Fig. 7. Stimulated adhesion of Jurkat E6 cells or human peripheral blood lymphocytes to ICAM-1 is abrogated by the guanine nucleotide exchange function-defective mutant cytohesin-1(E157K). Cytoplasmic Ig fusion constructs were expressed by recombinant vaccinia viruses in the respective cells, and the adhesion assay was performed as described in Materials and methods. (A) Adhesion of Jurkat E6 cells. (B) Adhesion of peripheral blood lymphocytes. (C) Immunoblot analysis showing expression of selected Ig fusions used in this experiment.

Fig. 2. Co-localization of cytohesin-1 with CD18 in the human lymphoblastoid cell line 721-LCL. 721-LCL cells were incubated with either anti-human CD18 mouse monoclonal antibody KIM185 (B) or anti-human CD29-antibody 4B7R (C) at 1 µg/ml for 10 min, followed by cross-linking with secondary goat anti-mouse Texas red-conjugated F(ab′)₂ fragment at 37°C. Cells were then plated on poly-l-lysine-coated slides as described in Materials and methods. For staining of endogenous cytohesin-1, cells subsequently were permeabilized and incubated with rat hybridoma supernatants containing the anti-cytohesin mAb. Detection of the rat immunoglobulin was performed with an FITC-conjugated anti-rat secondary reagent. Anti-cytohesin-1 staining is specific, because the isotype-matched rat control antibody CAD9 did not yield detectable fluorescence (A). Cross-species reactivity of the secondary reagents was not detectable (not shown).

Fig. 1. Specificity of anti-cytohesin-1 monoclonal antibody mAb7H2. (A) To demonstrate the specificity of mAb7H2, Ig fusions of human cytohesin-1 and human ARNO (cytohesin-2) were precipitated on protein A–Sepharose. In the subsequent immunoblot analysis, cytohesin-1, but not ARNO, is detected by mAb7H2. (B) Cytohesin-1 was precipitated from various human cell lines by mAb7H2, and protein expression was monitored by immunoblot analysis: the protein is present in U266 cells (lymphoblastoid myeloma line), 721-LCL cells (Burkitt lymphoma), Jurkat E6 cells (T-cell leukemia) and at reduced levels in HeLa cells (cervical carcinoma line). Hardly any, or no cytohesin-1 expression is found in HT29 cells (adenocarcinoma line) or in CV-1 cells (African green monkey kidney line). The upper band corresponds to the heavy chain of the monoclonal antibody.

Fig. 8. Cytohesin-1 induces spreading of Jurkat E6 cells on ICAM-1. At 8 h post-infection with recombinant vaccinia viruses, Jurkat E6 cells were plated on ICAM-1 for 20 min; non-adherent cells were washed off with HBSS. After fixation of adherent cells, phase-contrast micrographs (first panel, A1, B1, C1, D1 and E1) were taken. For subsequent laser-scanning microscopical analysis (second panel, A2, B2, C2, D2 and E2), Ig fusion proteins were stained with an FITC-labeled anti-Ig antibody. Expression of wild-type Ig–cytohesin-1 (C1 and C2) causes strong spreading of cells on ICAM-1. In contrast, cells do not spread when they express cytohesin-1(E157K) (D1 and D2), Ig–PHc (E1 and E2) or a control construct (B1 and B2), or if they are not infected (A1 and A2). In all these cases, cells retain a spherical shape, and thus do not spread on ICAM-1. The spreading index (F) of 50 cells per field was quantified as described in Materials and methods.

Fig. 6. Both cytohesin-1 and the guanine nucleotide exchange function-defective mutant cytohesin-1(E157K) induce the mAb24 epitope of LFA-1 in Jurkat cells. Half-maximal binding of the cells to soluble ICAM-1–Fc chimeric protein is not affected by overexpression of cytohesin-1, or mutant derivatives thereof. (A) Cytoplasmic Ig fusion proteins (as indicated) were expressed in Jurkat cells by recombinant vaccinia viruses, and the cells subsequently were fixed and permeabilized. Cytoplasmic expression of the chimeras is detected by an FITC-labeled, anti-human IgG antibody. The ‘control’ sample corresponds to uninfected cells. (B) mAb24 staining of aliquots from the cells shown in Figure 5A. Cells were incubated with 5 µg/ml mAb24 in HBSS in the presence of 2 mM EGTA. (C) Soluble ligand binding is not affected by cytohesin-1. The assay was carried out as described in Materials and methods. The input ICAM-1–Fc protein concentration is plotted versus the percentage of positive cells detected by flow cytometry.

Fig. 3. Mapping of the cytohesin-1-binding site within the cytoplasmic domain of CD18, using the yeast two-hybrid system. Deletion mutants of the CD18 cytoplasmic domain and control constructs were expressed as fusion proteins with the LexA DNA-binding domain (bait) and co-expressed with fusion proteins comprising the Sec7 domain of cytohesin-1 in the context of the B42 transactivation domain (prey). Deletion analysis shows that nine membrane-proximal amino acids of the CD18 cytoplasmic tail, including amino acids 723–731, retained the ability to interact with cytohesin-1 (++) compared with the strong interaction with the full-length cytoplasmic tail of CD18 (+++). In contrast, a triple mutation (WKA723–725TRG) of the N-terminal three amino acids of the CD18 cytoplasmic domain completely abrogates the interaction with cytohesin-1. No interaction (–) was detected with the LexA protein alone, or with the CD29 and β-7 integrin cytoplasmic domain fusion proteins. Interaction studies were performed in the yeast strain EGY48/JK103 and monitored by the generation of blue dye as a result of the interaction-dependent lacZ gene activity in yeast cells grown on medium containing X-gal and galactose. The strength of interaction was scored as (–) negative, positive (++) and strongly positive (+++).

