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• W2023004879 abstract "FcγRI requires both the intracellular domain of the α-chain and associated leukocyte Fc receptor (FcR) γ-chains for its biological function. We recently found the C terminus of periplakin to selectively interact with the cytoplasmic domain of the FcγRI α-chain. It thereby enhances the capacity of FcγRI to bind, internalize, and present antigens on MHC class II. Here, we characterized the domains involved in FcγRI-periplakin interaction using truncated and alanine-substituted FcγRI mutants and randomly mutagenized periplakin. This allowed us to design TAT peptides that selectively interfered with endogenous FcγRI-periplakin interactions. The addition of these peptides to FcγRI-expressing cells modulated FcγRI ligand binding, as assessed by erythrocyte-antibody-rosetting. These data support a dominant-negative role of C-terminal periplakin for FcγRI biological activity and implicate periplakin as a novel regulator of FcγRI in immune cells. FcγRI requires both the intracellular domain of the α-chain and associated leukocyte Fc receptor (FcR) γ-chains for its biological function. We recently found the C terminus of periplakin to selectively interact with the cytoplasmic domain of the FcγRI α-chain. It thereby enhances the capacity of FcγRI to bind, internalize, and present antigens on MHC class II. Here, we characterized the domains involved in FcγRI-periplakin interaction using truncated and alanine-substituted FcγRI mutants and randomly mutagenized periplakin. This allowed us to design TAT peptides that selectively interfered with endogenous FcγRI-periplakin interactions. The addition of these peptides to FcγRI-expressing cells modulated FcγRI ligand binding, as assessed by erythrocyte-antibody-rosetting. These data support a dominant-negative role of C-terminal periplakin for FcγRI biological activity and implicate periplakin as a novel regulator of FcγRI in immune cells. Leukocyte Fc receptors (FcRs) 1The 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. are membrane-expressed glycoproteins that bind the constant fragments (Fc) of immunoglobulins (1Daeron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1046) Google Scholar, 2Ravetch J.V. Bolland S. Annu. Rev. Immunol. 2001; 19: 275-290Crossref PubMed Scopus (1387) Google Scholar). 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 α-chain and promiscuous FcR γ-chains that are indispensable for tyrosine-based signals (3Ernst L.K. Duchemin A.M. Anderson C.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6023-6027Crossref PubMed Scopus (155) Google Scholar, 4Scholl P.R. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8847-8850Crossref PubMed Scopus (96) Google Scholar, 5Masuda M. Roos D. J. Immunol. 1993; 151: 7188-7195PubMed Google Scholar, 6Morton H.C. Van den Herik-Oudijk I.E. Vossebeld P. Snijders A. Verhoeven A.J. Capel P.J. Van de Winkel J.G.J. J. Biol. Chem. 1995; 270: 29781-29787Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). FcγRI (CD64, FcγRIa1) 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 (7Allen J.M. Seed B. Science. 1989; 243: 378-381Crossref PubMed Scopus (203) Google Scholar, 8Van de Winkel J.G.J. Cappel P.J.A. Human IgG Fc Receptors. Molecular Biology Intelligence Unit Series, Landes Bioscience, Austin, Texas1996Google Scholar). Products of related genes include FcγRIb and FcγRIc isoforms, but these specify low affinity IgG receptors if functionally expressed at all (9Ernst L.K. Van de Winkel J.G.J. Chiu I.M. Anderson C.L. J. Biol. Chem. 1992; 267: 15692-15700Abstract Full Text PDF PubMed Google Scholar, 10Porges A.J. Redecha P.B. Doebele R. Pan L.C. Salmon J.E. Kimberly R.P. J. Clin. Investig. 1992; 90: 2102-2109Crossref PubMed Scopus (40) Google Scholar, 11Ernst L.K. Duchemin A.M. Miller K.L. Anderson C.L. Mol. Immunol. 1998; 35: 943-954Crossref PubMed Scopus (32) Google Scholar, 12Van Vugt M.J. Reefman E. Zeelenberg I. Boonen G. Leusen J.H.W. Van de Winkel J.G.J. Eur. J. Immunol. 