Title: Direct Interaction between Endothelial Nitric-oxide Synthase and Dynamin-2
Abstract: Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, A23187, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed 35S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (Kd) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium. Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, A23187, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed 35S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (Kd) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium. Endothelial nitric-oxide synthase (eNOS)1 is a membrane-associated protein that is localized in the Golgi apparatus as well as within cholesterol-rich plasmalemmal vesicles, termed caveolae (1Sessa W.C. Garcia-Cardena G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 2Garcia-Cardena G. Oh P. Liu J. Schnitzer J. Sessa W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (572) Google Scholar, 3Shaul P. Smart E. Robinson L. German Z. Yuhanna I. Ying Y. Anderson R. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). The ability of eNOS to generate nitric oxide (NO) has traditionally been ascribed to agonist-stimulated calcium-dependent activation. However, during recent years several groups have demonstrated that activation of eNOS, both in conjunction with and independent of intracellular calcium flux, occurs through the allosteric binding of eNOS with neighboring regulatory proteins (4Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 5Michel J. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 6Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (850) Google Scholar). In this regard, several eNOS-associated proteins have been identified, including the caveolae coat protein, caveolin, calmodulin, Hsp 90, and the bradykinin-2 receptor (6Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (850) Google Scholar, 7Ju H. Venema V. Marrero M. Venema R. J. Biol. Chem. 1998; 273: 24025-24029Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8Garcia-Cardena G. Martasek P. Masters B.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 9Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Dynamin-2 is a large ubiquitously expressed GTP-binding protein that targets to Golgi membranes and colocalizes with caveolin within caveolae (10McClure S. Robinson P. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (74) Google Scholar). Although the function of dynamin is best characterized in membrane scission events (11McNiven M. Cell. 1998; 94: 151-154Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 12Henley J. Krueger E. Oswald B. McNiven M. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (613) Google Scholar, 13Oh P. McIntosh P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (545) Google Scholar, 14Jones S. Howell K. Henley J. Cao H. McNiven M. Science. 1998; 279: 573-577Crossref PubMed Scopus (269) Google Scholar), members of this family of proteins also modulate signaling pathways by means of distinct protein interactions (10McClure S. Robinson P. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (74) Google Scholar, 15Whistler J. von Zastrow M. J. Biol. Chem. 1999; 274: 24575-24578Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16Scaife R. Gout I. Waterfield M. Margolis R. EMBO J. 1994; 13: 2574-2582Crossref PubMed Scopus (77) Google Scholar, 17Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar). In support of this concept, dynamin-1 binds and regulates the calcium-sensitive phosphatase, calcineurin, and the dynamin family of proteins interact with the SH3 domains of a variety of signaling proteins by virtue of a proline-rich domain (16Scaife R. Gout I. Waterfield M. Margolis R. EMBO J. 1994; 13: 2574-2582Crossref PubMed Scopus (77) Google Scholar, 17Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar, 18Lai M. Hong J. Ruggiero A. Burnett P. Slepnev V. De Camilli P. Snyder S. J. Biol. Chem. 1999; 37: 25963-25966Abstract Full Text Full Text PDF Scopus (125) Google Scholar, 19Okamoto P. Herskovits J. Vallee R. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Wang L.-H. Sudhof T. Anderson R. