The Voltage-dependent Anion Channel Is a Receptor for Plasminogen Kringle 5 on Human Endothelial Cells
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m303172200
ISSN1083-351X
AutoresMario Gonzalez‐Gronow, Theodosia A. Kalfa, Carrie E. Johnson, Govind Gawdi, Salvatore V. Pizzo,
Tópico(s)S100 Proteins and Annexins
ResumoHuman plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1–3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1–3 or 4 has been identified as cell surface-associated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltage-dependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr252–Lys283, which is homologous to the primary sequence of VDAC residues Tyr224–Lys255. Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (Kd of 28 nm) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca2+ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5. Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1–3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1–3 or 4 has been identified as cell surface-associated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltage-dependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr252–Lys283, which is homologous to the primary sequence of VDAC residues Tyr224–Lys255. Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (Kd of 28 nm) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca2+ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5. Angiogenesis is essential for tumor growth (1Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar, 2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6089) Google Scholar, 3Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 4Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7217) Google Scholar). Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; K5, kringle five; Pg, plasminogen; SK, streptokinase; VDAC, voltage-dependent anion channel; HUVEC, human umbilical vein endothelial cells; FACS; fluorescence assisted cytometry scanning; pHi, intracellular pH; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; DSPM+, 2-(dimethylaminostyryl)-1-methyl-pyridinium ion. is a potent mitogen promoting endothelial cell proliferation (5Dusak B.A. Czerniak P. Sun T. Eidsvoog K. Dexter D.L. Yayon A. J. Natl. Cancer Inst. 1993; 85: 121-131Crossref PubMed Scopus (95) Google Scholar, 6Kim K.J. Li B. Winer J. Armanini M. Gillet N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3353) Google Scholar), whereas angiostatin inhibits this process in vitro and suppresses tumor growth in vivo (7O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3172) Google Scholar, 8O'Reilly M.S. Holmgren L. Chen C. Folkman J. Nat. Med. 1996; 2: 689-692Crossref PubMed Scopus (1151) Google Scholar). Angiostatin is a fragment of plasminogen (Pg) consisting of either the first three or four of its kringles (7O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3172) Google Scholar). Pg kringle 5 (K5) also suppresses growth factor-stimulated angiogenesis via cell cycle G1 arrest and induction of apoptosis (9Ji W.R. Barientos L.G. Llinas M. Gray H. Villareal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P.A. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar, 10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 11Cao Y. O'Reilly M.S. Marshall B. Flynn E. Ji R.W. Folkman J. J. Clin. Invest. 1998; 101: 1055-1063Crossref PubMed Scopus (240) Google Scholar, 12Lu H. Dhanabal M. Volk R. Waterman M.J.F. Ramchandran R. Knebelman B. Segal M. Sukhatme V.P. Biochem. Biophys. Res. Commun. 1999; 258: 668-673Crossref PubMed Scopus (72) Google Scholar); however, the cellular receptor(s) mediating these effects are unknown. K5 confers on Pg the capacity to bind to human umbilical vein endothelial cells (HUVEC) with high affinity (13Wu H.L. Wu I.S. Fang R.Y. Hau J.S. Wu D.H. Chang B.I. Lin T.M. Shi G.Y. Biochem. Biophys. Res. Commun. 