1999; 29: 143-149Crossref PubMed Scopus (0) Google Scholar). Besides a role in antigen clearance, FcγRI (a1) can potently enhance MHC class I and II antigen presentation in vitro and in vivo (13Gosselin E.J. Wardwell K. Gosselin D.R. Alter N. Fisher J.L. Guyre P.M. J. Immunol. 1992; 149: 3477-3481PubMed Google Scholar, 14Wallace P.K. Tsang K.Y. Goldstein J. Correale P. Jarry T.M. Schlom J. Guyre P.M. Ernstoff M.S. Fanger M.W. J. Immunol. Methods. 2001; 248: 183-194Crossref PubMed Scopus (52) Google Scholar, 15Heijnen 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. Van de Winkel J.G.J. J. Clin. Investig. 1996; 97: 331-338Crossref PubMed Scopus (170) Google Scholar, 16Curnow R.T. Cancer Immunol. Immunother. 1997; 45: 210-215Crossref PubMed Scopus (135) Google Scholar). These properties make FcγRI a candidate target for immunotherapy, and concepts are being developed to modulate immune responses by FcγRI-directed agents (16Curnow R.T. Cancer Immunol. Immunother. 1997; 45: 210-215Crossref PubMed Scopus (135) Google Scholar, 17Thepen T. Van Vuuren A.J. Kiekens R.C. Damen C.A. Vooijs W.C. Van De Winkel J.G.J. Nat. Biotechnol. 2000; 18: 48-51Crossref PubMed Scopus (74) Google Scholar, 18Van Spriel A.B. Van den Herik-Oudijk I.E. Van de Winkel J.G.J. J. Immunol. 2001; 166: 7019-7022Crossref PubMed Scopus (17) Google Scholar, 19Van Roon J.A. Van Vuuren A.J. Wijngaarden S. Jacobs K.M. Bijlsma J.W. Lafeber F.P. Thepen T. Van de Winkel J.G.J. Arthritis Rheum. 2003; 48: 1229-1238Crossref PubMed Scopus (47) Google Scholar). The potential of such therapeutic approaches supports further work to enlarge our knowledge of FcγRI biology. The FcR γ-chain has been studied in great detail and is critically important for FcγRI function; it stabilizes FcγRI α-chain surface expression in vivo (20Van Vugt M.J. Heijnen A.F. Capel P.J. Park S.Y. Ra C. Saito T. Verbeek J.S. Van de Winkel J.G.J. Blood. 1996; 87: 3593-3599Crossref PubMed Google Scholar) and mediates several key functions that require ITAM signaling motifs (21Indik Z.K. Hunter S. Huang M.M. Pan X.Q. Chien P. Kelly C. Levinson A.I. Kimberly R.P. Schreiber A.D. Exp. Hematol. 1994; 22: 599-606PubMed Google Scholar, 22Davis W. Harrison P.T. Hutchinson M.J. Allen J.M. EMBO J. 1995; 14: 432-441Crossref PubMed Scopus (94) Google Scholar, 23Van Vugt M.J. Van den Herik-Oudijk I.E. Van de Winkel J.G.J. Clin. Exp. Immunol. 1998; 113: 415-422Crossref PubMed Scopus (16) Google Scholar, 24Melendez A.J. Bruetschy L. Floto R.A. Harnett M.M. Allen J.M. Blood. 2001; 98: 3421-3428Crossref PubMed Scopus (40) Google Scholar). In addition, recent data show that the cytosolic domain of the FcγRI α-chain (FcγRI-CY) could transduce signals leading to cellular effector functions (25Van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. Van Dijk M.A. Leusen J.H.W. Geuze H.J. Van de Winkel J.G.J. Blood. 1999; 94: 808-817Crossref PubMed Google Scholar, 26Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). MHC class II antigen presentation assays using IIA1.6 cells co-expressing truncated FcγRI-CY mutants and “signaling-dead” FcR γ-chains indicated a motif for antigen presentation in the membrane proximal ∼34 aa of FcγRI-CY (25Van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. Van Dijk M.A. Leusen J.H.W. Geuze H.J. Van de Winkel J.G.J. Blood. 1999; 94: 808-817Crossref PubMed Google Scholar). Deletion of FcγRI-CY in the presence of functional FcR γ-chain lowered the kinetics of endocytosis and phagocytosis and abolished interleukin-6 production (26Edberg J.C. Yee A.M. Rakshit D.S. Chang D.J. Gokhale J.A. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 1999; 274: 30328-30333Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). FcγRI-CY signaling likely involves (de)phosphorylation of its serine residues and may include other mechanisms of post-translational modification (27Edberg J.C. Qin H. Gibson A.W. Yee A.M. Redecha P.B. Indik Z.K. Schreiber A.D. Kimberly R.P. J. Biol. Chem. 2002; 277: 41287-41293Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Thus far no protein effectors have been described that control FcγRI function by FcγRI-CY interaction. Filamin A (ABP-280) has been shown to bind FcγRI-CY, but no functional consequences are known for this interaction (28Ohta Y. Stossel T.P. Hartwig J.H. Cell. 1991; 67: 275-282Abstract Full Text PDF PubMed Scopus (81) Google Scholar). We recently found periplakin to selectively bind FcγRI-CY and to modulate ligand binding, receptor modulation, and antigen presentation via FcγRI. 