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Originally published online as doi:10.1189/jlb.0504305 on September 30, 2004

Published online before print September 30, 2004
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(Journal of Leukocyte Biology. 2005;77:80-89.)
© 2005 by Society for Leukocyte Biology

CD93 interacts with the PDZ domain-containing adaptor protein GIPC: implications in the modulation of phagocytosis

Suzanne S. Bohlson1, Mingyu Zhang, Christopher E. Ortiz and Andrea J. Tenner

Department of Molecular Biology and Biochemistry, University of California, Irvine

1 Correspondence: Department of Molecular Biology and Biochemistry, 2419 McGaugh Hall, University of California, Irvine, CA 92697. E-mail: sbohlson{at}uci.edu


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ABSTRACT
 
CD93 was originally identified as a myeloid cell-surface marker and subsequently associated with an ability to modulate phagocytosis of suboptimally opsonized immunoglobulin G and complement particles in vitro. Recent studies using mice deficient in CD93 have demonstrated that this molecule modulates phagocytosis of apoptotic cells in vivo. To investigate signal transduction mechanisms mediated by CD93, CD93 cytoplasmic tail (CYTO)-binding proteins were identified in a yeast two-hybrid screen. Fifteen of 34 positive clones contained a splice variant or a partial cDNA encoding GIPC, a PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein, shown previously to regulate cytoskeletal dynamics. A single clone of the N-terminal kinase-like protein p105 and an uncharacterized stem cell transcript also showed specificity for binding to the CYTO by yeast two-hybrid. Using the yeast two-hybrid system and an in vitro glutathione S-transferase fusion protein-binding assay, the binding of GIPC to the CYTO was shown to involve a newly identified class I PDZ-binding domain in the CD93 carboxyl terminus. Four positively charged amino acids in the juxtamembrane domain of CD93 were shown to be critical in stabilizing these interactions. Treatment of human monocytes with a cell-permeable peptide encoding the C-terminal 11 amino acids of CD93 resulted in an enhancement of phagocytosis, supporting the hypothesis that this protein-protein interaction domain is involved in the modulation of phagocytosis. These protein interactions may participate as molecular switches in modulating cellular phagocytic activity.

Key Words: complement • monocytes/macrophages • cell-surface molecules • adhesion molecules


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INTRODUCTION
 
Phagocytosis, or the cellular ingestion of large particles (>0.5 µm), is a critical first step in the initiation of an immune response, as well as a mechanism to maintain normal tissue homeostasis by the rapid clearance of apoptotic cells [1 ]. Phagocytosis is a complex biochemical process accompanied by the rapid redistribution of cellular membranes and cytoskeleton and is facilitated by numerous adaptor proteins. It is an actin-dependent mechanism that requires signal transduction processes similar to those required to mediate cellular adhesion and migration (for review, see ref. [2 ]), although specific biochemical pathways leading to uptake and processing of particles vary depending on the initial ligand-receptor interactions and microenvironment at the interface of the phagocytic cell and the particle. The ability to manipulate this process would be therapeutically beneficial in terms of host defense and tissue repair and remodeling.

A variety of ligands including fibronectin and P-selectin and a family of proteins including C1q, mannose-binding lectin (MBL), and surfactant protein A (SPA), designated defense collagens, have been shown to enhance phagocytosis of particles suboptimally opsonized with complement or antibody (for review, see ref. [3 ]), indicating complex regulation of this important function. In the process of characterizing this induced enhancement of phagocytic potential, a 126,000 Mr protein, now designated as CD93, was found to influence this activity. Although CD93 was initially characterized as a receptor or component of a receptor complex for soluble defense collagens [4 ], recent studies report that CD93 alone does not bind to C1q [5 ] and that CD93 is not required for the in vitro enhancement of phagocytosis by C1q [6 ], demonstrating that the mechanism of modulation of phagocytic activity by CD93 remains elusive. Anti-CD93 monoclonal antibodies (mAb) block the enhancement of phagocytosis mediated by defense collagens C1q, MBL, and SPA but not fibronectin [7 8 9 ]. Similarly, a polyclonal antibody generated against the last 11 amino acids in the cytoplasmic tail (C11; see Fig. 1A ) inhibits the C1q-mediated enhancement of phagocytosis when cells are transiently permeabilized in the presence of the antibody [10 , 11 ]. Furthermore, when immobilized on a surface, the immunoglobulin M (IgM) anti-CD93 mAb R3 enhances phagocytosis much like C1q [8 ], all consistent with the possibility that CD93 may be a negative regulator of phagocytosis. However, CD93-deficient mice were less competent at uptake of apoptotic cells injected intraperitoneally [6 ], and thus, the mechanism by which CD93 modulates phagocytosis remains elusive. These data do suggest that although CD93 is not required for the C1q-triggered enhancement of function, it may be part of a signaling complex that modulates the phagocytic process in vitro and in vivo.



