Nitric oxide regulates endocytosis by S-nitrosylation of dynamin

  1. Gaofeng Wang*,
  2. Nader H. Moniri*,†,
  3. Kentaro Ozawa†,‡,§,
  4. Jonathan S. Stamler‡,§, and
  5. Yehia Daaka*,,∥,**
  1. *Departments of Surgery, ‡Medicine, and Pharmacology and Cancer Biology and §Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710; and ∥Department of Pathology, Medical College of Georgia, Augusta, GA 30912

  1. Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 12, 2005 (received for review September 23, 2005)

 
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Abstract

The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the β2 adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both β2 adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both β2 adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.

The response of cells to their environment is partly determined by the complement of receptors expressed on the plasma membrane. Surface receptor expression is a function of a dynamic equilibrium between vesicle-confined receptor endocytosis (internalization) and receptor exocytosis (recycling and synthesis). The large GTPase dynamin plays a central role in the endocytotic budding of vesicles from the plasma membrane (1). Dynamin is thought to act as either a mechanochemical enzyme that executes vesiculation (2, 3) or, like other GTPases, to control downstream effector(s) that, in turn, mediate the vesicle fission (4). In particular, dynamin self-assembles to form a collar at the neck of invaginating pits and hydrolyzes GTP in the act of fission (5). Although dynamin activity can be regulated by phosphorylation (6–8) and by interactions with lipids (9) and partner proteins (10–12), the mechanisms of self-assembly and regulated GTP hydrolysis remain poorly understood.

Dynamin binds endothelial NO synthase (eNOS) (13), and emerging evidence suggests that eNOS may regulate vesicle trafficking (14, 15). It has been proposed that one key determinant of specificity in NO signaling is the interaction between NOSs and proteins that are targets of S-nitrosylation (16). We have therefore tested the hypothesis that NOS and its product NO regulate vesicular trafficking through S-nitrosylation of dynamin.

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Results

Activation of β2 adrenergic receptors (β2AR) with isoproterenol (ISO) initiates dynamin-dependent receptor internalization (17), which we used as a measure of vesicle trafficking from the plasma membrane. Although NO is ubiquitous in vivo, most studies examining receptor internalization have been carried out in model cell lines that do not exhibit measurable NOS activity [e.g., human embryonic kidney (HEK) and green monkey kidney fibroblast (COS) cells to allow for overexpression of proteins] (18, 19). To assess the possibility of NO-dependent regulation of receptor trafficking, we compared the rate of ISO-mediated β2AR internalization in native HEK cells and HEK cells that express NOS activity (HEK-eNOS) (20). ISO treatment induced time-dependent increases in β2AR internalization in both cell types (Fig. 1A), but the rate and magnitude of receptor endocytosis were significantly greater in the HEK-eNOS cells. Similar results were obtained by using HEK cells transiently cotransfected with expression plasmids encoding eNOS and β2AR; the expression of eNOS enhanced the rate of ISO-mediated β2AR internalization as compared with wild type cells (see Fig. 6, which is published as supporting information on the PNAS web site). Notably, the NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME) but not its inactive enantiomer d-NAME, markedly decreased the ISO-mediated internalization of β2AR in HEK-eNOS cells (Fig. 1B). Neither l-NAME nor d-NAME affected the ISO-induced β2AR internalization in native HEK cells (data not shown), and the addition of exogenous l-arginine to HEK-eNOS cells reversed the inhibitory effects of l-NAME on the ISO-induced β2AR internalization (see Fig. 7, which is published as supporting information on the PNAS web site). Furthermore, the slow-releasing NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-amonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) increased ISO-mediated β2AR internalization in native HEK cells that are deficient in NO but had no added effect in HEK-eNOS cells (Fig. 1C). The effect of NO is not restricted to β2AR internalization: stimulation with EGF promoted endocytosis of significantly more EGF receptors in the HEK-eNOS, compared with HEK cells (Fig. 1D), and, similar to the situation for β2AR, l-NAME inhibited the internalization (Fig. 1D). Furthermore, exposure to DETA-NO increased EGF-induced EGF receptor internalization in HEK but not HEK-eNOS cells (Fig. 1E). These data establish that NOS and its product NO enhance ligand-mediated internalization of both G protein-coupled receptors and receptor tyrosine kinases. They also suggest distinct mechanisms of receptor internalization in the presence and absence of NO.

Fig. 1.
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Fig. 1.

NOS regulates agonist-induced receptor internalization. (A) Internalization of β2AR in native HEK and HEK-eNOS cells. Cells were stimulated with ISO (10 μM) for the indicated time and receptor internalization determined by flow cytometry performed on cells transiently overexpressing Flag-epitope-tagged β2AR. (B) l-NAME inhibits ISO-mediated β2AR internalization in HEK-eNOS cells. Cells were pretreated overnight with l-NAME (1 mM) or its inactive enantiomer d-NAME (1 mM), followed by stimulation with ISO (10 μM) for 30 min. (C) NO potentiates the ISO-mediated β2AR internalization in native HEK cells. Cells were pretreated with DETA-NO for 4 h, followed by stimulation with ISO for an additional 30 min. (D) Internalization of EGF receptor in HEK and HEK-eNOS cells. Cells were treated, or not, with l-NAME, stimulated with EGF (5 ng/ml) for 30 min, and receptor internalization was determined by flow cytometry. (E) NO potentiates the EGF-induced internalization of EGF receptor in native HEK cells. Cells were pretreated with DETA-NO for 4 h followed by stimulation with EGF for an additional 30 min. Data in A–E are presented as percent decrease of total membrane-expressed receptor, and each point represents the mean ± SE of three to five experiments. *, P < 0.05 versus control sample.