2Beekman, 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. Periplakin represents a 195-kDa protein implicated in cornified envelope assembly and structural stability of epithelia (29Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (177) Google Scholar, 30Aho S. McLean W.H. Li K. Uitto J. Genomics. 1998; 48: 242-247Crossref PubMed Scopus (42) Google Scholar, 31Steinert P.M. Marekov L.N. Mol. Biol. Cell. 1999; 10: 4247-4261Crossref PubMed Scopus (125) Google Scholar, 32DiColandrea T. Karashima T. Maatta A. Watt F.M. J. Cell Biol. 2000; 151: 573-586Crossref PubMed Scopus (78) Google Scholar). Like other members of the plakin family (for review, see Refs. 33Leung C.L. Liem R.K. Parry D.A. Green K.J. J. Cell Sci. 2001; 114: 3409-3410PubMed Google Scholar and 34Leung C.L. Green K.J. Liem R.K. Trends Cell Biol. 2002; 12: 37-45Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) periplakin associates with the actin and intermediate filament cytoskeleton (32DiColandrea T. Karashima T. Maatta A. Watt F.M. J. Cell Biol. 2000; 151: 573-586Crossref PubMed Scopus (78) Google Scholar, 35Kazerounian S. Uitto J. Aho S. Exp. Dermatol. 2002; 11: 428-438Crossref PubMed Scopus (63) Google Scholar, 36Karashima T. Watt F.M. J. Cell Sci. 2002; 115: 5027-5037Crossref PubMed Scopus (69) Google Scholar). Periplakin has recently been suggested to be involved in signaling of protein kinase B (37Van den Heuvel A.P. de Vries-Smits A.M. Van Weeren P.C. Dijkers P.F. de Bruyn K.M. Riedl J.A. Burgering B.M. J. Cell Sci. 2002; 115: 3957-3966Crossref PubMed Scopus (53) Google Scholar) and G-proteins located downstream of the μ-opioid receptor in neurons (38Feng G.J. Kellett E. Scorer C.A. Wilde J. White J.H. Milligan G. J. Biol. Chem. 2003; 278: 33400-33407Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In the present study we characterized the molecular interaction between FcγRI and periplakin. We determined the periplakin binding domain of FcγRI and vice versa by progressive truncations and alanine-scanning mutagenesis of FcγRI-CY and random mutagenesis of periplakin. Peptides of these binding domains and the membrane-translocating TAT sequence (39Jones S.L. Wang J. Turck C.W. Brown E.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9331-9336Crossref PubMed Scopus (142) Google Scholar) were designed to interfere with FcγRI-periplakin interactions in IIA1.6 cells. FcγRI ligand binding was studied in EA-rosette assays. Generated Constructs—Yeast constructs FcγRI-CY (GenBank™ accession number L03418, bp 931–1125) and FcγRI-CY-truncated mutants (see Fig. 1) were generated by PCR and EcoRI/SalI-cloned into pGBT9 (Clontech, Palo Alto, CA). Alanine replacement of FcγRI 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™ accession number AF001691) 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-FcγRI and truncated mutants were cloned with HindIII/XbaI restriction into pcDNA3 (Invitrogen) as described in Van Vugt et al. (25Van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. Van Dijk M.A. Leusen J.H.W. Geuze H.J. Van de Winkel J.G.J. Blood. 1999; 94: 808-817Crossref PubMed Google Scholar). Periplakin clones 2.2, 3.4, and the mutated 2.2 (D1694G) were subcloned into pcDNA3.1 HISABC (Invitrogen). The murine FcR γ-chain with mutated ITAM (Y65F,Y76F) was expressed from pNUT (40Palmiter R.D. Behringer R.R. Quaife C.J. Maxwell F. Maxwell I.H. Brinster R.L. Cell. 1987; 50: 435-443Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 41Ra C. Jouvin M.H. Kinet J.P. J. Biol. Chem. 1989; 264: 15323-15327Abstract Full Text PDF PubMed Google Scholar). 