1992; 188: 703-711Crossref PubMed Scopus (22) Google Scholar). K5 also mediates binding of Pg to the Pg activator streptokinase (SK) (14Nihalani D. Sahni G. Biochim. Biophys. Res. Commun. 1995; 217: 1245-1254Crossref PubMed Scopus (31) Google Scholar, 15Lin L.F. Bhoung A. Reed G.L. Biochemistry. 2000; 39: 4740-4745Crossref PubMed Scopus (33) Google Scholar). Sequence similarities between SK and the mitochondrial human voltage-dependent anion channel (VDAC1) exist; specifically, the region comprising SK residues Tyr252–Lys283 are homologous to VDAC1 residues Tyr224–Lys255 (16McCabe K.M. Wheeler D.A. Adams V. Edward R.B. Biochem. Mol. Med. 1995; 56: 176-179Crossref PubMed Scopus (1) Google Scholar). We raised antibodies against peptides contained within these regions and used them to identify VDAC1 on the HUVEC surface by flow cytometry (FACS). Receptor binding assays demonstrated that K5 binds with high affinity to sites on these cells. K5 inhibits VEGF-stimulated HUVEC proliferation and induces a decrease in cytosolic pH and an increase in the potential of isolated mitochondria. Highly purified VDAC1 binds to K5 after reconstitution of the receptor into liposomes. Our data suggest that VDAC1 is a receptor for K5 on the cell surface. Materials—Culture media were from Invitrogen. Porcine pancreatic elastase, gastric mucosa pepsin, trypsin inhibitor, and pre-formed liposomes were from Sigma. Recombinant VEGF was from Calbiochem (San Diego, CA). Endothelial cell growth supplement was from Collaborative Research Inc. (Waltham, MA). 125I-Labeled Bolton-Hunter reagent was obtained from PerkinElmer Life Sciences. The 21-amino acid peptides EINNTDLISLEYKYVLKKGEK (Glu263–Lys283) of SK and KVNNSSLIGLGYTQTLKPGIK (Lys235–Lys255) of VDAC1 were from Research Genetics (Huntsville, AL). Fura-2/AM and bis(carboxyethyl)-carbonyl fluorescein and the 2-(dimethylaminostyryl)-1-methyl-pyridinium ion (DSPM+) were purchased from Molecular Probes, Inc. (Eugene, OR). Proteins—Human Pg was resolved into its isoforms, Pg 1 and 2 (17Deutsch D. Mertz B.T. Science. 1970; 170: 1095-1096Crossref PubMed Scopus (1670) Google Scholar, 18Gonzalez-Gronow M. Robbins K.C. Biochemistry. 1984; 23: 190-196Crossref PubMed Scopus (35) Google Scholar). Pg 2 was digested with elastase and fractionated by gel and affinity chromatography to obtain mini-Pg, followed by digestion of mini-Pg with pepsin to obtain K5 (10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 19Sottrup-Jensen L. Claeys H. Zajdel M. Petersen T.E. Magnusson S. Prog. Chem. Fibrinol. Thrombol. 1978; 3: 191-209Google Scholar, 20Thewes T. Ramesh V. Simplaceanu E.L. Llinas M. Biochim. Biophys. Acta. 1987; 912: 254-269Crossref PubMed Scopus (42) Google Scholar). Gel electrophoresis (10–20% gradient gel, nonreducing conditions) identified a doublet of ∼12 kDa, which was identified by mass spectrometry as K5. Amino-terminal sequence analysis yielded the sequence LPTVETPSEE, corresponding to Pg residues 450–459, confirming the identification of K5 (21Petersen T.E. Martzen M.R. Ichinose A. Davie E.W. J. Biol. Chem. 1990; 265: 6104-6109Abstract Full Text PDF PubMed Google Scholar). Reduction/alkylation of K5 was performed by incubating 20 μg of K5 with 1 mm dithiothreitol for 30 min followed by incubation with 5 mm iodoacetamide for 30 min, both at room temperature, and removal of these reagents by dialysis versus 10 mm Hepes, pH 7.5. Iodination of K5 was performed with 125I-labeled Bolton-Hunter reagent (specific activity, 500–700 cpm/ng). Antibodies—Antibodies to SK were raised in rabbits, and the IgG fraction specific against the SK sequence Glu263–Lys283 was purified by immunoaffinity on a resin containing this peptide conjugated to activated carboxyhexyl-Sepharose (Amersham Biosciences). The antibodies against the 21-amino acid sequence Lys235–Lys255 of VDAC1 conjugated to keyhole limpet hemocyanin (22Kagen A. Glick M. Jaffe B.B. Behrman H.R. Methods of Hormone Radioimmunoassay. Academic