present antigens on MHC class II. Here, we characterized the domains involved in FcRI-periplakin interaction using truncated and alanine-substituted FcRI mutants and randomly mutagenized periplakin. This allowed us to design TAT peptides that selectively interfered with endogenous FcRI-periplakin interactions. The addition of these peptides to FcRI-expressing cells modulated FcRI ligand binding, as assessed by erythrocyte-antibody-rosetting. These data support a dominant-negative role of C-terminal periplakin for FcRI biological activity and implicate periplakin as a novel regulator of FcRI in immune cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Leukocyte Fc receptors (FcRs)¹ are membrane-expressed glycoproteins that bind the constant fragments (Fc) of immunoglobulins (1, 2). FcR cross-linking can trigger a variety of cellular responses including phagocytosis, antigen presentation, and cytokine production. Most FcR exist as multisubunit complexes containing a unique ligand binding -chain and promiscuous FcR -chains that are indispensable for tyrosine-based signals (3–6).

FcRI (CD64, FcRIa1) is unique among multisubunit FcR due to a high affinity binding to human IgG, its limited myeloid cell distribution, and a relatively large intracellular domain (7, 8). Products of related genes include FcRIb and FcRIc isoforms, but these specify low affinity IgG receptors if functionally expressed at all (9–12). Besides a role in antigen clearance, FcRI (a1) can potently enhance MHC class I and II antigen presentation in vitro and in vivo (13–16). These properties make FcRI a candidate target for immunotherapy, and concepts are being developed to modulate immune responses by FcRI-directed agents (16–19). The potential of such therapeutic approaches supports further work to enlarge our knowledge of FcRI biology.

The FcR -chain has been studied in great detail and is critically important for FcRI function; it stabilizes FcRI -chain surface expression in vivo (20) and mediates several key functions that require ITAM signaling motifs (21–24). In addition, recent data show that the cytosolic domain of the FcRI -chain (FcRI-CY) could transduce signals leading to cellular effector functions (25, 26). MHC class II antigen presentation assays using IIA1.6 cells co-expressing truncated FcRI-CY mutants and "signaling-dead" FcR -chains indicated a motif for antigen presentation in the membrane proximal 34 aa of FcRI-CY (25). Deletion of FcRI-CY in the presence of functional FcR -chain lowered the kinetics of endocytosis and phagocytosis and abolished interleukin-6 production (26). FcRI-CY signaling likely involves (de)phosphorylation of its serine residues and may include other mechanisms of post-translational modification (27). Thus far no protein effectors have been described that control FcRI function by FcRI-CY interaction. Filamin A (ABP-280) has been shown to bind FcRI-CY, but no functional consequences are known for this interaction (28).

We recently found periplakin to selectively bind FcRI-CY and to modulate ligand binding, receptor modulation, and antigen presentation via FcRI.² Periplakin represents a 195-kDa protein implicated in cornified envelope assembly and structural stability of epithelia (29–32). Like other members of the plakin family (for review, see Refs. 33 and 34) periplakin associates with the actin and intermediate filament cytoskeleton (32, 35, 36). Periplakin has recently been suggested to be involved in signaling of protein kinase B (37) and G-proteins located downstream of the µ-opioid receptor in neurons (38).

In the present study we characterized the molecular interaction between FcRI and periplakin. We determined the periplakin binding domain of FcRI and vice versa by progressive truncations and alanine-scanning mutagenesis of FcRI-CY and random mutagenesis of periplakin. Peptides of these binding domains and the membrane-translocating TAT sequence (39) were designed to interfere with FcRI-periplakin interactions in IIA1.6 cells. FcRI ligand binding was studied in EA-rosette assays.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Generated Constructs—Yeast constructs FcRI-CY (GenBank^TM accession number L03418 [GenBank] , bp 931–1125) and FcRI-CY-truncated mutants (see Fig. 1) were generated by PCR and EcoRI/SalI-cloned into pGBT9 (Clontech, Palo Alto, CA). Alanine replacement of FcRI residues 311–325 was achieved by PCR-based cloning techniques (primer mutations and overlap extension PCR) and EcoRI/SalI insertion into pGBT9. pGAD-GH (Clontech) contained C-terminal periplakin (GenBank^TM accession number AF001691 [GenBank] ) clone 2.2 (bp 4620–5361) or 3.4 (bp 4207–5361). Glycine replacement of aspartic acid 1694 (D1694G) of periplakin clone 2.2 was achieved by substitution of periplakin bp Ala-5081 for Gly by PCR using an adjacent unique SpeI site of periplakin and a mutated primer. Mammalian expression constructs WT-FcRI and truncated mutants were cloned with HindIII/XbaI restriction into pcDNA3 (Invitrogen) as described in Van Vugt et al. (25). Periplakin clones 2.2, 3.4, and the mutated 2.2 (D1694G) were subcloned into pcDNA3.1 HISABC (Invitrogen). The murine FcR -chain with mutated ITAM (Y65F,Y76F) was expressed from pNUT (40, 41). PCR reagents were from PerkinElmer Life Sciences (Nieuwerkerk a/d IJssel, The Netherlands) except for primers (Isogen Bioscience, Maarssen, the Netherlands). All construct were verified by dideoxy sequencing using BigDye Terminators (Applied Biosystems, Warrington, UK) and analyzed on an ABI Prism® 3100 Genetic Analyzer (Applied Biosystems).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Definition of the periplakin binding site in FcRI. A, truncated FcRI constructs were tested in triplicate for interaction with periplakin clone 3.4 (aa 1372–1756) in yeast cells. Interactions are indicated by growth without histidine and -galactosidase activity. WT indicates WT-FcRI-CY; ND, not done. B, stable IIA1.6 cell transfectants expressing WT and truncated FcRI (green) were transiently transfected with periplakin clone 3.4 (red) and assessed by confocal microscopy. Merged images of two representative samples are shown, and yellow colors indicate co-localization. Experiments were performed at least three times, yielding essentially similar results.