Fig. 4. The sIg–CD18_cyt wild-type fusion protein, but not the sIg–CD18_tm/cyt-WKA723–725TRG mutant, binds to cytohesin-1 in vitro. Biochemical evaluation of an interaction between CD18 and cytohesin-1 was performed by an in vitro pull-down assay. The experiment employed single chain fusion proteins, expressed in COS cells, and purified His₆-cytohesin-1 from E.coli. (A) Schematic outline of the CD18 surface immunoglobulin fusion proteins used in this study (upper panel) and schematic of the pull-down experiment (lower panel). Surface immunoglobulin (sIg) chimeras comprised the CH2 and CH3 domains from human immunoglobulin G1, fused to the transmembrane and cytoplasmic domains of CD18. Surface expression was directed by the leader sequence of CD5 (the sketched domain sizes are not proportional to the actual amino acid sequences). (B) For pull-down of CD18 fusion proteins, 96-well microtiter plates were coated with the monoclonal antibody mAb7H2 to immobilize polyhistidine-tagged cytohesin-1 (lanes 1, 2 and 3) or cytohesin-1E157K (lanes 4 and 5), respectively. Subsequently, wells were incubated with lysates from COS cells expressing (lane 1) a surface Ig-control chimera bearing the transmembrane domain of CD7, (lanes 2 and 4) wild-type sIg–CD18_tm/cyt and (lanes 3 and 5) sIg–CD18_tm/cyt-WKA723–725TRG. Precipitated sIg proteins were collected and monitored by immunoblot analysis. Specific interaction of cytohesin-1 and cytohesin-1E157K with CD18 was confirmed by detection of the precipitated sIg–CD18_tm/cyt-wild-type fusion protein (lanes 2 and 4, upper left panel) with an anti-human IgG antibody. Neither sIg control protein (lane 1) nor the triple mutant of CD18, sIg–CD18_tm/cyt-WKA723–725TRG (lanes 3 and 5) revealed significant binding to recombinant cytohesin-1. A control was also performed with the mAb7H2 isotype-specific antibody CAD9. The amount of antibody-captured His₆-cytohesin-1 contained within each sample (middle panel) and expression of the sIg fusion proteins (lower panel) was verified by immunoblotting with mAb7H2 and anti-human IgG antibody, respectively.

Fig. 5. The adhesive capacity of the CD18(WKA723–725TRG) mutant is activated neither by overexpression of cytohesin-1 nor by stimulation through TCR- or phorbol ester-mediated signal transduction events. Assays were performed with HeLa cells expressing vaccinia virus-derived recombinant LFA-1 molecules, and with HeLa cells stably expressing LFA-1 (A). Additional adhesions were performed with hematopoietic, β-2 chain-deficient cell lines (B and C). (A) Upper left panel: Western blot analysis of recombinant vaccinia virus-derived CD18 and CD11a proteins. Wild-type α chain CD11a, wild-type CD18 or the mutant CD18(WKA723–725TRG) were expressed with recombinant vaccinia virus. Expression of CD18 is detected as a cluster of bands migrating between 100 and 120 kDa with the monoclonal antibody MEM48 under non-reducing conditions. CD11a was detected with the anti-CD11a antibody MHM24 (Dako) at 180 kDa. As a control, cells have been infected with a cytoplasmic Ig control protein. Upper right panel: an intracellular Ig–cytohesin-1 fusion protein is expressed in HeLa cells with the help of recombinant vaccinia viruses. The fusion protein was detected by flow cytometric analysis following fixation and permeabilization of the cells. Lower right panel: HeLa cells stably expressing either wild-type LFA-1 or the TRG triple mutant were established, and cell surface expression was measured by flow cytometry using the antibodies MEM48 (CD18) and MHM24 (CD11a). Lower left panel: adhesion assays with HeLa cells stably expressing wild-type and mutant versions of LFA-1 were performed. Overexpression of Ig–cytohesin-1, but not of the control construct, increases adhesion of LFA-1 wild-type-positive cells, but not of LFA-1 TRG mutant-positive cells. (B) Right panel: expression of either wild-type or mutant β-2 chain rescues cell surface expression of LFA-1 in β-2-deficient SKβ2.7 cells. Left panel: adhesion assay. Wild-type LFA-1, but not the CD18(WKA723–725TRG) mutant, responds to PMA stimulation in SKβ2.7 cells (gray bars). However, both receptors respond well to Mn²⁺ or to the activating anti-LFA-1 antibody MEM-48 (hatched or solid bars, respectively). (C) Right panel: expression of either wild-type or mutant β-2 chain rescues cell surface expression of LFA-1 in β-2-deficient LAD19 cells. Left panel: adhesion assay. Wild-type LFA-1, but not the CD18(WKA723–725TRG) mutant, responds to monclonal antibody OKT3 (anti-TCR)-mediated stimulation of adhesion to ICAM-1 in LAD19 cells (gray bars). However, both receptors respond to activating antibody MEM-48 (solid bars).