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  		Figure 1. CD93 has a 47 amino acid cytoplasmic tail and modulates phagocytosis. (A) The cytoplasmic tail contains a highly charged JX, and charge (+ or –) is indicated below the single-letter amino acid code. Shown in bold are potential sites of serine or tyrosine phosphorylation as identified using the NetPhos 2.0 server (Center for Biological Sequence Analysis, Technical University of Denmark). C11 (underlined) has been implicated in signal transduction [10 , 11 ], and this region contains a class I PDZ-binding domain (italics) as detected using Motif Scanner (Division of Signal Transduction, Harvard Institutes of Medicine, Boston, MA). CRD, Carbohydrate recognition domain; EGF, epidermal growth factor; TM, transmembrane domain. (B) Monocytes were incubated on immobilized anti-CD93 (R3) or isotype control mouse IgM (30 ug/ml) for 30 min prior to incubation with sheep erythrocytes suboptimally opsonized with IgG (EAIgG) for an additional 30 min. Images (representative of multiple experiments here and as reported in ref. [5 ]) were captured using an Axiovert 200 inverted microscope (Carl Zeiss Light Microscopy, Göttingen, Germany) at 40x magnification with an AxioCam (Zeiss) digital camera controlled by the AxioVision program (Zeiss). In this particular experiment, the phagocytic index for R3 was 2.5-fold higher for cells adherent to anti-CD93 (R3, right) when compared with cells adherent to the isotype control antibody (IgM, left).

CD93 is selectively expressed on myeloid cells, endothelial cells, platelets, and stem cells [12 ] and thus, may be a candidate target for regulation of the phagocytic process or of adhesion or membrane mobility in these specialized cells. The extracellular domain contains regions that suggest a function in cell-cell interactions and/or adhesion (see Fig. 1A ); however, the ligand(s) for CD93 has yet to be identified. In addition, the mechanisms by which anti-CD93 antibodies modulate defense collagen-triggered enhancement of phagocytosis and by which ligation of CD93 with immobilized anti-CD93 antibodies triggers enhanced phagocytosis remain to be elucidated. We hypothesized that ligation of CD93 triggers an intracellular signaling cascade leading to the enhancement of phagocytosis and that the cytoplasmic tail of CD93 is involved in this signaling cascade. Therefore, we performed a yeast two-hybrid screen to identify potential CD93 cytoplasmic tail (CYTO)-binding proteins and used glutathione S-transferase (GST) fusion proteins containing the full-length and mutated CYTO to validate the identified candidate protein interactions and to map the critical interaction sites within the 47 amino acid cytoplasmic tail. The interaction of one of these proteins, an adaptor protein, G{alpha} interacting protein (GAIP) interacting protein C terminus (GIPC), which has been shown in other systems to regulate migration [13 , 14 ], required a previously unidentified CD93 class I PSD-95/Dlg/ZO-1 (PDZ)-binding domain contained within C11. It is interesting that four charged amino acids in the juxtamembrane region of CD93 were also shown to be necessary for mediating a stable interaction between GIPC and CD93. Human monocytes treated with a cell-permeable C11 peptide were more phagocytic than monocytes treated with a control peptide, suggesting that this domain within the CYTO may be critical in modulating functional activity.


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MATERIALS AND METHODS
 
Mice
CD93-deficient mice on a C57BL/6 background (n=6) were a generous gift from Drs. Marina Botto and Mark Walport (Imperial College, London). Mice were genotyped by polymerase chain reaction (PCR) as described [6 ], and in some experiments, genotyping was confirmed by Western blot (data not shown).

Reagents and antibodies
All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Tat-C11 (RKKRRQRRRNQYSPTPGTDC) and Tat-C11 reverse (RKKRRQRRRCDTGPTPSYQN) were purchased from Alpha Diagnostics International (San Antonio, TX). Peptides were dissolved in 1% acetic acid and brought to a final concentration of 2.5 mM in phagocytosis buffer [RPMI 1640 (Gibco-Invitrogen, Carlsbad, CA) with 10 mM Hepes, pH 7.4, 5 mM MgCl2, and 100 units/ml penicillin G sodium/100 ug/ml streptomycin sulfate (Gibco-Invitrogen)]. Peptides were aliquoted and stored at –70°C until used. Human serum albumin (HSA), distributed by FFF Enterprises (Temecula, CA), was a product of the American Red Cross.

Rabbit polyclonal anti-GIPC was kindly provided by Dr. Marilyn Gist-Farquhar (University of California, San Diego). Rabbit polyclonal anti-p105 was kindly provided by Dr. Gustav Lienhard (Dartmouth Medical School, Hanover, NH). A rabbit polyclonal antibody against the C11 was generated as described previously [11 ]. Control mouse IgM and anti-CD93 R3 (IgM) were purified as described previously [4 ]. Peroxidase-conjugated secondary antibodies [F(ab')2 fragments] and fluorescein isothiocyanate (FITC) anti-rabbit F(ab')2 were from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-mouse Alexa 647 was kindly provided by Dr. Elizabeth Head (University of California, Irvine).

