Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6448-6453, June 1996 Cell Biology Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: Implications for nitric oxide signaling (endothelial nitric oxide synthase/signal transduction/vascular biology/N-myristoylation) GUILLERMO GARC1A-CARDENA*, PHIL OHt, JIANwEI LIu*, JAN E. SCHNITZERt, AND WILLIAM C. SESSA*t *Molecular Cardiobiology Program and Department of Pharmacology, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536; and tDepartment of Pathology, Harvard Medical School, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215 Communicated by Vincent T. Marchesi, Yale Univeristy, New Haven, CT, March 13, 1996 (received for review February 5, 1996) ABSTRACT The membrane association of endothelial insoluble membranes (TIM), suggesting that caveolae are nitric oxide synthase (eNOS) plays an important role in the signal processing centers (2-11). Additionally, caveolae have biosynthesis of nitric oxide (NO) in vascular endothelium. been implicated in other important cellular functions, includ- Previously, we have shown that in cultured endothelial cells ing endocytosis, potocytosis, and transcytosis (12, 13). and in intact blood vessels, eNOS is found primarily in the Endothelial nitric oxide synthase (eNOS) is a peripheral perinuclear region of the cells and in discrete regions of the membrane protein that metabolizes L-arginine to nitric oxide plasma membrane, suggesting trafficking of the protein from (NO). NO is a short-lived free radical gas involved in diverse the Golgi to specialized plasma membrane structures. Here, physiological and pathological processes. Endothelial-derived we show that eNOS is found in Triton X-100-insoluble mem- NO is an important paracrine mediator of vascular smooth branes prepared from cultured bovine aortic endothelial cells muscle tone and is an inhibitor of leukocyte adhesion and and colocalizes with caveolin, a coat protein of caveolae, in platelet aggregation (14, 15). As an autocrine mediator, NO cultured bovine lung microvascular endothelial cells as de- has been implicated in the modulation of growth factor signals termined by confocal microscopy. To examine if eNOS is and indeed in caveolae, we purified luminal endothelial cell cellular proliferation (16-18). Regulation of NO signaling plasma membranes and their caveolae directly from intact, in endothelial cells occurs largely at the level of eNOS activity perfused rat lungs. eNOS is found in the luminal plasma controlled by cofactors and targeting of eNOS to specific membranes and is markedly enriched in the purified caveolae. intracellular membranes (19). Because palmitoylation of eNOS does not significantly influ- eNOS is dually acylated by cotranslational N-myristoylation ence its membrane association, we next examined whether this and posttranslational cysteine palmitoylation (20-23). N- modification can affect eNOS targeting to caveolae. Wild-type myristoylation of eNOS is necessary for membrane association eNOS, but not the palmitoylation mutant form of the enzyme, and for subsequent palmitoylation at cysteines 15 and/or 26 as colocalizes with caveolin on the cell surface in transfected NIH determined in broken cell lysates (23). Functionally, in cul- 3T3 cells, demonstrating that palmitoylation of eNOS is tured aortic endothelial cells, intact blood vessels and heter- necessary for its targeting into caveolae. These data suggest ologous expression systems, eNOS is expressed primarily in the that the subcellular targeting ofeNOS to caveolae can restrict Golgi region of cells and such localization is necessary for NO signaling to specific targets within a limited microenvi- optimal stimulated release of NO from intact cells (24). The ronment at the cell surface and may influence signal trans- importance of eNOS cysteine palmitoylation is less clear duction through caveolae. because mutation of the palmitoylation sites inhibits protein palmitoylation but does not significantly influence eNOS The compartmentalization of plasma membrane proteins is enzyme activity, in vitro or partitioning into high speed mem- critical for specificity of intracellular signaling pathways. In- brane fractions (23), suggesting that palmitoylation may serve tegral membrane proteins, such as hormone receptors, are another function, such as specific membrane targeting. Here anchored in biological membranes by hydrophobic stretches of we show that eNOS is targeted to caveolae, in vivo and in vitro, amino acids comprising transmembrane domains, whereas via palmitoylation of the protein on cysteines 15 and 26. These other proteins, such as G-proteins, are associated and targeted observations provide molecular evidence for a