Confocal Microscopy—For co-localization studies, clone 3.4, clone 2.2, and clone 2.2-D1694G were transiently transfected in IIA1.6 cells stably expressing WT-FcRI, FcRI-342, FcRI-332, or the tailless FcRI-315 (numbers refer to the last C-terminal residue present in FcRI-CY) and the mutated murine FcR -chain cultured as described in Van Vugt et al. (25). Dead cells were removed 24 h post-transfection by Ficoll gradient centrifugation. After an additional 24 h, viable cells were adhered to poly-L-lysine (Sigma)-coated glass slides. Cells were fixed for 30–60 min in 3% paraformaldehyde, quenched for 5 min in 20 mM NH₄Cl, and blocked for 30 min in PBS with 0.1% saponin, 0.2% bovine serum albumin, 5% mouse serum, 5% goat serum (blocking buffer). FcRI was stained directly by CD64 monoclonal antibody (mAb) 10.1-FITC (Serotec, Oxford, UK) in blocking buffer. Periplakin was stained by incubation with polyclonal rabbit serum 5117 (a kind gift of Dr. B. Burgering, Laboratory of Physiological Chemistry and Center for Biomedical Genetics, University Medical Center Utrecht (37)), rinsing in phosphate-buffered saline, and subsequent incubation with goat--rabbit CY3 (Jackson Laboratories, West Grove, PA). Double staining of mIgG1-FITC (Dako, Glostrup, Denmark) and pre-immune serum 5117 in combination with goat--rabbit-CY3 served as negative controls as well as staining on mock-transfected cells. The slides were rinsed extensively, mounted in Mowiol containing 2.5% DABCO (1,4-diazabicyclo(2.2.2)octane; Sigma), and examined with a 63x planapo objective on a Leitz DMIRB fluorescence microscope (Leica, Voorburg, The Netherlands) interfaced with a Leica TCS4D confocal laser microscope (Leica).

Random Mutagenesis of C-terminal Periplakin—pGAD-GH clone 2.2 served as a PCR template in the presence of dITP and limiting amounts of dATP or dATP and dTTP to generate a pool of randomly mutated periplakin PCR products. This method was adapted from a protocol described by Spee et al. (43). Specific PCR characteristics were: 10 ng of pGAD-GH clone 2.2, 10 pmol per primer (pGAD-GH2, 5'-agatcctagaactag-3', and pGAD-GH3, 5'-gaattgtaatacgac-3'), 2 units of AmpliTaq Gold, 1x Gold buffer, 8 mM MgCl₂,30 µM dATP, 30 or 200 µM dTTP, 200 µM dCTP, dGTP, and dITP in a final volume of 50 µl. The PCR program consisted of an initial 5-min incubation at 95 °C and 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 4 min at 72 °C. PCR products were purified, and 100 ng of PCR product with 500 ng of EcoRI/ApaI-restricted, gel-purified pGAD-GH and 2 µg of pGBT9-FcRI-CY was transformed by 1 M sorbitol, 10 mM Bicine, 3% ethylene glycol in yeast cells. Yeast cells were plated on complete supplement mixture medium without leucine and tryptophan to select for functional plasmids. After 3 days, colonies were lifted and tested for loss of interaction by the absence of -galactosidase activity. Plasmids were prepared from -galactosidase-negative colonies and sequenced. Sequences were aligned using BioEdit software (www.mbio.ncsu.edu/BioEdit/bioedit.html).