Press, New York1979: 328-329Google Scholar) were prepared in rabbits by COVANCE (Denver, PA). The IgG fraction specific to VDAC1 was purified by immunoaffinity on a resin containing the VDAC1 peptide conjugated to carboxyhexyl-Sepharose. The monoclonal antibody 20B12 against human mitochondrial VDAC1 was from Molecular Probes, Inc. Endothelial Cell Proliferation Assay—HUVEC from Clonetics (San Diego, CA) were grown in Dulbecco's modified Eagle's medium containing 20% bovine serum, 100 units/ml penicillin/streptomycin, 2.5 μg/ml amphotericin B, 2 mm glutamine, 5 units/ml sodium heparin, and 200 μg/ml endothelial cell growth supplement (23Morales D.E. McGowan K.A. Grant D.S. Mashewari S. Bhartiya D. Cid M.C. Kleinman H.K. Schnaper H.W. Circulation. 1995; 91: 755-763Crossref PubMed Google Scholar). The cells were washed with phosphate-buffered saline and dispersed in a 0.05% trypsin solution. The cells were resuspended in medium (25 × 103 cells/ml) and plated in 96-well culture plates (0.2 ml/well). After 24 h at 37 °C, the medium was replaced with 0.2 ml of Dulbecco's modified Eagle's medium, 5% bovine serum, 1% antibiotics, and the test samples were applied. Cell proliferation was determined at 24 h using bromodeoxyuridine labeling and a colorimetric immunoassay (Roche Applied Science). The results were expressed as percentages of control proliferation determined in the presence of VEGF (10 ng/ml) and the absence of K5. Flow Cytometry—HUVEC were detached from the culture flasks (75 cm2) by incubation for 5 min at 37 °C with Ca2+ and Mg2+-free phosphate-buffered saline containing 4 mm EDTA and pelleted. The cells (1 × 107/ml) were washed with phosphate-buffered saline before resuspension in ice-cold Phenol Red-free Hanks' balanced salt solution (HBSS), 1% BSA, 0.3 mg/ml goat IgG, and 0.01% NaN3 (staining buffer). The cell suspensions (100 μl) were incubated 30 min with dilutions of rabbit polyclonal anti-human SK peptide IgG, anti-human VDAC1 peptide IgG, or the murine anti-human mitochondrial VDAC1 monoclonal antibody. The cells were washed with ice-cold staining buffer, pelleted, and resuspended in 100 μl of ice-cold staining buffer. The cell suspensions were incubated in the dark with an AF488-conjugated for 30 min to goat anti-rabbit or mouse IgG from Molecular Probes, Inc. The cells were washed twice with ice-cold staining buffer, resuspended in ice-cold 1% paraformaldehyde, and stored in the dark at 4 °C until analysis by FACS. The mean relative fluorescence after excitation at λ = 495 nm was determined for each sample on a FACSVantage SE flow cytometer (BD Biosciences) and analyzed with CELLQUEST® software (BD Biosciences). Ligand Binding Analysis—The cells were grown in tissue culture plates until the monolayers were confluent. The cells were washed in HBSS. The binding assays were performed at 4 °C in RPMI 1640 containing 2% BSA. Increasing concentrations of 125I-K5 were incubated with cells for 60 min in 96-well strip plates. Free and bound ligand were separated by aspirating the incubation mixture and washing the cell monolayers rapidly thrice with RPMI 1640 containing 2% BSA. The wells were stripped from the plates and radioactivity determined. The bound ligand was calculated after subtraction of nonspecific binding measured in the presence of 50 mm p-aminobenzamidine. The Kd and B max of K5 were determined by fitting the data directly to the Langmuir isotherm using the statistical program SYStat® for Windows. Antibody Binding Studies—The binding assays were performed in HUVEC grown in 96-well strip plates. The cells were washed in HBSS and incubated with increasing concentrations of 125I-labeled anti-human VDAC1 peptide IgG for 90 min at 25 °C in RPMI 1640 containing 2% BSA. The cells were rinsed with RPMI 1640, and the wells stripped from the plates were inserted in plastic tubes to determine radioactivity. IgG bound was calculated after subtraction