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Based on previously published studies indicating that both eNOS and dynamin-2 colocalize with caveolin and reside within similar membranes compartments (1Sessa W.C. Garcia-Cardena G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 2Garcia-Cardena G. Oh P. Liu J. Schnitzer J. Sessa W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (572) Google Scholar, 12Henley J. Krueger E. Oswald B. McNiven M. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (613) Google Scholar, 13Oh P. McIntosh P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (545) Google Scholar, 14Jones S. Howell K. Henley J. Cao H. McNiven M. Science. 1998; 279: 573-577Crossref PubMed Scopus (269) Google Scholar), we examined whether these two proteins might interact in a functional manner. Here we demonstrate that pools of eNOS and dynamin bind within cells in a manner regulated by ionic stringency and intracellular signals and that in vitro the proteins bind directly in a stoichiometric and high affinity manner. Additionally, the interaction of dynamin with eNOS potentiates eNOS catalysis in cells and in vitro. These studies identify a new NOS-associated protein and provide a novel function for dynamin-2. All tissue culture and transfection reagents were obtained from Life Technologies, Inc. eNOS mAb and pAb were obtained from Transduction Laboratories (Lexington, KY). Golgi 58-kDa protein was obtained from Sigma. The dynamin-2 pAb (dyn2T) has been previously described (12Henley J. Krueger E. Oswald B. McNiven M. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (613) Google Scholar). This antibody recognizes the proline-rich COOH-terminal tail unique to dynamin-2 and specifically precipitates this dynamin isoform from brain lysates. Calcium ionophore, A23187, was obtained from Sigma.3H-Labeled l-arginine and35S-labeled methionine were obtained from Amersham Pharmacia Biotech. Bovine aortic endothelial cells (BAEC), Clone 9 cells (rat hepatocyte cell line) (14Jones S. Howell K. Henley J. Cao H. McNiven M. Science. 1998; 279: 573-577Crossref PubMed Scopus (269) Google Scholar), and stably transfected eNOS-GFP ECV 304 cells (21Sowa G. Liu J. Papapetropoulos A. Rex-Haffner M. Hughes T. Sessa W. J. Biol. Chem. 1999; 274: 22524-22531Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) were used in these studies. The BAEC used in this study were between P2 and P5 and were obtained from Clonetics (San Diego, CA). BAEC were cultured in EGM, Clone 9 cells in Ham's F-12K medium, and ECV 304 cells in Dulbecco's modified Eagle's medium with 400 μg/ml G418. Media were supplemented with 10% fetal bovine serum, l-glutamine (1 mm), penicillin (100 IU/ml), and streptomycin (100 μg/ml). For transfections, Clone 9 cells were plated on glass coverslips in 12-well plates and cotransfected with a bovine eNOS expression vector (pcDNA3) and a rat dynamin-2 expression vector (pEGFP-N1) (22Cao H. Garcia F. McNiven M. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (338) Google Scholar). eNOS-GFP ECV 304 cells were plated in 100-mm dishes and cotransfected with a rat dynamin-2 expression vector (pcDNA3). Transfections were performed using LipofectAMINE (Life Technologies, Inc.) as per the manufacturer's protocol. Thirty six hours after transfection, Clone 9 cells were prepared for immunofluorescence microscopy, and eNOS-GFP ECV 304 cells (21Sowa G. Liu J. Papapetropoulos A. Rex-Haffner M. Hughes T. Sessa W. J. Biol. Chem. 1999; 274: 22524-22531Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) were prepared for NOS activity assay and immunoprecipitation analysis. Recombinant eNOS protein was purified fromEscherichia coli as described previously (23Martasek P. Liu Q. Liu J. Roman L. Gross S. Sessa W. Masters B. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar, 24Roman L. Sheta E. Martasek P. Gross S. Liu Q. Masters B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar). In brief, bovine eNOS in the plasmid, pCW was coexpressed with pGroELS plasmid into protease-deficient E. coli. eNOS was purified from extracts using a 2′,5′-ADP Sepharose 4B column. Typically 2.5–4 mg of eNOS was recovered from each preparation, yielding a single major band as determined by SDS-PAGE. Purified eNOS was stabilized withl-arginine (0.5 mm) and 5-fold molar excess of BH4. A cDNA construct encoding full-length GST-dynamin-2 fusion protein was created by subcloning dynamin-2 cDNA into the GST fusion protein vector, pGEX-1. GST-dynamin and GST constructs were transformed into BL21 (DE3) and induced with isopropyl-1-thio-β-d-galactopyranoside (1 mm) overnight and lysed by sonication with lysozyme 200 μg/ml in a buffer containing 20 mm Hepes (pH 7.2), 100 mm KCl, 2 mm MgCl, 1 mm dithiothreitol, 2 μm leupeptin, 1 mm PMSF. Samples were resonicated after addition of Triton X-100 to a final concentration of 1%. Cell debris was removed by centrifugation, and supernatant was mixed with glutathione-Sepharose beads and agitated for 2 h at 4 °C. Samples were centrifuged at 500 rpm, and pellets were washed three times in phosphate-buffered saline with 1% Triton X-100. Specificity and quality of GST-dynamin was assessed by Coomassie staining of SDS-PAGE gels and Western blotting of transferred proteins as depicted in Fig. 4 A. [35S]Methionine eNOS was translated in rabbit reticulocyte lysates using the TnT Coupled Reticulocye Lysate System (Promega, Madison, WI). The reaction mix, containing bovine eNOS DNA (or alternatively negative control containing empty vector), SP6 RNA polymerase promoter, and [35S]methionine, was incubated at 30 °C for 90 min. Translation products were examined by SDS-PAGE analysis and autoradiography of dried gels. Binding assays using [35S]methionine eNOS were performed as described below. BAEC and Clone 9 cells (48 h after cotransfection with eNOS and GFP-dynamin) were fixed in 2% paraformaldehyde. Double-labeling immunofluorescence was performed in BAEC as previously described in liver endothelial cells (25Shah V. Haddad F. Garcia-Cardena G. Frangos J. Mennone A. Groszmann R. Sessa W. J. Clin. Invest. 1997; 100: 2923-2930Crossref PubMed Scopus (266) Google Scholar), by incubating cells with eNOS pAb and Golgi 58-kDa protein mAb, dynamin pAb and Golgi 58-kDa protein mAb, or alternatively eNOS mAb and dynamin pAb. Immunofluorescence in Clone 9 cells transfected with GFP-dynamin was performed by incubating cells in eNOS mAb only. Primary antibodies were detected using fluorescein isothiocyanate- and Texas Red-coupled secondary antibodies. Washes were performed with phosphate-buffered saline, 0.1% bovine serum albumin after both primary and secondary antibody incubation. Cells were mounted in Anti-fade (Molecular Probes, Eugene, OR) and visualized using a confocal microscope (LSM 510, Zeiss, Germany). BAEC were homogenized in a lysis buffer (50 mm Tris-HCl, 0.1 mm EGTA, 0.1 mm EDTA, 2 μmleupeptin, 1 mm PMSF, 1% (v/v) Nonidet P-40, 0.1% SDS, 0.1% deoxycholate (pH 7.5)). In some experiments, BAEC were incubated in media containing 10 μm A23187 or equal volume of vehicle (Me2SO) for 10 min prior to lysis. Protein quantification of samples was performed using the Bio-Rad protein assay. eNOS immunoprecipitation was performed by incubating 1-ml aliquots of detergent-soluble protein lysate with excess eNOS mAb overnight (1:200 dilution) or alternatively with equal concentration of mouse IgG after preclearing of samples with Pansorbin as previously described (26Shah V. Toruner M. Haddad F. Cadelina G. Papapetropoulos A. Sessa W. Groszmann R. Gastroenterology. 1999; 117: 1222-1228Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Alternatively, dynamin immunoprecipitation was performed by incubating lysates with excess dynamin pAb (5 μg/ml) overnight. Immunocomplexes were bound by incubating protein samples with protein A beads for 1 h at 4 °C. Triplicate samples of bound proteins were extensively washed in a buffer (50 mmTris-HCl, 0.1 mm EGTA, 0.1 mm EDTA, 2 μm leupeptin, 1 mm PMSF) containing 0 mm NaCl, 100 mm NaCl, or alternatively 1m NaCl. Bound proteins were eluted by boiling samples in Laemmli loading buffer. Gel electrophoresis of proteins and Western blotting were performed as previously described (25Shah V. Haddad F. Garcia-Cardena G. Frangos J. Mennone A. Groszmann R. Sessa W. J. Clin. Invest. 