EA-rosetting—Human erythrocytes were prepared by Ficoll/Hypaque density centrifugation, stored in sterile Alsever at 4 °C, and used within 2 weeks. Erythrocytes were fluorescently labeled using the PKH26 fluorescent cell linker kit (Sigma) according to the manufacturer's protocol and opsonized by hybridoma supernatant containing mIgG2a anti-human glycophorin A for 1 h at 2 x 10⁸ erythrocytes/ml at 4 °C (44). Erythrocytes were washed twice with Hepes-buffered RPMI 1640 medium (Invitrogen) at 4 °C. Subsequently, 5 x 10⁶ erythrocytes were resuspended with 1 x 10⁵ cells in 50 µl of RPMI in round-bottom 96-well plates, incubated for 60 min at 4 °C, and resuspended after a 30-min incubation. EA-rosettes were fixed by the addition of 3% paraformaldehyde for 30 min. Cells were diluted 2–3-fold in Hepes-buffered RPMI 1640 medium and analyzed by flow cytometry. Cells and free erythrocytes were distinguished by their scatter patterns and autofluorescence in the FL1 channel. The percentage of cells that were FL-2-positive was expressed as percentage of EA-rosettes. FcRI surface expression was measured on a FACScalibur flow cytometry system (BD Biosciences) using the F(ab')₂ fragment of CD64 mAb H22 (Ref. 45; a kind gift of Dr. T. Keler, Medarex, Annandale, NJ) and goat F(ab')₂ anti-human k-light chain-FITC (Southern Biotech, Birmingham, AL).

TAT Peptides—Fusion peptides of the protein transduction domain of TAT (39, 46) and the binding domains of FcRI for periplakin (TAT-FcRI) or vice versa (TAT-PPL) were from Eurogentec (Herstal, Belgium). The sequence of TAT-FcRI is YGRKKRRQRRRGVTIRKELKRKKKWDLEI (29-mer), and that of TAT-PPL is YGRKKRRQRRRGKLRSQECDWEEISVK (27-mer). Peptides were >95% pure and had standard N and C termini. Control TAT-peptide (YGRKKRRQRRRG) consisted of the TAT sequence above and was a kind gift of Dr. P. Coffer (Dept. of Pulmonary Diseases, University Medical Center Utrecht). For transduction, cells were washed twice in Hepes-buffered RPMI 1640 medium containing 5 mM EDTA (EDTA buffer) to remove free extracellular calcium. Cells were incubated for 30 min in 10 µM TAT peptide at 37 °C in EDTA buffer at 5 x 10⁶ cells/ml. Cells were diluted in ice-cold EDTA buffer, washed at 4 °C in buffer without EDTA, and used in EA-rosette assays. Aliquots were analyzed by flow cytometry for FcRI surface expression and cell viability by annexin V and propidium iodide staining (Roche Applied Science). Periplakin expression was assessed by Western blot using periplakin-recognizing rabbit serum 5117 after transient expression of full-length periplakin (construct kindly provided by Dr. F. Watt, Keratinocyte Laboratory, London Research Institute). For control experiments, TAT peptides were FITC-labeled (Molecular Probes, Leiden, The Netherlands), pulsed as described above, and analyzed by confocal microscopy. Surface IgG was stained by an anti-mouse IgG mAb CY-3 conjugated (Jackson).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of the Periplakin Interaction Domain within FcRI—To pinpoint the binding domain of FcRI for periplakin, we tested progressive truncations of FcRI-CY for interaction with C-terminal periplakin in yeast two-hybrid binding assays (Fig. 1). The minimal binding domain consisted of the membrane-proximal 17 amino acids of FcRI-CY, as demonstrated by growth of yeast colonies on histidine-depleted media after transformation with FcRI-327 and periplakin (residues 1372–1756). However, in -galactosidase assays a slightly larger domain of FcRI (22 residues, FcRI-332) was required for periplakin interaction in yeast cells. Removal of the N-terminal valine (residue 311) or more (312 + 313) completely abrogated periplakin interaction. However, residues of the GAL4 DNA binding domain directly upstream of the N terminus of FcRI-CY did not contribute to the interaction, as a stretch of six glycines in between the GAL4 DNA binding domain and FcRI-CY left binding to periplakin intact (data not shown).

We next mapped the binding domain of FcRI for periplakin in IIA1.6 transfectants. Stable transfectants of WT-FcRI, FcRI-342, FcRI-332, and FcRI-315 were co-transfected with C-terminal periplakin clone 3.4 and assessed after 48 h without further stimulation. WT-FcRI and FcRI-342 co-localized with c-terminal periplakin, showing both proteins to be present at similar sites in cells. Although periplakin localized to the (sub)plasma membrane area, co-localization of FcRI-332 and tail-less FcRI (FcRI-315) with C-terminal periplakin was abrogated, suggesting a loss of interaction.