Cell culture
Human peripheral blood monocytes were isolated by counterflow elutriation using a modification of the technique of Lionetti et al. [15 ] as described [16 ]. Human embryonic kidney (HEK)293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Invitrogen) with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin G sodium/100 ug/ml streptomycin sulfate (Gibco-Invitrogen), 1x nonessential amino acids (Gibco-Invitrogen), and 10 mM Hepes, pH 7.4. Human umbilical vein endothelial cells (HUVEC) were provided by Dr. Chris Hughes (University of California, Irvine) and cultured on 1% gelatin-coated flasks in RPMI (Gibco-Invitrogen) with 10% fetal calf serum (FCS), 100 units/ml penicillin G sodium/100 ug/ml streptomycin sulfate (Gibco-Invitrogen), 10 mM Hepes, pH 7.4, and 1% endothelial cell growth supplement (Sigma Chemical Co.). Mouse bone marrow (BM)-derived macrophages were isolated as described previously [17 ]. Briefly, BM was collected from adult femurs and following removal of adherent cells, cultured in DMEM 10% FCS with 10 mM Hepes, pH 7.4, 100 units/ml penicillin G sodium/100 ug/ml streptomycin sulfate, and supplemented with 20% L929 conditioned media (as a source of macrophage-colony stimulating factor) for 7 days prior to use in phagocytosis experiments. Yeast media including YPD, minimal SD base, and amino acid drop-out supplements were from Clontech (Palo Alto, CA). Bacto agar was from Difco (Sparks, MD).

Immunofluorescence
HUVEC were plated on sterile gelatin-coated coverslips at 5 x 104/well in HUVEC growth media to allow for adherence to the coverslip. Cells were washed once in cold RPMI and incubated with 5 ug/ml anti-CD93 R139 (or isotype control) for 45 min at 4°C. Following two 5-min washes with RPMI and two 5-min washes with phosphate-buffered saline (PBS), cells were incubated with Alexa 647 anti-mouse IgG for 30 min at 4°C. After washing, cells were fixed with 3.7% formaldehyde for 10 min and following washing, permeabilized with 0.1% Triton X-100. Cells were washed once with PBS and blocked for 1 h with 2% normal donkey serum (Jackson ImmunoResearch Laboratories) in PBS. Cells were stained with 5 ug/ml polyclonal anti-GIPC or control rabbit IgG for 1 h, washed three times with PBS, and stained with secondary FITC anti-rabbit (1:500) for 45 min. Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and analyzed by confocal microscopy (BioRad, Hercules, CA). Z serial section images (0.5 µm) were taken.

Phagocytosis assay
Phagocytosis assays were performed essentially as described previously [8 ]. Briefly, human monocytes were resuspended in phagocytosis buffer at 2.5 x 105 cells/ml and mouse macrophages at 1.25 x 105/ml. In some experiments, 30 µM tat-peptides were added to and gently mixed with cells. Cells (250 µl) were added immediately to protein-coated eight-well Lab Tek chamber slides (Nalgene, Naperville, IL), centrifuged at 70 g for 3 min, and allowed to adhere for 30 min at 37°C 5% CO2. Sheep erythrocytes, suboptimally opsonized with IgG, were used as targets and prepared as described previously [18 ]. Targets (107 in 100 µl) were added and after centrifuging at 70 g for 3 min, incubated for an additional 30 min at 37°C 5% CO2. Uningested targets were lysed, and cells were fixed in 1% glutaraldehyde in PBS. Cells were visualized using a modified Giemsa stain (Sigma Chemical Co.), and at least 200 cells/well were counted. Percent phagocytosis is the number of cells ingesting at least one target/total number of cells scored x 100. Phagocytic index is the number of ingested targets per 100 cells counted. Paired student’s t-test, two-tailed, was performed to assess statistical significance.

Yeast two-hybrid screening
The yeast two-hybrid screening was performed essentially as described [19 ]. The yeast strain Y190 and the bait plasmid pAS1-CYH2 and all irrelevant bait plasmids were a generous gift from Dr. Marian Waterman (University of California, Irvine). The human CTYO was amplified by PCR from the full-length cDNA in pcDNA3.1 [8 ] using the following primers: 5'-CGCGGATCCTGTATCGCAAGCGGAGAGCGAAG and 3'-TGCGGTCGACCTCACTTTCAGCAGTCTGTCC. The PCR product was subcloned into pGEM-T (Promega, Madison, WI) and sequenced (University of California, Irvine, DNA core facility), and the BamHI-SalI fragment containing the CYTO was cloned into pAS1-CYH2 3' of the GAL4 DNA-binding domain (pAS1-CYH2-cyto). Y190 was transformed with pAS1-CYH2-cyto using the Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, CA). Y190, expressing the CYTO, was transformed with CsCl2-banded DNA generated from one round of amplification of the human peripheral blood leukocyte MATCHMAKER cDNA library in pACT2 (Clontech, Palo Alto, CA). The transformation protocol followed was from Clontech (MATCHMAKER GAL4 Two-Hybrid System 3 and Libraries User Manual PT3241-1) for large scale (sequential). pACT2 plasmids from LacZ-positive clones growing on –His/–Leu/–Trp plus 25 mM 3-amino-1,2,4-triazole were isolated by growth on –Leu followed by growth on –Leu plus 2.5 ug/ml cyclohexamide to eliminate the pAS1 plasmid, and DNA was isolated using the YEASTMAKER yeast plasmid isolation kit (Clontech). Electrocompetent DH10S Escherichia coli were transformed with DNA recovered from the yeast, and the DNA was recovered from E. coli and sequenced using the pACT forward 5' primer described previously [19 ]. Sequence analyses were performed by online BLAST searches. To generate the GIPC-PDZ domain construct, the PDZ domain was amplified by PCR using a GIPC N-terminal-deleted splice variant in pACT2 isolated in the yeast two-hybrid screen as a template and the pACT forward 5' primer and 3'-CTCGAGTCACTTCAGCGTGAAGGTAC. The PCR product was subcloned into pGEM and ligated into pACT2 using EcoRI and XhoI. Irrelevant baits encoding functional Gal4 DNA-binding domain fusion proteins were the kind gift of Dr. Marian Waterman, and several are described in ref. [20 ].