of nonspecific binding measured in the presence of 50 μm nonlabeled IgG. The B max of the anti-VDAC1 IgG was then calculated. Measurements of Intracellular Free Ca2+ Concentration and Cytosolic pHi—HUVEC [Ca2+]i was measured by digital imaging microscopy using the fluorescent indicator Fura-2/AM (24Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1993; 268: 20791-20794Abstract Full Text PDF PubMed Google Scholar). For measurements of pHi, HUVEC were incubated overnight in Dulbecco's modified Eagle's medium on glass coverslips and then washed with HBSS with 0.1 m sodium bicarbonate, pH 7.1. The cells were incubated for 20 min with 2 μm 2′,7′-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in HBSS, rinsed with buffer thrice, and placed on the fluorescent microscope stage. Intracellular pH (pHi) was measured by a digital video imaging technique in cells stimulated by the ligands, which were added after obtaining a stable base line (25Prpic V. Yu S.F. Figuereido F. Hollenbach P.W. Gawdi G. Herman B. Uhing R.J. Adams D.O. Science. 1989; 244: 469-471Crossref PubMed Scopus (61) Google Scholar). Gel Electrophoresis—Electrophoresis was performed in 0.1% SDS employing a discontinuous Laemmli buffer system (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). The gels were stained with 0.25% Coomassie Brilliant Blue R-250. Transfer to nitrocellulose membranes was carried out by the Western blot method (27Towbin H. Staehlin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4355Crossref PubMed Scopus (44923) Google Scholar). The dye-conjugated M r markers (Bio-Rad) used were of M r 38,100, 28,400, 18,200, 9,200, and 4,300. Purification of VDAC1 From 1-LN Cells—It is difficult to obtain large numbers of cultured HUVEC; however, we found that 1-LN cells are a good source of this protein. 1-LN cells were grown in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin G, and 100 ng/ml streptomycin in 20 culture flasks (150 cm2). After detaching with 10 mm EDTA in HBSS and pelleting, the cells were suspended in 10 ml of 20 mm Hepes, pH 7.2, 0.25 m sucrose containing the proteinase inhibitors (each at 0.5 mg/ml) antipapain, bestatin, chymostatin, trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64), leupeptin, pepstatin, o-phenanthroline, and aprotinin. The cells were lysed by sonication on ice (five 10-s bursts with 30-s intervals). The homogenate was centrifuged at 800 × g for 15 min, followed by centrifugation at 50,000 × g for 1 h. The pellet containing cell membranes was resuspended in 20 mm Tris-HCl, pH 8.0, containing 1% (v/v) Triton X-100 to solubilize membranes and centrifuged at 50,000 × g for 30 min to remove insoluble materials. VDAC was sequentially purified to homogeneity using gel filtration on Sephadex G-150 and immunoaffinity chromatography with an anti-VDAC peptide IgG conjugated to Sepharose 4-B (see Fig. 1C). Incorporation of VDAC1 into Liposomes and Binding of K5 to the Reconstituted Receptor—Purified VDAC1 was reconstituted into liposomes (28Lichtenberg D. Barenholz Y. Glick D. Methods in Biochemical Analysis. Wiley, New York1988: 337-462Google Scholar, 29Barenholz Y. Amsalen S. Gregoriadis D. Liposome Technology. CRC Press, Boca Raton, FL1993: 517-616Google Scholar) as follows. 50 μl of a suspension of liposomes (8 μm l-α-phosphatidylcholine, 8 μm phosphatidylethanolamine, 8 μm β-oleoyl-γ-palmitate and 6.9 μm cholesterol) in 5% Me2SO were mixed with VDAC1 (5 μg) and incubated with agitation for 30 min at room temperature. The concentration of Me2SO was reduced to 0.5% with 50 mm Tris-HCl, pH 7.4. After the addition of 125I-K5 (10 nm) and incubation for another 30 min at room temperature, the mixture was filtered through a Sephadex G-75 column (55 × 2 cm). To study inhibition of K5 binding to VDAC1 reconstituted into liposomes, the mixture was incubated with the specific anti-VDAC1 IgG for 30 min at room temperature before the