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). Yeast Two-hybrid Protein Interaction Assays—A GAL4-based yeast two-hybrid system (Clontech) was used to assess interactions between truncated FcγRI molecules and periplakin. Bait and prey plasmids were transformed by 1 m sorbitol, 10 mm Bicine, 3% ethylene glycol (42Klebe R.J. Harriss J.V. Sharp Z.D. Douglas M.G. Gene (Amst.). 1983; 25: 333-341Crossref PubMed Scopus (372) Google Scholar) into yeast strain YGHI. Protein-protein interactions were reported by yeast growth on medium without leucine, tryptophan, histidine, and expression of β-galactosidase, indicated by blue staining of yeast colonies after replica filter lifting, N2 snap-freezing, and incubation for 2–4 h in Z-buffer (60 mm Na2HPO4, 60 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4; Sigma) containing 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Roche Applied Science). 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-FcγRI, FcγRI-Δ342, FcγRI-Δ332, or the tailless FcγRI-Δ315 (numbers refer to the last C-terminal residue present in FcγRI-CY) and the mutated murine FcR γ-chain cultured as described in Van Vugt et al. (25Van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. Van Dijk M.A. Leusen J.H.W. Geuze H.J. Van de Winkel J.G.J. Blood. 1999; 94: 808-817Crossref PubMed Google Scholar). 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). FcγRI 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 (37Van den Heuvel A.P. de Vries-Smits A.M. Van Weeren P.C. Dijkers P.F. de Bruyn K.M. Riedl J.A. Burgering B.M. J. Cell Sci. 2002; 115: 3957-3966Crossref PubMed Scopus (53) Google Scholar)), rinsing in phosphate-buffered saline, and subsequent incubation with goat-α-rabbit CY3 (Jackson Laboratories, West Grove, PA). Double staining of mIgG1-FITC (Dako, Glostrup, Denmark) and pre-immune serum 5117 in combination with goat-α-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 63× 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. (43Spee J.H. de Vos W.M. Kuipers O.P. Nucleic Acids Res. 1993; 21: 777-778Crossref PubMed Scopus (137) Google Scholar). 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, 1× 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-FcγRI-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 β-galactosidase activity. Plasmids were prepared from β-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 × 108 erythrocytes/ml at 4 °C (44Boot J.H. Geerts M.E. Aarden L.A. J. Immunol. 1989; 142: 1217-1223PubMed Google Scholar). Erythrocytes were washed twice with Hepes-buffered RPMI 1640 medium (Invitrogen) at 4 °C. Subsequently, 5 × 106 erythrocytes were resuspended with 1 × 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. FcγRI surface expression was measured on a FACScalibur flow cytometry system (BD Biosciences) using the F(ab′)2 fragment of CD64 mAb H22 (Ref. 45Graziano R.F. Tempest P.R. White P. Keler T. Deo Y. Ghebremariam H. Coleman K. Pfefferkorn L.C. Fanger M.W. Guyre P.M. J. Immunol. 1995; 155: 4996-5002PubMed Google Scholar; 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 (39Jones S.L. Wang J. Turck C.W. Brown E.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9331-9336Crossref PubMed Scopus (142) Google Scholar, 46Bracke M. Lammers J.W. Coffer P.J. Koenderman L. Blood. 2001; 97: 3478-3483Crossref PubMed Scopus (34) Google Scholar) and the binding domains of FcγRI for periplakin (TAT-FcγRI) or vice versa (TAT-PPL) were from Eurogentec (Herstal, Belgium). The sequence of TAT-FcγRI 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 × 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 FcγRI 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). Identification of the Periplakin Interaction Domain within FcγRI—To pinpoint the binding domain of FcγRI for periplakin, we tested progressive truncations of FcγRI-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 FcγRI-CY, as demonstrated by growth of yeast colonies on histidine-depleted media after transformation with FcγRI-Δ327 and periplakin (residues 1372–1756). However, in β-galactosidase assays a slightly larger domain of FcγRI (22 residues, FcγRI-Δ332) 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 FcγRI-CY did