1997; 100: 2923-2930Crossref PubMed Scopus (266) Google Scholar), using eNOS mAb and dynamin pAb. Membranes were stained with Ponceau S or gels with Coomassie Blue to confirm equal protein loading. Densitometric analysis of autoradiographs was performed using Scion Image from the National Institutes of Health. Increasing concentrations of recombinant eNOS proteins (60–300 nm) were incubated overnight at 4 °C with GST-dynamin beads or, alternatively, GST beads alone in immunoprecipitation buffer in the absence of detergents. Bound proteins were washed in a buffer containing 50 mmTris (pH 7.7), 200 mm NaCl, and 1 mm EDTA (8Garcia-Cardena G. Martasek P. Masters B.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Bound proteins were eluted with Laemmli buffer and used for gel electrophoresis. In vitro binding of GST-dynamin with35S-eNOS was examined by incubating GST-dynamin beads (60–600 nm) or GST beads alone, with a fixed concentration of in vitro translated eNOS (3 μl of rabbit reticulocyte lysate) or, alternatively, by incubating increasing concentrations ofin vitro translated eNOS (1–12 μl of rabbit reticulocyte lysate) with a fixed concentration of GST-dynamin beads, in 300 μl of a buffer containing 50 mm Tris-HCl, 0.1 mm EDTA overnight. Bound proteins were washed three times with a buffer containing 50 mm Tris-HCl, 200 mmNaCl, 1 mm EDTA and then eluted and used for gel electrophoresis. Binding studies with GST-dynamin and recombinant eNOS were analyzed by SDS-PAGE and Western blot analysis, whereas binding studies with GST-dynamin and 35S-eNOS were examined by SDS-PAGE and autoradiography of dried gels. Quantification of autoradiographs was performed by densitometric analysis using Scion Image. Estimation of equilibrium dissociation constant (Kd) was performed by incubating a fixed concentration of GST-dynamin beads (5 nm) with purified recombinant eNOS (0–320 nm) premixed with proportionate volume of 35S-eNOS (0–16 μl) used as a radiolabel tracer (the protein concentration of the radiolabel tracer is negligible compared with the protein concentration of the recombinant protein). Bound radioactive counts were measured directly by scintillation counting. Quantitation of bound and free 35S-eNOS allowed for determination of bound and free recombinant eNOS.Kd was calculated by Scatchard plot analysis of bound and free levels of recombinant eNOS. NOS activity from recombinant eNOS protein and eNOS-GFP EVC 304 cell lysates was assessed by measuring the conversion of 3H-labeled l-arginine to3H-labeled l-citrulline in the presence and absence of l-nitroarginine methyl ester (l-NAME) as described previously (6Garcia-Cardena G. Fan R. Shah V. Sorrentino R. Cirino G. Papapetropoulos A. Sessa W.C. Nature. 1998; 392: 821-824Crossref PubMed Scopus (850) Google Scholar, 26Shah V. Toruner M. Haddad F. Cadelina G. Papapetropoulos A. Sessa W. Groszmann R. Gastroenterology. 1999; 117: 1222-1228Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). GST-dynamin or, alternatively, GST alone was eluted from glutathione-Sepharose beads with reduced glutathione, and the eluted protein was incubated with 15 pmol of purified recombinant eNOS protein in molar ratios of 0:1, 0.5:1, 1:1, and 2:1 as described by Ju et al. (4Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Cell lysates were prepared from eNOS-GFP ECV 304 cells cotransfected with 3 or 6 μg of dynamin-2 DNA using the buffer described above for Western blot and separated into 200-μg aliquots for NOS assay. To determine NOS activity, triplicate samples of recombinant proteins or duplicate samples of cell lysate were incubated with a buffer containing 1 mm NADPH, 3 μmtetrahydrobiopterin, 100 nm calmodulin, 2.5 mmCaCl2, 10 μm l-arginine andl-[3H]arginine (0.2 μCi) at 37 °C for 20 min in the presence and absence of 1 mm l-NAME. The reaction mix was terminated by the addition of 1 ml of cold stop buffer (20 mm Hepes, 2 mm EDTA, 2 mm EGTA (pH 5.5)) and passed over a Dowex AG 50WX-8 resin column. Radiolabeled counts per min of generatedl-citrulline were measured and used to determinel-NAME inhibitable NOS activity. All data are given as mean ± S.E. Data were analyzed using paired and unpaired Student'st tests and one-way analysis of variance. Both eNOS and dynamin appear to reside within similar subcellular compartments based on previous studies examining subcellular localization of each of the proteins (2Garcia-Cardena G. Oh P. Liu J. Schnitzer J. Sessa W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (572) Google Scholar, 12Henley J. Krueger E. Oswald B. McNiven M. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (613) Google Scholar, 13Oh P. McIntosh P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (545) Google Scholar, 14Jones S. Howell K. Henley J. Cao H. McNiven M. Science. 1998; 279: 573-577Crossref PubMed Scopus (269) Google Scholar). To determine whether eNOS and dynamin colocalize within cells, we performed confocal immunofluorescence microscopy in Clone 9 cells heterologously expressing eNOS and dynamin as well as in BAEC which express both proteins endogenously. As seen in Fig.1 A, in Clone 9 cells transiently transfected with eNOS and GFP-dynamin, NOS protein is detected predominantly in a perinuclear distribution. GFP-dynamin is also detected in a perinuclear distribution with additional fluorescent signal detected within distinct punctate areas within the cytoplasm and plasma membrane (Fig. 1 B). As seen in the merged image (Fig.1 C), there is colocalization of the two proteins in a perinuclear distribution. To examine the subcellular distribution and colocalization of endogenously expressed eNOS and dynamin, we next performed double labeling immunofluorescence microscopy in BAEC. In BAEC, eNOS is detected in a perinuclear pattern with some protein detected within distinct regions of plasma membrane (Fig.1 D). In Fig. 1 E, immunolocalization of dynamin in BAEC demonstrates a similar distribution of protein as observed in Clone 9 cells, and as observed in the merged image in Fig.1 F, colocalization of the two proteins in BAEC is observed in predominantly a perinuclear pattern (small arrow) as well as in specific domains of plasma membrane (large arrow). Next, we further examined the nature of this prominent perinuclear area of colocalization in BAEC by examining the colocalization of eNOS and alternatively dynamin, with the Golgi marker, Golgi 58-kDa protein. eNOS and Golgi 58-kDa protein (Fig. 1, G and H, respectively) are detected in a perinuclear distribution and colocalization of proteins is detected, indicating that pools of eNOS protein reside within Golgi membranes as previously noted (Fig.1 I, small arrow) (1Sessa W.C. Garcia-Cardena G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 27Andries L. Brutsaert D. Sys S. Circ. Res. 1998; 82: 195-203Crossref PubMed Scopus (119) Google Scholar). Note that distinct pools of eNOS in plasma membrane do not colocalize with Golgi 58-kDa protein, demonstrating the specificity of Golgi 58-kDa protein mAb (Fig. 1 I, large arrow). Next we examined localization of dynamin (Fig. 1 J) and Golgi 58-kDa protein (Fig. 1 K) in BAEC. As seen in Fig. 1 L, pools of dynamin also colocalize with Golgi 58-kDa protein. No fluorescence was detected in negative control slides in which serum was substituted for the primary antibody or in cells incubated with secondary antibody alone (data not shown). These studies suggest that eNOS and dynamin colocalize predominantly within Golgi membranes of cells. Based on the prominent colocalization of eNOS and dynamin particularly within BAEC, we next examined whether the two proteins interact biochemically. For this purpose, detergent-soluble lysates were prepared from BAEC, and eNOS protein was immunoprecipitated under nondenaturing conditions. As seen in Fig. 2 A, immunoprecipitation of eNOS coimmunoprecipitates dynamin (lane labeled 0 mm NaCl). As seen in Fig. 2 B, immunoprecipitation of dynamin conversely coimmunoprecipitates eNOS from BAEC extracts (lane labeled0 mm NaCl). As observed in Fig. 2, Aand B, coprecipitation of eNOS and dynamin is maintained in the presence of an increase in the ionic strength of the wash buffer to 100 mm NaCl (lane labeled 100 mm