To assess the relative contribution of residues within FcRI-CY for periplakin interaction, alanine-scanning mutagenesis was applied to FcRI-CY residues 311–325, and proteins were assessed for interaction with periplakin in yeast cells. Notably, substitutions were found that either abrogated or improved the interaction between FcRI and periplakin (Fig. 2A). Alanine replacement of plasma membrane-proximal residues abrogated the interaction, except for Thr-312, whereas single substitutions of the stretch of positively charged residues (KRKKK) and Asp-324 apparently led to a better interaction, except for the last lysine. The increase in -galactosidase activity may reflect a better-stabilized interaction with periplakin. By alanine substitution of the proximal part of FcRI-CY, we targeted a sequence that is largely conserved from mouse to man (Fig. 2B). However, we did not detect any interaction between murine FcRI-CY and human periplakin. The absence of Glu-316 and Trp-323 in the mouse sequence might contribute to the differences between these species.

View larger version (40K): [in this window] [in a new window]   FIG. 2. Alanine-scanning mutagenesis of FcRI and interaction with periplakin. A, site-directed mutagenesis was applied to FcRI residues 311–325, and mutants were tested for interaction with periplakin clone 3.4 in yeast cells (shown in triplicate). Growth of yeast cells on medium lacking leucine and tryptophan (-LT), leucine, tryptophan, histidine (-LTH), and -galactosidase activity are shown. One representative experiment is shown (n = 2). B, sequence alignment of FcRI from five different species. Both human and mouse FcRI are truncated at the C terminus, where no significant alignment was observed. Alanine substitutions that abrogate or enhance the interaction are indicated by - or +, respectively. Green residues indicate hydrophobicity/aromaticity; blue indicates hydrophilicity/basicity; red indicates acidity; yellow indicates aliphaticity.

Identification of the Interaction Domain of Periplakin for FcRI—Random mutagenesis of periplakin (schematically shown in Fig. 3A) was chosen as a tool to define the periplakin domain that interacts with FcRI. We prepared a library of mutated clone 2.2 cDNAs by PCR amplification in the presence of dITP and screened for loss of interaction with FcRI in yeast cells (Table I). A 2-fold increase of colonies that acquired a loss-of-interaction phenotype was achieved by the addition of dITP in the PCR. FcRI-periplakin interaction was lost in 43% (68/158), and 57.3% (55/96) of yeast colonies by lowering dATP and dATP/dTTP concentrations, respectively. By (partially) sequencing plasmids from 41 colonies, a total of 65 missense mutations and 8 premature stop codons were identified. The most C-terminal-located stop codon was introduced after residue 1688, indicating that residues 1510–1688 of periplakin do not contain a full binding site for FcRI. Only one of the missense mutants had a single amino acid substitution (aspartic acid at position 1694 into glycine (D1694G)) that resulted in a disturbed interaction (Fig. 3B). We confirmed this observation by site-directed mutagenesis of D1694G in the original periplakin 2.2 construct. Interactions were mitigated both in yeast cells (data not shown, n = 2) and transfected IIA1.6 cells (Fig. 3C). The D1694G-mutated clone 2.2 staining was more dominant in the cytoplasm and sometimes followed a filamentous pattern.

View larger version (36K): [in this window] [in a new window]   FIG. 3. Schematic representation of FcRI binding site in periplakin. A, domain structure of periplakin as adapted from DiColandrea et al. (32). Numbers indicate domain boundaries (aa). Periplakin clones 3.4 and 2.2 code for 384 and 247 c-terminal residues, respectively. In clone 2.2 the postulated binding domain for FcRI is indicated by a white box. B, alignment of postulated FcRI and periplakin binding sites. + and - indicate residue charge; an open box marks a potential negative charge. Asterisks and letters refer to substituted residues of periplakin in the random mutagenesis PCR. D1694G is indicated in red. C, periplakin clone 2.2 or D1694G-mutated clone 2.2 (red) was transiently transfected in stably FcRI (green)-expressing IIA1.6 cells with WT FcR -chains or ITAM-mutated FcR -chains and analyzed by confocal microscopy. Merged images of two representative examples are shown per group; yellow indicates co-localization. Experiments were performed three times with identical results.

Flow Cytometric Analysis of EA-rosetting—Our previous work documented the ligand binding capacity of FcRI to be increased by C-terminal periplakin transfection. Here, we developed a quick assay to measure FcRI-ligand binding via the use of EA-rosetting by flow cytometry (i.e. the percentage of cells that bound mIgG2a-sensitized erythrocytes, upper right panel in Fig. 4B). Non-bound cells and FL2-labeled erythrocytes were found in the lower right and upper left quadrants, respectively. We observed 5% background binding to untransfected IIA1.6 cells or unsensitized erythrocytes (data not shown). A 2.5-fold increase in binding of mIgG2a-sensitized erythrocytes to FcRI was observed upon co-expression of C-terminal periplakin in IIA1.6 cells (Fig. 4C). Binding appeared independent of small differences in surface levels of FcRI (Fig. 4A) and FcR -chain signals. Similar differences were observed when EA-rosetting was scored by light microscopy (data not shown, n = 4).