novel consensus to subcellular membranes by the co- or posttranslational lipid sequence that may be sufficient for localization of proteins to modifications N-myristoylation, palmitoylation, or prenylation caveolae and suggest a role for NO in modulating signal (1). Signals initiated at the cell surface can propagate via transduction through these plasma membrane compartments. soluble intracellular messengers, changes in protein phosphor- ylation, protein-protein interactions, and protein-lipid inter- actions. MATERIALS AND METHODS The specialization of plasma membrane microdomains is a Materials and Antibodies. Dulbecco's modified Eagle's potential mechanism for integrating extracellular signals into medium (DMEM), glutamine, trypsin-EDTA, penicillin/ intracellular messages. For example, caveolae are plasma streptomycin, and fetal bovine serum (FBS) were from membrane invaginations composed primarily of glycosphingo- GIBCO. Rabbit polyclonal antibodies to eNOS and caveolin lipids, cholesterol, and the integral membrane protein, caveo- were from Transduction Laboratories (Lexington, KY); eNOS lin. Several proteins involved in signal transduction, such as monoclonal antibody (mAb) was provided by J. Pollock (Med- inositol 1,4,5-triphosphate receptors, calcium ATPase, mem- ical College of Georgia) and e-COP mAb was provided by M. bers of the src family of nonreceptor tyrosine kinases, G proteins, G protein-coupled membrane receptors, and gan- Krieger (Massachusetts Institute of Technology). gliosides have been found in caveolae or in Triton X-100- Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; BLMVEC, bovine lung microvascular endothelial cells; TIM, The publication costs of this article were defrayed in part by page charge Triton X-100 insoluble membranes; BAEC, bovine aortic endothelial payment. This article must therefore be hereby marked "advertisement" in cells; WT, wild type; TGN, trans-Golgi network. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 6448 Cell Biology: Garcl'a-Cardefia et al. Proc. Natl. Acad. Sci. USA 93 (1996) 6449 Cell Culture. Bovine aortic endothelial cells (BAEC) and positively charged colloidal silica particles that coated the bovine lung microvascular endothelial cells (BLMVEC) were luminal endothelial cell surface of the pulmonary vasculature. obtained and grown in tissue culture as described (21, 25). NIH Silica coating followed by cross-linking created a stable silica 3T3 cells were grown in DMEM supplemented with 10% pellicle that specifically marked luminal plasma membranes (vol/vol) FBS, L-glutamine (1 mM), penicillin (100 units/ml), and allowed for purification of these membranes from tissue streptomycin (100 ,ug/ml), and tetrahydrobiopterin (100 ,uM; homogenates by centrifugation. The sedimented pellets (P) ref. 26). Subconfluent NIH 3T3 cells were transiently trans- contained highly purified endothelial cell luminal plasma fected with bovine wild-type (WT) or C15/26S palmitoylation membranes with associated caveolae and showed ample en- mutant eNOS cDNAs subcloned into the mammalian expres- richment ofvarious endothelial cell surface markers relative to sion vector, pcDNA3 (23), according to the standard calcium the starting whole lung homogenates (H; refs. 4 and 11). phosphate precipitation method. After overnight transfection, Caveolae were removed from P by shearing during homoge- cells were trypsinized and cultured onto gelatin coated cov- nization at 4°C in the absence of Triton X-100. These homo- erslips for confocal, immunofluorescence microscopy. genates were subjected to sucrose density centrifugation to Triton X-100 Solubility of Endothelial Proteins. One yield two collected fractions; a low density fraction of a 100-mm dish of confluent BAEC (passage 2-4) was used for homogenous population of biochemically and morphologically each sample. Each dish was washed two times with cold distinct caveolar vesicles (V'). Protein samples from each phosphate-buffered saline (PBS) and cells were released by fraction (5 ,ug) were separated by SDS/PAGE (5-15% gradi- incubating in MBS (125 mM NaCl/20 mM Mes, pH 6.0) plus ent gels) and electrotransfered to nitrocellulose membranes 0.02% EDTA for 10 min on ice. Cells were collected by for immunoblotting with antisera to eNOS and caveolin as centrifugation for 5 min at 1000 x g at 4°C. The pellets were described above. suspended in 0.5 ml of MBS plus 1% Triton X-100 and incubated on ice for 30 min. The samples were Dounce homogenized (20 strokes) and centrifuged for 5 min at 16,000 RESULTS x g at 4°C (10). The supernatant fraction was collected and Triton X-100 insolubility was considered a hallmark of cy- designated the Triton-soluble fraction. The pellet was resus-