ß-Galactosidase assays
Filter lift assays were performed as described [19 ]. Briefly, yeast colonies were absorbed onto 0.45 µm nitrocellulose filters (Fisher Scientific, Pittsburgh, PA) and lysed by immersion in liquid nitrogen. Yeast were incubated for 1 h in Z buffer (113 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0) and 38.6 mM ß-mercaptoethanol, pH 7.0, with 1 mg/ml X-Gal and were analyzed for color change every 15 min. To quantitate ß-galactosidase activity using o-nitrophenyl ß-D-galactopyranoside as a substrate, transformed yeast were grown to log phase and assayed according to the protocol from Clontech (Yeast Protocols Handbook PT3024-1). Samples were processed in duplicate or triplicate.

Cell lysis
HEK293T cells, which do not express endogenous CD93 (data not shown), were lysed in 1% octyl-ß-D-glucopyranoside (Calbiochem, La Jolla, CA), 100 mM NaCl, 10 mM triethanolamine, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 1 µg/ml pepstatin for 1 h on ice. Lysates were centrifuged at 4°C, 14,000 rpm, for 15 min to remove insoluble material.

Western blot
Proteins were resolved by 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) for 2–3 h at 300 mAmps. Blots were blocked overnight with 3–5% milk in Tris-buffered saline-Tween 20 (TBST; 50 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween) at 4°C. Blots were incubated with primary antibodies for 2 h at room temperature, washed three times for 5 min each in TBST, incubated with secondary antibodies for 1 h, washed three times for 15 min each, and developed using enhanced chemiluminescence (Amersham, Piscataway, NJ).

GST pull-down assay
An equal amount of HEK293T cell lysate was incubated overnight or 2 h at 4°C with GST or a GST-CYTO fusion protein immobilized on glutathione beads. Bound proteins were eluted with 2.1 mg/ml glutathione in 50 mM Tris, pH 8.0, for 2 h at 4°C, resolved by 8% or 10% SDS-PAGE, transferred to PVDF, and probed with rabbit anti-GIPC or anti-p105. Band densities were quantified using National Institutes of Health (NIH; Bethesda, MD) Image software (rsb.info.nih.gov/nih-image/download.html) and normalized to GST fusion protein loading. Generation of GST-CYTO, CYTO-C11, 4ala-C11, and CYTO-juxtamembrane domain (JX) is described elsewhere (M. Zhang, S. S. Bohlson, Marisela Dy, and A. J. Tenner, manuscript in preparation). To generate the 4ala mutant, CD93 in pCDNA3.1 was used as a template, and the following primers were used to amplify the cytoplasmic tail with a four alanine substitution in the juxtamembrane region: 5'-TATGCGGCCGCGGCAGCGAAGAGGGAGGAGAAG-3' and 5'-CTAGAAGGCACAGTCGAGGCTG-3'. The PCR product was digested with NotI and subcloned into pcDNA3.1 digested with EcoRV and NotI. The product was removed from pcDNA3.1 by EcoRI/NotI and cloned into pGEX-4T using the same sites and sequenced (UCI DNA core facility).


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RESULTS
 
GIPC, BM046, and p105 are CYTO-binding proteins
Ligation of CD93 with immobilized antibodies triggers enhanced phagocytosis (Fig. 1B and ref. [8 ]), and polyclonal antibodies directed at the cytoplasmic tail inhibit enhanced phagocytosis [10 , 11 ]. Therefore, these data suggest that the cytoplasmic tail of CD93 is involved in the modulation of phagocytic activity. To explore the mechanism by which the cytoplasmic tail of CD93 could modulate phagocytosis, a yeast two-hybrid screen of a peripheral blood leukocyte cDNA library was performed to identify candidate CYTO-binding proteins. Five independent screens of over 5 million clones yielded 34 positive clones as assessed by growth on selective media (-tryptophan, -leucine, -histidine with 25 mM 3-aminotriazole) and presence of ß-galactosidase activity as determined by colony filter lift assay (Table 1A ). The positive clones were sequenced and subsequently analyzed by BLAST (National Center for Biotechnology Information, National Library of Medicine, NIH). Clones containing proper reading frames were identified as potential interactors and further screened to verify the ability to activate yeast reporter genes in the presence of bait but not in the absence of bait. Four positive clones from this screen (each encoding a distinct gene product) were tested for ß-galactosidase activity against six irrelevant baits as a specificity control. Three of the four clones encoding GIPC, BM046, and p105 displayed specificity for the CYTO, whereas one clone (encoding RanBP) did not (Table 1B) . RanBP was not further characterized.