addition of 125I-K5. The kinetic parameters of K5 binding to VDAC1 on reconstituted liposomes were performed on large unilamellar liposomes (0.4 μm in diameter) prepared by extrusion of multilamellar vesicles through 0.4-μm defined polycarbonate filters (Nucleopore, Pleasanton, CA) (30Mayer L.D. Hope M.J. Cullis P.R. Biochim. Biophys. Acta. 1986; 858: 161-168Crossref PubMed Scopus (1571) Google Scholar). For these experiments, proteoliposomes containing VDAC1 or BSA were prepared by mixing the proteins (50 μg) in 2.5 mm Hepes, pH 7.4, 145 mm NaCl, and 0.3 mm N-dodecyl-β-d-maltopyranoside with N-dodecyl-β-d-maltopyranoside saturated (0.6 mm) liposomes at a 1:3 volume ratio of protein preparations to liposomes (31Rigaud J.L. Pitard B. Levy D. Biochim. Biophys. Acta. 1995; 1231: 223-246Crossref PubMed Scopus (403) Google Scholar). The detergent was removed after three 2-h incubations at 4 °C of the proteoliposomes with 10 mg of Biobeads SM2 (Bio-Rad) followed by three 30-min centrifugations at 100,0000 × g. Phospholipid phosphate was then determined (32Bottcher C.F.J. Van Gent C.M. Fries C. Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (851) Google Scholar). To assess the orientation and amount of VDAC1 incorporated, the liposomes (10 μm phospholipid) in 2.5 mm Hepes, pH 7.4, were incubated with increasing amounts of 125I-labeled anti-VDAC1 IgG for 1 h at room temperature followed by filtration over 0.1-μm pore size VM-MultiScreen filters (Millipore Corp., Bedford, MA). After three rinses with 200 μl of 2.5 mm Hepes, pH 7.4, the filters were stripped from the plates, and radioactivity was determined. Increasing concentrations of 125I-K5 were incubated for 1 h at room temperature with VDAC1 proteoliposomes, BSA proteoliposomes, or empty liposomes (10 μm phospholipid) in 2.5 mm Hepes, pH 7.4, containing 145 mm NaCl. Filtration and determination of kinetic parameters were carried out as described above. Preparation of Mitochondria—Mitochondria from 1-LN cells were isolated (33Rickwood D. Wilson M.T. Darley Usmar V.M. Mitochondria: a Practical Approach. Portland Press, London1987: 1-16Google Scholar), and the protein levels were estimated using the bicinchonic acid method (34Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18645) Google Scholar). Mitochondrial Membrane Potential—Membrane potential (ΔΨ) was determined at room temperature using DSMP+, a fluorescent indicator of membrane potential (35Mewes H.W. Rafael J. FEBS Lett. 1981; 131: 7-10Crossref PubMed Scopus (43) Google Scholar). The assay consisted of a final volume of 2 ml containing sucrose (250 mm), Hepes (10 mm), EGTA (2.5 mm) pH 7.4, mitochondria (0.2 mg) cellular protein, DSPM + 2 (nmol), rotenone (1 μg), sodium succinate (10 μm), pH 7.4, and increasing concentrations of K5. The mixture was incubated for 20 min with K5 prior to addition of DSPM+. The fluorescence intensity was measured (excitation λ = 489 nm, emission λ = 566 nm) in a Shimadzu RF-5301PC spectrofluorometer (Shimadzu Corporation, Kyoto, Japan). Maximal fluorescence in the absence of K5 was obtained with mitochondria incubated with DSPM+ alone. The results are the means of fluorescence determinations from three experiments. Analyses of Sequence Similarities between SK and Human VDAC1—The regions of sequence similarity between streptokinase and human VDAC1 were identified by the BLAST program provided by the Swiss Institute of Bioinformatics (Fig. 1A). The topology prediction for helical transmembrane proteins was solved with use of the hidden Markov model, also provided by the Swiss Institute of Bioinformatics, and shows a loop through the outer mitochondrial membrane spanning VDAC1 residues 247QTLKPGIKL255 (Fig. 1B). We raised rabbit antibodies to the SK peptide 263EINNTDLISLEYKYVLKKGEK283 and the VDAC1 peptide 236KVNNSSLIGLGYTQTLKPGIK255. A Coomassie Brilliant Blue stain of the purified VDAC (Fig. 1C, lane 1) shows a major band