not contribute to the interaction, as a stretch of six glycines in between the GAL4 DNA binding domain and FcγRI-CY left binding to periplakin intact (data not shown). We next mapped the binding domain of FcγRI for periplakin in IIA1.6 transfectants. Stable transfectants of WT-FcγRI, FcγRI-Δ342, FcγRI-Δ332, and FcγRI-Δ315 were co-transfected with C-terminal periplakin clone 3.4 and assessed after 48 h without further stimulation. WT-FcγRI and FcγRI-Δ342 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 FcγRI-Δ332 and tail-less FcγRI (FcγRI-Δ315) with C-terminal periplakin was abrogated, suggesting a loss of interaction. To assess the relative contribution of residues within FcγRI-CY for periplakin interaction, alanine-scanning mutagenesis was applied to FcγRI-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 FcγRI 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 β-galactosidase activity may reflect a better-stabilized interaction with periplakin. By alanine substitution of the proximal part of FcγRI-CY, we targeted a sequence that is largely conserved from mouse to man (Fig. 2B). However, we did not detect any interaction between murine FcγRI-CY and human periplakin. The absence of Glu-316 and Trp-323 in the mouse sequence might contribute to the differences between these species. Together, these results pointed to a periplakin binding domain of FcγRI-CY in juxtaposition to the plasma membrane. Periplakin binding to FcγRI-CY in yeast required a minimal motif of 17 residues within FcγRI-CY, but significant co-localization was only observed when 32 residues of FcγRI-CY were present. This discrepancy was observed consistently and indicated that both systems differ in FcγRI-periplakin binding requirements. For further studies with blocking peptides, we utilized the membrane-proximal 17 residues of FcγRI as a blocking domain for FcγRI-periplakin interaction. Identification of the Interaction Domain of Periplakin for FcγRI—Random mutagenesis of periplakin (schematically shown in Fig. 3A) was chosen as a tool to define the periplakin domain that interacts with FcγRI. 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 FcγRI 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. FcγRI-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 FcγRI. 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.Table IRandom PCR mutagenesis of periplakinScreen no.Control PCRMutagenesis PCRStop codonsI [dATP] ↓27.5% (53/193)43.0% (68/158)1644, 1649, 1663, 1666, 1676II [dATP] + [dTTP] ↓29.6% (28/95)57.3% (55/96)1653, 1681, 1688 Open table in a new tab Six substitutions were located within the 15 residues adjacent to Asp-1694 (1687-KLRSQECDWEEISVK-1701). At four positions residue charge was affected, and two times a proline was inserted (K1687E,K1687D, L1688P, D1694G, E1696G, S1699P, K1701E; Fig. 3B). When this domain was aligned to the proximal part of FcγRI-CY (in opposite direction), several parts exhibited clear opposing electrostatic charges that may well contribute to the interaction between both proteins (Fig. 3B). However, it is hard to assign specific periplakin residues besides Asp-1694 of being located at the binding interface, as the tertiary structure of these domains is unknown. These data supported periplakin 1687-KLRSQECDWEEISVK-1701 to be part of the FcγRI binding domain of periplakin, and we hypothesized that a peptide with this sequence may block such interaction. Flow Cytometric Analysis of EA-rosetting—Our previous work documented the ligand binding capacity of FcγRI to be increased by C-terminal periplakin transfection. Here, we developed a quick assay to measure FcγRI-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 FcγRI 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 FcγRI (Fig. 4A) and FcR γ-chain signals. Similar differences were observed when EA-rosetting was scored by light microscopy (data not shown, n = 4). Modulation of EA-rosetting by TAT-Periplakin—Peptides containing a TAT motif and the postulated binding domains of FcγRI and periplakin were designed to block intracellular FcγRI-periplakin