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  		Table 1. Yeast Two-Hybrid Screen Identifies GIPC, BM046, and p105 As Specifically Binding the CD93 CYTO

Sequence analysis of the 34 positive clones recovered from the yeast screen identified 15 as encoding a splice variant [14 ] or partial cDNA of GIPC, a 333 amino acid protein containing a single, central PDZ domain and a C-terminal acyl carrier protein (ACP) domain [21 ] (Fig. 2A ). GIPC has been shown in previous studies to interact with the PDZ-binding domains of a variety of transmembrane proteins (for review, see ref. [22 ]). A single, partial cDNA was identified as BM046, a CD34+ stem cell transcript [23 ] of interest, as CD93 is also expressed on this stem cell population [24 ]. BM046 is homologous to the Rho-GAP ARGHAP9 [25 ] as a result of the presence of a central PH domain (Fig. 2A) . An additional clone containing a partial cDNA of p105, a protein with an N-terminal serine kinase-like domain, also reacted positively and specifically with the CYTO in the two-hybrid screen. The N-terminus of p105 has homology to a serine kinase domain. However, the amino acid residues within the expected active site suggest that the protein has no actual kinase activity, perhaps serving as a decoy serine kinase [26 ]. This finding is of interest, as multiple potential sites for serine phosphorylation within the CYTO have been identified using NetPhos 2.0 (Fig. 1A , bold). Western blot analysis determined that GIPC and p105 are expressed in monocytes and HUVEC, cells that express high levels of CD93 (Fig. 2B) . These proteins are also found in other cell types that do not express CD93, such as lymphocytes (Fig. 2B) . Analysis of cellular localization of CD93 and GIPC was performed in HUVEC cells by confocal microscopy. CD93 was localized to the membrane, whereas GIPC was distributed on the membrane, cytoplasm, and nucleus. CD93 and GIPC colocalized on the cell membrane (Fig. 2C) .



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  		Figure 2. CYTO-binding proteins. (A) Schematic of CD93-binding proteins identified by yeast two-hybrid. Portion of cDNA in isolated clones is indicated by underline, and the number of clones is in parentheses. PH, Plextrin homology domain. (B) Western blot analysis demonstrates GIPC and p105 expression in cell types that express high levels of CD93. Cell lysate (10 µg) from peripheral blood monocytes, lymphocytes, or HUVEC was separated by 10% SDS-PAGE and transferred to PVDF. The blot was probed with rabbit anti-p105 antibody, rabbit anti-GIPC antibody, or rabbit anti-CD93 C11. (C) HUVEC were double-labeled with anti-CD93 (R139, red) and anti-GIPC (polyclonal, green) and analyzed by confocal microscopy. Single images demonstrate cellular localization of the respective proteins, and a merged image demonstrates colocalization (yellow) in the 0.5-µm section.

Deletion of the CD93 class I PDZ-binding domain eliminates the interaction between GIPC and CD93; however, the GIPC-PDZ domain is not sufficient to mediate this interaction
The class I family of PDZ-binding domains has been described as preferring the C-terminal sequence Thr/Ser-X-hydrophobic. Deletion of the coding sequences for the C-terminal four amino acids of CD93 (GTDC) in the yeast two-hybrid bait plasmid eliminated the interaction of this construct with GIPC in yeast as quantified by ß-galactosidase activity (Fig. 3A ). The lack of ß-galactosidase activity was not a result of a lack of protein expression, as bait (CYTO or CYTO-PDZ) and prey (GIPC) plasmids were well expressed in yeast as demonstrated by Western blot analysis (data not shown). In contrast, deletion of the CD93 class I PDZ-binding domain did not eliminate the interaction between CD93 and p105 or BM046, which was expected, as these proteins do not contain PDZ domains (Fig. 3B) . It is interesting that the interaction between GIPC and CD93 is likely stronger in yeast compared with the interaction with p105 or BM046, as ß-galactosidase activity was substantially higher in cells transformed with GIPC. Taken together, these data suggest that the interaction between GIPC and CD93 is mediated via the PDZ-binding domain of CD93 binding to the PDZ domain of GIPC. However, expression of the GIPC-PDZ domain alone was not sufficient to mediate the interaction between GIPC and CD93, as a prey plasmid containing the PDZ domain of GIPC (i.e., lacking the N- and C-terminal domains, Fig. 2A ) did not induce ß-galactosidase activity in the presence of the full-length CYTO (Fig. 3C) . Protein expression (bait and prey) was confirmed by Western blot (data not shown). The GIPC-PDZ domain has been shown to be sufficient for mediating interactions with other class I PDZ binding domain-containing proteins, including, for example, GAIP [21 ] and the insulin growth factor (IGF)-1 receptor [14 ]. These data suggest that regions other than the PDZ-binding domain of CD93 stabilize the interaction between CD93 and GIPC.