of M r ∼32,000. A blot binding assay with a rabbit anti-VDAC1 (peptide Lys235–Lys255) IgG shows reactivity with this protein (Fig. 1C, lane 2). Similarly, the purified VDAC1 showed reactivity with the anti-SK (peptide Glu263–Lys283) IgG (Fig. 1C, lane 3), confirming the structural relatedness between VDAC1 and SK. However, the purified VDAC1 did not show any reactivity with 125I-K5 when electroblotted to a nitrocellulose membrane (Fig. 1C, lane 4). Binding of K5 to VDAC1 Incorporated into Liposomes—The experiments described above demonstrate a significant impact on the ability of purified receptor to bind to K5; therefore, the purified VDAC1 was incorporated into liposomes and gel filtration on Sephadex G-75 employed to identify and separate the reactants (Fig. 2). 125I-K5 eluted at a column volume of 100–120 ml (Fig. 2A). When 125I-K5 was incubated with solubilized VDAC1 (2 μg), the radiolabeled material eluted in the same fractions as above, suggesting no reactivity between K5 and solubilized receptor (Fig. 2B). When VDAC1 was incorporated into liposomes and then reacted with K5, the radiolabeled material eluted from the column as two peaks, one of them corresponding to the void volume where VDAC1 elutes and the other corresponding to the elution volume of unreacted K5 (Fig. 2C). These data indicate that K5 binds to VDAC1 when this receptor is incorporated into a lipid membrane. Binding of K5 to membrane-incorporated VDAC1 was inhibited by anti-VDAC1 (peptide Lys235–Lys255) IgG, demonstrating that this is the region responsible for binding to K5 (Fig. 2D). K5 binds to VDAC1 proteoliposomes (Fig. 3A) in a dose-dependent manner with high affinity (Kd of 22 ± 3.1 nm). The binding is specific for VDAC1 because control proteoliposomes prepared with BSA or empty liposomes show little specific binding (Fig. 3A). Binding of 125I-K5 to VDAC1 proteoliposomes is inhibited by unlabeled K5 or anti-VDAC1 IgG (Fig. 3B), suggesting that VDAC1 is a receptor for K5. Analyses of VDAC1 on the Cell Surface of HUVEC by Flow Cytometry—As determined by FACS, HUVEC reacted with an antibody against the SK peptide (Fig. 4A) as well as an antibody against the VDAC1 peptide (Fig. 4B) or a murine antibody against human mitochondrial VDAC1 (Fig. 4C), as expected because mitochondrial and plasma membrane VDAC1 share the same primary structure (35Mewes H.W. Rafael J. FEBS Lett. 1981; 131: 7-10Crossref PubMed Scopus (43) Google Scholar). FACS analysis of HUVEC reacted with K5 (0.1 μm) prior to reaction with antibody against the VDAC1 peptide (Fig. 4D) shows inhibition of binding of this antibody, suggesting that both K5 or the IgG compete for the same binding site. Taken together, these experiments show that VDAC1 is not only expressed on the surface of HUVEC but also establishes the structural relationship between SK and VDAC1 hypothesized by McCabe et al. (16McCabe K.M. Wheeler D.A. Adams V. Edward R.B. Biochem. Mol. Med. 1995; 56: 176-179Crossref PubMed Scopus (1) Google Scholar). Inhibition of Endothelial Cell Proliferation by K5—K5 inhibited VEGF-dependent HUVEC proliferation in a dose-dependent manner (Fig. 5A). As observed previously (10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), the antiendothelial cell proliferation of K5 was abolished after reduction/alkylation of the protein, suggesting that the formation of appropriate disulfide bridges is essential to maintain its activity. Binding of K5 to HUVEC—K5 binds to these cells in a dose-dependent manner with high affinity (Kd of 28 ± 1.37 nm) and to a large number of sites (12.6 ± 0.56 × 105 binding sites/cell) (Fig. 5B). The value of the Kd is comparable with that determined for binding of K5 to VDAC1 reconstituted in proteoliposomes. Electrophoretic separation of proteins in a HUVEC lysate followed by a blot binding assay with a rabbit anti-VDAC1 IgG (Fig. 5B, inset) shows only one band of M r ∼32,000. Binding of K5 to