interaction. FcγRI surface expression and periplakin levels were unaffected by the addition of TAT peptides (Fig. 5, A and B; n = 3). Similarly, cell viability assessed by annexin V and propidium iodide-staining remained intact (data not shown, n = 3). To visualize cell transduction with the TAT peptides, FITC-conjugated TAT-FcγRI 311–327 (TAT-FcγRI), TAT-periplakin 1687–1701 (TAT-PPL), and non-TAT peptides were incubated with IIA1.6 cells (n = 3). Intracellular accumulation of FITC was observed only with the TAT peptides, consistent with their intracellular delivery (Fig. 5C). Subsequently, transfectants were pulsed with TAT peptides and incubated with mIgG2a-sensitized erythrocytes to assess the modulating capacity of these peptides on FcγRI-ligand interaction. TAT-PPL increased the interaction of IIA1.6 cells with mIgG2a-sensitized erythrocytes to levels observed in C-terminal periplakin transfection experiments (Fig. 5D, n = 3). TAT-FcγRI slightly increased the capacity of FcγRI to form EA-rosettes, but differences were not significant, possibly due to an inability of this peptide to transduce cells as efficiently as TAT-PPL or to adapt to an appropriate tertiary structure. Similarly, a peptide consisting of only the TAT sequence did not affect EA-rosetting ability. When C-terminal periplakin was co-expressed with FcγRI none of the peptides modulated EA-rosetting. These data support that the interaction between FcγRI and endogenous periplakin was effectively blocked by C-terminal periplakin. In this report we studied the molecular interaction between FcγRI and periplakin. Minimal binding domains were defined and generated as TAT peptides to disrupt intracellular FcγRI-periplakin interactions. TAT-PPL transduction enhanced the capacity of FcγRI 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 FcγRI by preventing FcγRI-CY binding to endogenous periplakin (Fig. 6). This suggests that endogenous periplakin somehow decreases FcγRI-ligand binding. We showed that the proximal part of FcγRI-CY binds periplakin. Receptor truncation experiments in yeast cells indicated FcγRI C-terminal residues 333–374 to be fully dispensable for the interaction of FcγRI-CY with periplakin. FcγRI-Δ327, albeit less efficient than WT-FcγRI, bound periplakin, as indicated by growth on histidine-depleted media showing the 17 membrane-proximal residues of FcγRI to be minimally required for interaction. In transfected cells, FcγRI-CY-dependent co-localization with periplakin was observed. Notably, however, a larger motif of FcγRI (FcγRI-Δ342) was required than in the yeast system. The discrepancy between yeast and mammalian cells for the minimal requirements of interaction between FcγRI and periplakin might be explained by differences in protein folding in the two systems or associated molecules like the FcR γ-chain or filamin A in IIA1.6 cells. This might account for the non-functional TAT-FcγRI peptide as well. The presence of the WT or ITAM-mutated FcR γ-chain did not influence co-localization under the conditions tested. FcγRI-CY harbors an antigen presentation motif present in FcγRI-Δ342 and not in FcγRI-Δ332 (25Van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. Van Dijk M.A. Leusen J.H.W. Geuze H.J. Van de Winkel J.G.J. Blood. 1999; 94: 808-817Crossref PubMed Google Scholar). In IIA1.6 cells, we observed co-localization of FcγRI-Δ342, but not FcγRI-Δ332, with periplakin, consistent with a possible regulatory role for periplakin in MHC class II antigen presentation. We recently found that IFN-γ, a cytokine regulating proteins important for MHC class I and II antigen presentation (for review, see Ref. 47Boehm U. Klamp T. Groot M. Howard J.C. Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2493) Google Scholar), up-regulated both periplakin and FcγRI expression in monocytes and PMN. Recently, also PMN were shown to functionally express MHC class II (48Radsak M. Iking-Konert C. Stegmaier S. Andrassy K. Hansch G.M. Immunology. 