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  		Figure 3. The PDZ-binding domain of CD93 is required for GIPC binding, but the PDZ domain of GIPC is not sufficient to mediate this interaction. (A) ß-Galactosidase activity (quantified as described in Materials and Methods) in yeast transformed with the full-length cytoplasmic tail bait construct (CYTO, solid bars) or a bait construct lacking the PDZ-binding domain (CYTO-PDZ, open bars) with GIPC. Bars represent an average plus standard deviation of triplicates and are representative of four similar experiments. Background ß-galactosidase activity (from baits in the absence of prey) was minimal (<4%) and subtracted from the values shown. (B) p105 and BM046 interact with the full-length cytoplasmic tail bait construct and the PDZ-binding domain deletion mutant. Bars represent the average of triplicates plus standard deviation. Data are representative of two similar experiments. (C) A GIPC clone lacking N- and C-termini in the yeast prey plasmid pACT2, GIPC-PDZ, was tested along with the parent clone (a splice variant of GIPC identified by the yeast two-hybrid assay) for the ability to activate ß-galactosidase by colony filter lift in the presence (or absence) of the CYTO bait construct (n=2). The yeast transformants were scored as described in Table 1 .

GIPC binding to the CYTO requires the positively charged JX of CD93
To confirm the binding data generated by yeast two-hybrid as well as to map the sites in the CYTO required for interactions with the above-identified proteins, a series of GST fusion proteins was constructed (Fig. 4A ). GST fusion protein pull-down experiments were performed using extracts from HEK293T cells. An interaction between GIPC and a GST fusion protein encoding the CYTO but not with GST alone was detected (Fig. 4B) . This specific interaction was reproducible using a variety of ionic strength conditions (0, 10, 40, 150 mM NaCl, data not shown) and following overnight (Fig. 4B) and 2-h (data not shown) incubations. Deletion of C11 from the cytoplasmic tail decreased the interaction between GIPC and CD93-CYTO (Fig. 4B) ; however, it was not completely ablated, as was seen in the yeast two-hybrid assay (Fig. 3A) , which likely results from different binding conditions in the yeast system versus the mammalian cell lysate system. However, the decreased binding is consistent with the hypothesis that the C11 domain is necessary for optimal GIPC binding. It is interesting that deletion of the highly charged juxtamembrane region of CD93 also decreased the interaction with GIPC (CYTO-JX, Fig. 4B ). To further define the residues in the JX region responsible for stabilizing the interaction with GIPC, four charged residues in the JX (RKRR) were changed to alanine (4ala, Fig. 4A ). This mutant also showed diminished binding to GIPC in the GST pull-down assay, showing that this region is important in mediating the interaction. It is important that deletion of C11 in combination with substitution of four charged residues in the juxtamembrane region with alanine (4ala-C11) completely eliminated the interaction with GIPC (Fig. 4B) . These data suggest that the class I PDZ-binding domain found within C11 and the highly charged juxtamembrane region of CD93 contribute to GIPC binding to the cytoplasmic tail.



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  		Figure 4. Mapping the site in the CYTO required for interacting with GIPC. (A) A panel of GST fusion proteins containing the CYTO or specific mutants as noted was constructed. (B) Lysates of HEK293T cells (in 100 mM NaCl, 1% octylglucoside, as described in Materials and Methods), transiently transfected with flag-tagged mouse GIPC, were used in GST pull-down assays. Bound proteins were eluted with glutathione and subjected to SDS-PAGE and Western blot analysis. Rabbit anti-GIPC was used to detect GIPC bound to different GST fusion proteins. Blots were scanned and analyzed using NIH Image software. Bars represent average plus standard deviation of band density normalized to total GST fusion protein loading from two separate experiments. A blot from a single experiment is shown below the histogram.

Similar experiments were performed with endogenous p105, another protein identified in the yeast two-hybrid interaction screen. As predicted from the observations in the yeast system, the interaction between CD93 and p105 was maintained in the absence of the CD93 PDZ-binding domain (CYTO-C11). However, this interaction was completely dependent on the highly charged JX. No interaction was detected with the most minimal mutation in this region (4ala; Fig. 5 ). Analysis of interactions with BM046 in this system was not possible as a result of lack of available BM046 antibodies.



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  		Figure 5. Mapping the site in the CYTO required for interacting with endogenous p105. The GST pull-down assays were performed as described in Figure 4 . Blots were probed with anti-p105 (1 ug/ml) and analyzed as described in Figure 4 (n=2). A blot from one representative experiment is shown.