NaCl). However, when beads are washed in the presence of 1 m NaCl, the interaction between the two proteins is markedly diminished suggesting an ionic strength protein-protein interaction (lane labeled 1m NaCl). We next examined the specificity of binding and pools of bound protein in cells, by substituting nonimmune sera for eNOS mAb during the immunoprecipitation and by comparing the pools of dynamin bound to eNOS with the total pool of dynamin in the lysate, respectively. As seen in Fig. 2C, ∼5% of the dynamin pool contained in the post-immunoprecipitation lysate (lane labeledpost IP that contains 20 μl from the 1 ml of lysate) is bound to eNOS (lane IP) as assessed by the similar dynamin Western blot signal intensity in these two lanes. (Similar results were obtained when the pre-IP lysate (not shown) was analyzed for dynamin in place of the post-IP lysate as the total pool of dynamin is not markedly reduced by eNOS immunoprecipitation.) Also, as seen in lane NIS, neither eNOS nor dynamin is detected when mouse IgG is substituted for eNOS mAb during the immunoprecipitation procedure. These studies indicate that fractions of eNOS and dynamin coexist in a complex within cells. To determine whether binding between eNOS and dynamin within cells is regulated by intracellular signals, we stimulated BAEC with the calcium ionophore, A23187 (10 μm), an agonist which promotes NO release (1Sessa W.C. Garcia-Cardena G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Control cells were treated with vehicle. Detergent-soluble lysates were prepared, and eNOS protein was quantitatively immunoprecipitated. As seen in lanes labeled IP, from the representative blot (Fig. 3 A), eNOS immunoprecipitated from lysates of BAEC incubated with the ionophore coimmunoprecipitate markedly greater levels of dynamin as compared with equal amounts of eNOS immunoprecipitated from cells treated with vehicle (compare the intensity of the dynamin Western blot signal in lanes labeled IP under vehicle and A23187). As seen in the lanes labeled Post IP, after immunoprecipitation, eNOS is barely detected within 20 μl of the remaining supernatant, whereas the majority of the cellular pool of dynamin remains unbound to eNOS. In Fig. 3 B, when the series of experiments (n = 3) are examined as a group by densitometric analysis, a significant increase in dynamin bound to eNOS is detected after stimulation of cells with A23187 (* indicatesp < 0.05). Coprecipitation between eNOS and dynamin from cell lysates may occur directly or through an adapter protein. To determine whether eNOS and dynamin are able to bind directly, we next examined whether eNOS in solution binds to dynamin in the form of a GST fusion protein. Fig.4 A demonstrates the purity and specificity of the GST dynamin fusion protein. In the left panel, enrichment of a single 26-kDa protein eluted from GST beads, and the enrichment of a 130-kDa protein eluted from GST-dynamin beads is observed by Coomassie staining of an SDS-PAGE gel. Theright panel of Fig. 4 A demonstrates the specific Western blot signal for dynamin that is detected from purified GST-dynamin beads but not GST beads alone. To determine whether eNOS and dynamin bind directly in vitro and to quantify the relative pools of bound protein, we incubated GST dynamin beads or alternatively GST beads alone with increasing concentrations of purified recombinant eNOS protein in solution (60–300 nm). As seen in the representative Western blot in Fig. 4 B, left panel, specific binding of purified eNOS with GST dynamin is detected, whereas GST beads alone do not coprecipitate eNOS from solution. Additionally, at a 1:1 molar ratio of GST-dynamin to eNOS (lane labeled 150 nm eNOS in Fig. 4 B), a majority of the pool of available eNOS protein is bound to dynamin (compare lane labeled 150 nm eNOS to lane input,which represents the available eNOS protein in the 150 nmsample). Further increase in the available concentration of eNOS results in a smaller increase in eNOS binding (compare lanelabeled 300 nm eNOS to lane 150 nm cm;1 eNOS). These observations are quantified in Fig. 4 B, right panel, in which the