View larger version (39K): [in this window] [in a new window]   FIG. 4. Flow cytometric analysis of FcRI-mediated EA-rosette formation. A, stable subcloned IIA1.6 transfectants expressing FcRI/WT FcR -chain, FcRI/mutated FcR -chain (two independent subclones), and FcRI/mutated FcR -chain/periplakin clone 3.4 (two independent subclones) were assessed for FcRI expression using F(ab')2 fragments of the CD64 mAb H22 and goat F(ab')2 anti-human light chain FITC by flow cytometry. Black lines represent FcRI staining, and dotted lines represent isotype control. B, erythrocytes (RBC) were FL2-labeled and sensitized with mIgG2a -glycophorin A mAb. Subsequently, FcRI transfectants and sensitized erythrocytes were incubated, and EA-rosetting was assessed by flow cytometry as the percentage of cells bound to mIgG2a-erythrocytes. Lower right quadrants contain non-bound transfectants (that differ in size and FL-1 autofluorescence from erythrocytes), upper left quadrants contain non-bound erythrocytes, upper right quadrants contain transfectants bound to erythrocytes. C, analysis of EA-rosetting of transfectants using erythrocytes sensitized with two concentrations of mIgG2a -glycophorin A mAb. One representative experiment out-of-four is shown; error bars indicate S.D.

View larger version (24K): [in this window] [in a new window]   FIG. 5. Modulation of EA-rosetting by TAT-peptides. A, FcRI surface expression of stable transfectants expressing FcRI and the ITAM-mutated FcR -chain with or without periplakin clone 3.4 after TAT peptide transduction. FcRI expression was measured with F(ab')2 fragments of the CD64 mAb H22 and goat F(ab')2 anti-human light chain FITC by flow cytometry. Filled histograms are non-transduced cells, black lines represent TAT-periplakin-transduced cells, gray lines represent TAT-FcRI-transduced lines. B, periplakin levels after TAT peptide transduction. Full-length periplakin was transiently overexpressed, and periplakin levels were assessed by Western blot. Tubulin staining was used as a loading control. C, transduction of IIA1.6 cells with FITC-conjugated peptides. Non-TAT control peptide and TAT peptides were FITC-conjugated and pulsed into cells as described under "Experimental Procedures." The images contain identical numbers of cells. Surface IgG on IIA1.6 cells was CY-3-labeled in the insert by a CY-3 conjugated mAb recognizing mIgG. D, transfectants were pulsed with TAT peptides and incubated with erythrocytes (RBC) sensitized with different amounts of mIgG2a -glycophorin A. EA-rosette capacity was scored by flow cytometry. One representative experiment of three is shown. Error bars indicate S.D.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this report we studied the molecular interaction between FcRI and periplakin. Minimal binding domains were defined and generated as TAT peptides to disrupt intracellular FcRI-periplakin interactions. TAT-PPL transduction enhanced the capacity of FcRI to form EA-rosettes in transfected IIA1.6 cells without affecting receptor expression levels (Fig. 5). Because this effect mimicked stable transfection of C-terminal periplakin, it is likely that C-terminal periplakin and TAT-PPL regulate FcRI by preventing FcRI-CY binding to endogenous periplakin (Fig. 6). This suggests that endogenous periplakin somehow decreases FcRI-ligand binding.

View larger version (46K): [in this window] [in a new window]   FIG. 6. Schematic showing the proposed regulatory mechanism of C-terminal periplakin and TAT-periplakin on FcRI function. A, endogenous periplakin (light gray) binds the periplakin binding site (white box) of FcRI (black). B, the addition of TAT-periplakin (small light gray boxes) functionally "out-competes" endogenous periplakin binding to FcRI. C, transfected C-terminal periplakin (hatched light gray boxes) blocks endogenous periplakin-FcRI interaction similar to TAT-periplakin. D, both C-terminal periplakin and endogenous periplakin are blocked by excess TAT-periplakin.

FcRI-CY harbors an antigen presentation motif present in FcRI-342 and not in FcRI-332 (25). In IIA1.6 cells, we observed co-localization of FcRI-342, but not FcRI-332, with periplakin, consistent with a possible regulatory role for periplakin in MHC class II antigen presentation. We recently found that IFN-, a cytokine regulating proteins important for MHC class I and II antigen presentation (for review, see Ref. 47), up-regulated both periplakin and FcRI expression in monocytes and PMN. Recently, also PMN were shown to functionally express MHC class II (48). If periplakin is involved in antigen presentation, its role may be to "fine-tune" responses, as tailless FcRI was shown capable of mediating antigen presentation, although less efficiently when a functional FcR -chain was present (25).