A cell-permeable peptide encoding C11 modulates monocyte phagocytosis
Previous studies showed that mouse macrophages [10 ] and rat microglia [11 ], transiently permeabilized in the presence of an anti-C11 antibody, lost the ability to respond to C1q with an enhancement of phagocytosis of suboptimally opsonized particles. These data suggested that the cytoplasmic tail and particularly, the C11 region (Fig. 1) were involved in modulating the enhancement of phagocytosis. Data presented here identifying the class I PDZ-binding domain within C11 further support a role for this region of the cytoplasmic tail in the regulation of phagocytic function, as this region of the tail is capable of facilitating interactions with PDZ domain-containing proteins. Therefore, a peptide encoding the cell-permeable region of the human immunodeficiency virus tat protein [27 ] followed by C11 was tested for its effect on phagocytosis. Although the control peptide (tat-C11-reverse) triggered some increase in phagocytic ability (two- to fourfold), it was not statistically significant when compared with the no-peptide control (P>0.05). Cells treated with tat-C11 were significantly more phagocytic than control cells (no peptide or tat-C11 reverse-treated) in three separate experiments when assessed as the percent of monocytes engaged in phagocytic activity (Fig. 6A , % phagocytosis) or as the number of targets ingested per phagocyte (Fig. 6B , phagocytic index). These results support the hypothesis that C11 is involved in the modulation of phagocytosis. It is interesting that this enhanced phagocytosis with the C11 peptide was also demonstrated with CD93-deficient BM macrophages (Fig. 6C and 6D) , showing that the C11 peptide is functioning independent of endogenous CD93.



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  		Figure 6. Modulation of phagocytic activity by CD93 C11. Human monocytes (A and B) or CD93–/– murine BM-derived macrophages (C and D) were incubated on HSA-coated (4 ug/ml) slides in the presence of 30 µM tat-C11, tat-C11-reverse, or no peptide, as described in Materials and Methods. Cells were further incubated with EAIgG for 30 min and following lysis of extracellular EAIgG, stained with modified Giemsa. (A and C) Percent phagocytosis is the number of phagocytes ingesting at least one EAIgG/total number of phagocytes (at least 200 per well) x 100. (B and D) Phagocytic index is the number of ingested EAIgG per 100 cells. (A and B) Bars represent the fold increase in percent phagocytosis or phagocytic index over the no-peptide control (plus standard deviation) from three separate experiments, each performed in duplicate. *, P= 0.05 (paired students t-test, two-tailed), comparing tat-C11 to tat-C11-reverse. (C and D) Values are the average plus standard deviation from two separate experiments, performed in duplicate. *, P = 0.043; **, P = 0.028.


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DISCUSSION
 
In this study, a class I PDZ-binding domain within the CD93 C11 domain (Fig. 1) was identified, and the PDZ domain-containing protein GIPC was shown to be a candidate CD93-interacting protein by the yeast two-hybrid interaction screen. In addition, this study demonstrates that the C11 domain and the highly charged JX of CD93 are required to mediate an effective interaction between CD93 and GIPC in mammalian cell lysates (Fig. 4) . Delivery of a cell-permeable C11 peptide triggered enhanced phagocytic activity in human monocytes and BM-derived macrophages (from wild-type and CD93–/– mice) in response to suboptimally opsonized targets (Fig. 6) , suggesting that this C11 motif is a functionally important domain. Previous studies had shown that blocking C11 with antibody in live cells inhibits phagocytosis [10 , 11 ]. These data suggest that upon stimulation, the C11 domain of the CYTO plays a critical role in the modulation of phagocyte activity by mediating protein-protein interactions, perhaps with a protein containing a PDZ domain.

To determine a mechanism by which the cytoplasmic tail of CD93 regulated phagocytosis, CYTO-binding proteins were identified using a yeast two-hybrid screen. GIPC, the predominant interactor identified in the screen, required the class I PDZ-binding domain located at the very C terminus of CD93 for optimal binding. Songyang et al. [28 ] described the class I PDZ-binding domain using an oriented peptide library approach to investigate binding specificities of multiple PDZ domains. Although the –2 position in CD93 (Thr) conforms to the preferred class I PDZ-binding domain specificity, the 0 position in CD93 is a polar residue (cysteine). Recently, Tani and Mercurio [29 ] showed that GIPC could interact with the integrin {alpha}6B subunit, which contains a polar serine residue at position 0. GIPC has also been shown to bind to the IGF-1 receptor [14 ], which similar to CD93, contains a cysteine at position 0. It is interesting that GIPC binding to the CYTO also required a four amino acid-charged sequence in the juxtamembrane region of the CYTO for optimal binding. GIPC has been shown to interact with other molecules, including myosin VI [30 ] and TrkA [31 ], independently of the GIPC-PDZ domain. Our data suggest that the interaction between GIPC and CD93 requires the PDZ-binding domain as well as the C-terminal ACP domain of GIPC and not the N-terminus of GIPC (as N-terminal-deleted splice variants were isolated in the yeast two-hybrid screen).