2000; 101: 521-530Crossref PubMed Scopus (143) Google Scholar). If periplakin is involved in antigen presentation, its role may be to “fine-tune” responses, as tailless FcγRI was shown capable of mediating antigen presentation, although less efficiently when a functional FcR γ-chain was present (25Van Vugt M.J. Kleijmeer M.J. Keler T. Zeelenberg I. Van Dijk M.A. Leusen J.H.W. Geuze H.J. Van de Winkel J.G.J. Blood. 1999; 94: 808-817Crossref PubMed Google Scholar). Alanine substitution of individual FcγRI-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 FcγRI binding to periplakin. Remarkably, most single substitutions of the large positive KRKKK stretch and Asp-324 seemed to facilitate binding between FcγRI and periplakin. Overall sequence similarity of FcγRI with mouse FcγRI 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 FcγRI shares significant similarity with human FcγRI. In mice, seven allelic variants of FcγRI have been described, from which three have altered amino acids in the proximal part of the intracellular domain (49Gavin A.L. Leiter E.H. Hogarth P.M. Immunogenetics. 2000; 51: 206-211Crossref PubMed Scopus (17) Google Scholar). Although no functional polymorphisms have been assigned to the cytosolic tail of human FcγRI, amino acid substitutions that influence periplakin interaction might have an effect on FcγRI function in vivo. Notably, the human FcγRI b and c isoforms have identical cytosolic tails to FcγRIa1 but contain asparagine at position 324 instead of aspartic acid (11Ernst L.K. Duchemin A.M. Miller K.L. Anderson C.L. Mol. Immunol. 1998; 35: 943-954Crossref PubMed Scopus (32) Google Scholar). 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 FcγRI binding domain within periplakin (Fig. 3). Furthermore, the effect of TAT-PPL on FcγRI function implies this region to be essential (Fig. 5). Within this sequence aspartic acid at position 1694 diminished binding to FcγRI-CY. The negatively charged residues in this region of periplakin align with the cationic residues in FcγRI-CY, and mutations found in periplakin align with critical residues of FcγRI in yeast (Fig. 3). This part of periplakin also binds vimentin and is a conserved structure among plakin family members (36Karashima T. Watt F.M. J. Cell Sci. 2002; 115: 5027-5037Crossref PubMed Scopus (69) Google Scholar). However, periplakin residues 1687 and 1689 are exclusively found in periplakin and might facilitate selective binding of FcγRI to periplakin in immune cells. In addition, protein kinase B and the μ-opioid receptor can bind to the C terminus of periplakin (37Van den Heuvel A.P. de Vries-Smits A.M. Van Weeren P.C. Dijkers P.F. de Bruyn K.M. Riedl J.A. Burgering B.M. J. Cell Sci. 2002; 115: 3957-3966Crossref PubMed Scopus (53) Google Scholar, 38Feng G.J. Kellett E. Scorer C.A. Wilde J. White J.H. Milligan G. J. Biol. Chem. 2003; 278: 33400-33407Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). No clear sequence similarity exists between FcγRI and other periplakin-binding proteins. Thus far, it is not known whether other proteins beside FcγRI 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 FcγRI 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 FcγRI (data not shown, n = 3). COS cells express considerable levels of endogenous periplakin, and FcγRI monomeric IgG binding assays in these cells showed that FcγRI-CY lowered ligand affinity by ∼2-fold (50Miller K.L. Duchemin A.M. Anderson C.L. J. Exp. Med. 1996; 183: 2227-2233Crossref PubMed Scopus (69) Google Scholar), supporting the hypothesis that FcγRI-CY-periplakin binding decreases FcγRI interaction with monomeric IgG. This report describes FcγRI function to be enhanced by reagents that target the receptor intracellular tail. This might lead to the development of selective reagents that regulate FcγRI function for immunotherapy. FcγRI-directed immunotherapy may be enhanced by TAT-PPL, resulting in increased treatment efficacies when applied in combination." @default.
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• W2023004879 title "Modulation of FcγRI (CD64) Ligand Binding by Blocking Peptides of Periplakin" @default.
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