Alanine substitution of individual FcRI-CY residues 311–325 that affected binding to periplakin were found largely conserved from mouse to man (Fig. 2). Residues located directly adjacent to the plasma membrane and more downstream (Lys-322, Trp-323, and Leu-325) abrogated FcRI binding to periplakin. Remarkably, most single substitutions of the large positive KRKKK stretch and Asp-324 seemed to facilitate binding between FcRI and periplakin. Overall sequence similarity of FcRI with mouse FcRI or other activable human FcR receptors is low (maximally 20%), and no obvious overlapping domains are present. These could not interact with periplakin in yeast two-hybrid studies,² although the membrane =proximal region of mouse FcRI shares significant similarity with human FcRI.

In mice, seven allelic variants of FcRI have been described, from which three have altered amino acids in the proximal part of the intracellular domain (49). Although no functional polymorphisms have been assigned to the cytosolic tail of human FcRI, amino acid substitutions that influence periplakin interaction might have an effect on FcRI function in vivo. Notably, the human FcRI b and c isoforms have identical cytosolic tails to FcRIa1 but contain asparagine at position 324 instead of aspartic acid (11). This cytosolic tail variant exhibited increased interaction with periplakin in yeast two-hybrid binding studies (data not shown). However, the functional relevance of these isoforms is not known at present.

The random mutagenesis PCR suggested periplakin residues 1687–1701 to be part of the FcRI binding domain within periplakin (Fig. 3). Furthermore, the effect of TAT-PPL on FcRI function implies this region to be essential (Fig. 5). Within this sequence aspartic acid at position 1694 diminished binding to FcRI-CY. The negatively charged residues in this region of periplakin align with the cationic residues in FcRI-CY, and mutations found in periplakin align with critical residues of FcRI in yeast (Fig. 3). This part of periplakin also binds vimentin and is a conserved structure among plakin family members (36). However, periplakin residues 1687 and 1689 are exclusively found in periplakin and might facilitate selective binding of FcRI to periplakin in immune cells. In addition, protein kinase B and the µ-opioid receptor can bind to the C terminus of periplakin (37, 38). No clear sequence similarity exists between FcRI and other periplakin-binding proteins. Thus far, it is not known whether other proteins beside FcRI can compete for binding to periplakin in immune cells or bind simultaneously to form complexes.

Thus far we have not succeeded in demonstrating modulation of FcRI binding to monomeric IgG by periplakin. Although we observed a small increase in a stable transfectant containing C-terminal periplakin, TAT-PPL did not consistently modulate monomeric IgG binding to FcRI (data not shown, n = 3). COS cells express considerable levels of endogenous periplakin, and FcRI monomeric IgG binding assays in these cells showed that FcRI-CY lowered ligand affinity by 2-fold (50), supporting the hypothesis that FcRI-CY-periplakin binding decreases FcRI interaction with monomeric IgG.

This report describes FcRI function to be enhanced by reagents that target the receptor intracellular tail. This might lead to the development of selective reagents that regulate FcRI function for immunotherapy. FcRI-directed immunotherapy may be enhanced by TAT-PPL, resulting in increased treatment efficacies when applied in combination.

	FOOTNOTES

 
^* This work was supported by Dutch Science Foundation (NWO) Grant 901-07-229 (to J. H. W. L.) and by Medarex Europe (to J. M. B. and J. E. B.). 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.

^|| To whom correspondence should be addressed: Immunotherapy Laboratory, Dept. of Immunology, Lundlaan 6, KC.02-085.2, University Medical Center Utrecht, 3584 EA, Utrecht. Tel.: 31-30-2504268; Fax: 31-30-2504305; E-mail: J.H.W.Leusen{at}lab.azu.nl.

¹ The abbreviations used are: FcR, Fc receptor; Fc, constant fragments; aa, amino acids; CY, cytosolic tail; ITAM, immunoreceptor tyrosine-based activation motif; mAb, monoclonal antibody; PPL, periplakin; WT, wild type; Bicine, N,N-bis(2-hydroxyethyl)glycine; MHC, major histocompatibility complex; FITC, fluorescein isothiocyanate; EA, erythrocyte-antibody.