Although specific GIPC binding to the CYTO was readily demonstrated in two independent systems (yeast two-hybrid and GST pull-down assays), and GIPC and CD93 were colocalized in the plasma membrane, an interaction between GIPC and CD93 in resting cells could not be demonstrated by coimmunoprecipitation under conditions that did coimmunoprecipitate GIPC with the IGF-1 receptor (data not shown). This would be consistent with a transient or induced interaction between GIPC and CD93 in cells. It is interesting that a CD93 homologue, endosialin, also contains a C-terminal class I PDZ-binding domain [32 ]. In addition, the human CD93 C11 domain (containing the class I PDZ-binding domain) is completely conserved in mouse and rat [10 , 33 ], all consistent with the hypothesis that this is a functionally important domain. The cytoplasmic tail of CD93 is similar to the cytoplasmic tail of CD44, a highly glycosylated adhesion molecule well-characterized for its involvement in the modulation of inflammation (for review, see ref. [34 ]). Notably, anti-CD44 antibodies have demonstrated a role for CD44 in a variety of inflammatory disease states; however, the knockout mouse displays a surprisingly mild phenotype [35 ] similar to the moderately affected, CD93-deficient mouse [6 ]. Also, anti-CD44 antibodies stimulate an enhancement in uptake of apoptotic cells [36 , 37 ]. It is interesting that the CD44 and CYTO contain highly charged juxtamembrane regions characteristic of binding ezrin, radixin, and moesin (ERM) family members, proteins that link transmembrane receptors to the actin cytoskeleton via a direct interaction with actin [38 ]. Indeed we have demonstrated that this region of the CYTO is also capable of mediating interactions with ERM family members (M. Zhang, S. S. Bohlson, Marisela Dy, and A. J. Tenner, manuscript in preparation). Furthermore, the cytoplasmic tails of CD44 and CD93 contain no intrinsic enzymatic activity but do contain C-terminal PDZ-binding domains. The importance of the coordination between the JX and PDZ-binding domain of these molecules in modulating their adhesive/phagocytic functions remains to be determined.

The tissue-expression pattern of CD93 (endothelial cells, peripheral blood monocytes, neutrophils, stem cells, and platelets) as well as the extracellular domain architecture suggest that CD93 is an adhesion molecule (Fig. 1 and refs. [4 , 12 , 39 ]). Recently, McGreal et al. [5] described an adhesive function for CD93 by demonstrating that the CRD of CD93 was recognized by the mNI-11 antibody. When CD93 is ligated in fluid phase with mNI-11, lipopolysaccharide-stimulated U937 cells (monocyte-like) undergo homotypic aggregation, and this aggregation is inhibited with antibodies to lymphocyte function-associated antigen-1 or intercellular adhesion molecule-1 [40 ]. Furthermore, immobilized mNI-11 promoted rapid spreading of HUVEC, and this was completely inhibited by cytochalasin D (indicating involvement of the actin cytoskeleton) [41 ]. Adhesion molecules and particularly integrins have been shown to be required for optimal phagocyte activity, even in the absence of ligand [42 ]. Therefore, it is possible that the modulation of phagocytosis observed with CD93 may reflect a more general adhesive function of this molecule.

GIPC has been shown to interact with itself [30 ] and the cytoskeleton-associated proteins myosin VI, {alpha}-actinin, and the kinesin family member KIF-1B [30 ]. It is interesting that myosin VI is the only isoform of myosin that moves toward the pointed ends of actin filaments, as would be required for moving vesicles away from the plasma membrane during an endocytic process, and myosin VI has been implicated in the regulation of endocytosis [43 , 44 ]. GIPC has also been shown to localize to the endocytic compartment of proximal tubule epithelium and to associate with megalin, the most abundant endocytic receptor in this type of epithelial cell [45 ], all suggesting a role for GIPC in the regulation of endocytosis. Another GIPC-interacting protein, {alpha}-actinin, has been shown to be associated with the phagocytic cup [46 ]; therefore, it will be important to determine where GIPC and GIPC-associated proteins localize during phagocytosis. It is interesting that overexpression of wild-type GIPC has been shown to inhibit migration of ECV cells in a wounding assay [13 ] and in a separate study, to inhibit migration of MCF-7 cells in response to serum [14 ]. These studies are of particular interest, as they suggest a role for GIPC in the regulation of migration, a process similar to phagocytosis and requiring dynamic reorganization of membrane and cytoskeleton. Future studies should determine if GIPC is involved in CD93-mediated modulation of adhesion/phagocytosis and elucidate the mechanism by which CD93 mediates its effect.


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ACKNOWLEDGEMENTS
 
This work was supported by NIH Grants AI-41090 and 5T32 CA 09054. The authors thank Donald Chung, Xiomara Fernandez, Mallary Greenlee, and Krissy Yamamoto for technical assistance, Drs. Marina Botto and Mark Walport for CD93-deficient mice, Dr. Marian Waterman for yeast two-hybrid reagents and guidance, Dr. Marilyn Gist-Farquhar (University of California, San Diego) for the anti-GIPC polyclonal antibody, Dr. Gustav Lienhard (Dartmouth Medical School) for the anti-p105 antibody, Dr. Chris Hughes for HUVEC, and Marisela Dy and Richard Silva for assistance in generating GST fusion proteins.

Received May 25, 2004; revised July 28, 2004; accepted September 6, 2004.


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