HUVEC is inhibited by Pg, Pg peptides containing K5, or by an IgG fraction against VDAC1 peptide showing structural relatedness to SK (Fig. 5C). Binding of Anti-VDAC1 Peptide IgG to HUVEC— 125I-Labeled anti-VDAC1 IgG bind to HUVEC in a dose-dependent manner to a large number of sites (B max of 11.6 × 105 binding sites/cells) (Fig. 5D). This value is comparable with that determined for the binding of K5 to HUVEC, suggesting VDAC1 as a unique receptor for K5 on the cell surface. Effect of K5 Binding on HUVEC [Ca2+]i and pHi—We also investigated whether K5 binding to HUVEC produced changes in [Ca2+]i or pHi and compared these changes with those produced by Pg 2. Pg 2 (100 nm) added to HUVEC induces a transient rise in [Ca2+]i lasting about for 90 s before returning to base line (Fig. 6A). Pg 2 also induced a rise in pHi, which was continuous for 400 s (Fig. 6B). A similar concentration (100 nm) of K5 induced a small a rise in [Ca2+]i (Fig. 6C) and produced a continuous decrease in pHi during the same time period (Fig. 6D). Incubation of HUVEC with K5 followed by Pg 2 shows a decreased stimulation in [Ca2+]i (Fig. 6E); however, the decrease in pHi induced by K5 is abolished after the addition of Pg (Fig. 6F). Incubation of HUVEC with anti-VDAC1 peptide IgG prior to the addition of K5 causes no change in [Ca2+]i (Fig. 6G) or pHi (Fig. 6H and Table I).Table IEffects induced by Pg 2 and K5 on HUVFCLigandChanges in [Ca2+]iChanges in pHinMPg 2+450+0.17K5+100-0.20K5 + Pg2+10+0.07K5 + anti-VDAC1+10+0.07These data were obtained from Fig. 6 Open table in a new tab These data were obtained from Fig. 6 Effect of K5 on Mitochondrial Membrane Potential—The fluorescent dye DSPM+ was used as an indicator of a coupled membrane potential (ΔΨ) (35Mewes H.W. Rafael J. FEBS Lett. 1981; 131: 7-10Crossref PubMed Scopus (43) Google Scholar). We observed that K5 induces a concentration-dependent increase in ΔΨ with mitochondria isolated from 1-LN cells (Fig. 7). In search of an endothelial cell receptor for K5, a potent suppressor of growth factor-stimulated angiogenesis (9Ji W.R. Barientos L.G. Llinas M. Gray H. Villareal X. DeFord M.E. Castellino F.J. Kramer R.A. Trail P.A. Biochem. Biophys. Res. Commun. 1998; 247: 414-419Crossref PubMed Scopus (94) Google Scholar, 10Cao Y. Chen A. An S.S.A. Ji R.W. Davidson D. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 11Cao Y. O'Reilly M.S. Marshall B. Flynn E. Ji R.W. Folkman J. J. Clin. Invest. 1998; 101: 1055-1063Crossref PubMed Scopus (240) Google Scholar, 12Lu H. Dhanabal M. Volk R. Waterman M.J.F. Ramchandran R. Knebelman B. Segal M. Sukhatme V.P. Biochem. Biophys. Res. Commun. 1999; 258: 668-673Crossref PubMed Scopus (72) Google Scholar), we investigated the functional relationship between SK and VDAC1, two proteins displaying sequence homologies (16McCabe K.M. Wheeler D.A. Adams V. Edward R.B. Biochem. Mol. 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The integrity of Tyr512 is required for reactivity of K5 with its ligands (41Thewes T. Constantine K. Byeon I.J.L. Llinas M. J. Biol. Chem. 1990; 265: 3906-3915Abstract Full Text PDF PubMed Google Scholar). The same residue is also essential for the interaction of K5 with Pg residue Lys50, which stabilizes Pg in a closed conformation when the protein is in the circulation (42Ponting C.P. Holland S.K. Cederholm-Williams S.A. Marshall J.M. Brown A.J. Spraggon G. Blake C.C.F. Biochim. Biophys. Acta. 1992; 1159: 155-161Crossref PubMed Scopus (42) Google Scholar). Endothelial cell proliferation is preceded by an increase in cytosolic pHi, leading to angiogenesis and the repair of injured endothelial cells (43Komatsu S. Sawada S. Tamagaki T. Tsuda Y. Kono Y. Higaki T Imamura H. Tada Y Yamasaki S. Toratani A. Sato T. Akamatsu N. Tsuji H. Nakagawa M J. Pharmacol. Toxicol. 1999; 41: 33-41Crossref PubMed Scopus (8) Google Scholar). 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