² Beekman, J. M., Bakema, J. E., van de Winkel, J. G. J., and Leusen, J. H. W. (2004) Proc. Natl. Acad. Sci. U.S.A., in press.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Daeron, M. (1997) Annu. Rev. Immunol. 15, 203-234[CrossRef][Medline] [Order article via Infotrieve]
2. Ravetch, J. V., and Bolland, S. (2001) Annu. Rev. Immunol. 19, 275-290[CrossRef][Medline] [Order article via Infotrieve]
3. Ernst, L. K., Duchemin, A. M., and Anderson, C. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6023-6027[Abstract/Free Full Text]
4. Scholl, P. R., and Geha, R. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8847-8850[Abstract/Free Full Text]
5. Masuda, M., and Roos, D. (1993) J. Immunol. 151, 7188-7195[Abstract]
6. Morton, H. C., Van den Herik-Oudijk, I. E., Vossebeld, P., Snijders, A., Verhoeven, A. J., Capel, P. J., and Van de Winkel, J. G. J. (1995) J. Biol. Chem. 270, 29781-29787[Abstract/Free Full Text]
7. Allen, J. M., and Seed, B. (1989) Science 243, 378-381[Abstract/Free Full Text]
8. Van de Winkel J. G. J., and Cappel P. J. A. (1996) Human IgG Fc Receptors, Molecular Biology Intelligence Unit Series, Landes Bioscience, Austin, Texas
9. Ernst, L. K., Van de Winkel, J. G. J., Chiu, I. M., and Anderson, C. L. (1992) J. Biol. Chem. 267, 15692-15700[Abstract/Free Full Text]
10. Porges, A. J., Redecha, P. B., Doebele, R., Pan, L. C., Salmon, J. E., and Kimberly, R. P. (1992) J. Clin. Investig. 90, 2102-2109[Medline] [Order article via Infotrieve]
11. Ernst, L. K., Duchemin, A. M., Miller, K. L., and Anderson, C. L. (1998) Mol. Immunol. 35, 943-954[CrossRef][Medline] [Order article via Infotrieve]
12. Van Vugt, M. J., Reefman, E., Zeelenberg, I., Boonen, G., Leusen, J. H. W., and Van de Winkel, J. G. J. (1999) Eur. J. Immunol. 29, 143-149[CrossRef][Medline] [Order article via Infotrieve]
13. Gosselin, E. J., Wardwell, K., Gosselin, D. R., Alter, N., Fisher, J. L., and Guyre, P. M. (1992) J. Immunol. 149, 3477-3481[Abstract]
14. Wallace, P. K., Tsang, K. Y., Goldstein, J., Correale, P., Jarry, T. M., Schlom, J., Guyre, P. M., Ernstoff, M. S., and Fanger, M. W. (2001) J. Immunol. Methods 248, 183-194[CrossRef][Medline] [Order article via Infotrieve]
15. Heijnen, I. A., Van Vugt, M. J., Fanger, N. A., Graziano, R. F., de Wit, T. P., Hofhuis, F. M., Guyre, P. M., Capel, P. J., Verbeek, J. S., and Van de Winkel, J. G. J. (1996) J. Clin. Investig. 97, 331-338[Medline] [Order article via Infotrieve]
16. Curnow, R. T. (1997) Cancer Immunol. Immunother. 45, 210-215[CrossRef][Medline] [Order article via Infotrieve]
17. Thepen, T., Van Vuuren, A. J., Kiekens, R. C., Damen, C. A., Vooijs, W. C., and Van De Winkel, J. G. J. (2000) Nat. Biotechnol. 18, 48-51[CrossRef][Medline] [Order article via Infotrieve]
18. Van Spriel, A. B., Van den Herik-Oudijk, I. E., and Van de Winkel, J. G. J. (2001) J. Immunol. 166, 7019-7022[Abstract/Free Full Text]
19. Van Roon, J. A., Van Vuuren, A. J., Wijngaarden, S., Jacobs, K. M., Bijlsma, J. W., Lafeber, F. P., Thepen, T., and Van de Winkel, J. G. J. (2003) Arthritis Rheum. 48, 1229-1238[CrossRef][Medline] [Order article via Infotrieve]
20. Van Vugt, M. J., Heijnen, A. F., Capel, P. J., Park, S. Y., Ra, C., Saito, T., Verbeek, J. S., and Van de Winkel, J. G. J. (1996) Blood 87, 3593-3599[Abstract/Free Full Text]
21. Indik, Z. K., Hunter, S., Huang, M. M., Pan, X. Q., Chien, P., Kelly, C., Levinson, A. I., Kimberly, R. P., and Schreiber, A. D. (1994) Exp. Hematol. 22, 599-606[Medline] [Order article via Infotrieve]
22. Davis, W., Harrison, P. T., Hutchinson, M. J., and Allen, J. M. (1995) EMBO J. 14, 432-441[Medline] [Order article via Infotrieve]
23. Van Vugt, M. J., Van den Herik-Oudijk, I. E., and Van de Winkel, J. G. J. (1998) Clin. Exp. Immunol. 113, 415-422[CrossRef][Medline] [Order article via Infotrieve]
24. Melendez, A. J., Bruetschy, L., Floto, R. A., Harnett, M. M., and Allen, J. M. (2001) Blood 98, 3421-3428[Abstract/Free Full Text]
25. Van Vugt, M. J., Kleijmeer, M. J., Keler, T., Zeelenberg, I., Van Dijk, M. A., Leusen, J. H. W., Geuze, H. J., and Van de Winkel, J. G. J. (1999) Blood 94, 808-817[Abstract/Free Full Text]
26. Edberg, J. C., Yee, A. M., Rakshit, D. S., Chang, D. J., Gokhale, J. A., Indik, Z. K., Schreiber, A. D., and Kimberly, R. P. (1999) J. Biol. Chem. 274, 30328-30333[Abstract/Free Full Text]
27. Edberg, J. C., Qin, H., Gibson, A. W., Yee, A. M., Redecha, P. B., Indik, Z. K., Schreiber, A. D., and Kimberly, R. P. (2002) J. Biol. Chem. 277, 41287-41293[Abstract/Free Full Text]
28. Ohta, Y., Stossel, T. P., and Hartwig, J. H. (1991) Cell 67, 275-282[CrossRef][Medline] [Order article via Infotrieve]
29. Ruhrberg, C., Hajibagheri, M. A., Parry, D. A., and Watt, F. M. (1997) J. Cell Biol. 139, 1835-1849[Abstract/Free Full Text]
30. Aho, S., McLean, W. H., Li, K., and Uitto, J. (1998) Genomics 48, 242-247[CrossRef][Medline] [Order article via Infotrieve]
31. Steinert, P. M., and Marekov, L. N. (1999) Mol. Biol. Cell 10, 4247-4261[Abstract/Free Full Text]
32. DiColandrea, T., Karashima, T., Maatta, A., and Watt, F. M. (2000) J. Cell Biol. 151, 573-586[Abstract/Free Full Text]
33. Leung, C. L., Liem, R. K., Parry, D. A., and Green, K. J. (2001) J. Cell Sci. 114, 3409-3410[Free Full Text]
34. Leung, C. L., Green, K. J., and Liem, R. K. (2002) Trends Cell Biol. 12, 37-45[CrossRef][Medline] [Order article via Infotrieve]
35. Kazerounian, S., Uitto, J., and Aho, S. (2002) Exp. Dermatol. 11, 428-438[CrossRef][Medline] [Order article via Infotrieve]
36. Karashima, T., and Watt, F. M. (2002) J. Cell Sci. 115, 5027-5037[CrossRef][Medline] [Order article via Infotrieve]
37. Van den Heuvel, A. P., de Vries-Smits, A. M., Van Weeren, P. C., Dijkers, P. F., de Bruyn, K. M., Riedl, J. A., and Burgering, B. M. (2002) J. Cell Sci. 115, 3957-3966[Abstract/Free Full Text]
38. Feng, G. J., Kellett, E., Scorer, C. A., Wilde, J., White, J. H., and Milligan, G. (2003) J. Biol. Chem. 278, 33400-33407[Abstract/Free Full Text]
39. Jones, S. L., Wang, J., Turck, C. W., and Brown, E. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9331-9336[Abstract/Free Full Text]
40. Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., Maxwell, I. H., and Brinster, R. L. (1987) Cell 50, 435-443[CrossRef][Medline] [Order article via Infotrieve]
41. Ra, C., Jouvin, M. H., and Kinet, J. P. (1989) J. Biol. Chem. 264, 15323-15327[Abstract/Free Full Text]
42. Klebe, R. J., Harriss, J. V., Sharp, Z. D., and Douglas, M. G. (1983) Gene (Amst.) 25, 333-341[CrossRef][Medline] [Order article via Infotrieve]
43. Spee, J. H., de Vos, W. M., and Kuipers, O. P. (1993) Nucleic Acids Res. 21, 777-778[Free Full Text]
44. Boot, J. H., Geerts, M. E., and Aarden, L. A. (1989) J. Immunol. 142, 1217-1223[Abstract]
45. Graziano, R. F., Tempest, P. R., White, P., Keler, T., Deo, Y., Ghebremariam, H., Coleman, K., Pfefferkorn, L. C., Fanger, M. W., and Guyre, P. M. (1995) J. Immunol. 155, 4996-5002[Abstract]
46. Bracke, M., Lammers, J. W., Coffer, P. J., and Koenderman, L. (2001) Blood 97, 3478-3483[Abstract/Free Full Text]
47. Boehm, U., Klamp, T., Groot, M., and Howard, J. C. (1997) Annu. Rev. Immunol. 15, 749-795[CrossRef][Medline] [Order article via Infotrieve]
48. Radsak, M., Iking-Konert, C., Stegmaier, S., Andrassy, K., and Hansch, G. M. (2000) Immunology 101, 521-530[CrossRef][Medline] [Order article via Infotrieve]
49. Gavin, A. L., Leiter, E. H., and Hogarth, P. M. (2000) Immunogenetics 51, 206-211[CrossRef][Medline] [Order article via Infotrieve]
50. Miller, K. L., Duchemin, A. M., and Anderson, C. L. (1996) J. Exp. Med. 183, 2227-2233[Abstract/Free Full Text]

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