It has been reported that eNOS interacts with dynamin 2 and that the interaction may activate eNOS (13). The region of dynamin 2 that binds eNOS involves a proline-rich domain that is conserved across dynamin isoforms (1). We found that eNOS coprecipitates with dynamin 1 from HEK-eNOS cells (Fig. 2A Upper) and that stimulation with ISO increased the amount of dynamin that is precipitated by eNOS (normalized for amounts of eNOS). In reciprocal immunoprecipitation experiments, ISO promoted an increase in the amount of dynamin that was precipitated by limiting amounts of antibody (normalized for eNOS) (Fig. 2 A Lower). The stimulation with ISO induced a 3-fold increase in eNOS–dynamin complex formation (see Fig. 8, which is published as supporting information on the PNAS web site) and ≈5% of total eNOS coprecipitated with dynamin. We used a control antibody to demonstrate specificity of the eNOS–dynamin interaction that is induced by ligand (see Fig. 9, which is published as supporting information on the PNAS web site). Together, these results suggest that dynamin might self-assemble on eNOS. Because ISO is known to increase eNOS activity (21), we explored the possibility that formation of the eNOS–dynamin complex may be NO-dependent. Indeed, we observed that the NOS inhibitor l-NAME reduced both basal- and ISO-mediated interactions between dynamin and eNOS (Figs. 2 A and 8). In cultured neurons, dynamin concentrates near the plasma membrane (22), and we found that l-NAME inhibited the ISO-induced redistribution of dynamin 1 to the particulate fraction (Fig. 2B). Thus, the expression of dynamin in the particulate fraction and dynamin's interaction with eNOS appear to depend on NO. More generally, our results indicate that agonist-stimulated assembly of dynamin is directly regulated by endogenously produced NO.

Fig. 2.
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Fig. 2.

Dynamin interacts with eNOS and is S-nitrosylated. (A) NOS-activity-dependent interaction between dynamin 1 and eNOS. HEK-eNOS cells expressing dynamin 1 were exposed (+), or not (–), to l-NAME (10 mM) for 48 h and treated (+), or not (–), with ISO (10 μM) for 10 min at 37°C. Note that l-arginine is present in the cell culture medium at a concentration of 0.4 mM. (Upper) eNOS was immunoprecipitated (IP), and coprecipitated dynamin was detected by protein immunoblotting (IB). Amounts of immunoprecipitated eNOS are also shown (IB: eNOS). (Lower) Dynamin 1 was immunoprecipitated with limiting amounts of antibody (see Materials and Methods), and coprecipitated eNOS was detected by protein immunoblotting. The amount of immunoprecipitated dynamin 1 is also shown (IB: dynamin). (B) Agonist-mediated, NOS-dependent redistribution of endogenous dynamin 2. HEK-eNOS cells were pretreated with l-NAME (10 mM) for 48 h, then exposed to ISO (10 μM) for 10 min, followed by isolation of plasma membranes. Dynamin expression was determined by protein immunoblotting. The membrane was stripped of Ig and reblotted with anti-actin antibodies to verify equal protein loading. (C) S-nitrosylation of purified dynamin 1 protein. Purified proteins (40 μg) were mixed with DEA-NO (10, 100 μM), GSNO (10, 100 μM), or GSH (100 μM, used as a control) for 20 min, and protein S-nitrosylation (SNO) was determined by using the biotin switch assay. Membranes were stripped and reblotted with anti-dynamin 1 antibody to show that protein loading was equivalent in each lane. CN, control. (D) Dynamin is S-nitrosylated in situ. HEK cells were treated with 1 mM DETA-NO (which is estimated to produce submicromolar steady-state concentrations of NO over 24 h) for the indicated times, immunoprecipitated with anti-nitrosocysteine antibodies, and immunoblotted for dynamin 1. Total cell lysates were used to demonstrate equal protein loading (Lower). (E) Agonist-mediated S-nitrosylation of endogenous dynamin 2. HEK-eNOS cells were treated with ISO (10 μM) at 37°C, and cell lysates were subjected to SNO analysis. ISO treatment induced the S-nitrosylation of endogenous dynamin 2, which was identified by stripping the filter and reblotting it with anti-dynamin antibodies. The gel shows two additional S-nitrosylated proteins. The uppermost band was unaffected by ISO and was used as an internal control. The images in A–E are representative of three experiments.


NO exerts many cellular effects by S-nitrosylation, i.e., through covalent attachment to cysteine thiols in target proteins (to form an S-nitrosothiol; SNO) (23), and the selectivity of S-nitrosylation may be conferred through direct interactions between NOSs and their targets (16). To determine whether dynamin is a substrate for S-nitrosylation (18), we exposed the purified protein to two different classes of NO donor. Both diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NO), a synthetic NO donor that releases NO at controlled rates, and the endogenous NO donor S-nitrosoglutathione (GSNO) readily S-nitrosylated dynamin, as determined by using the biotin switch assay (Fig. 2C). Furthermore, loading of dynamin with guanine nucleotide consistently promoted its S-nitrosylation (GTPγS > GDP) compared with the nucleotide-free form (data not shown). Binding of GTP may, therefore, serve to allosterically promote S-nitrosylation of dynamin.

We conducted a series of experiments designed to increase confidence in the physiological relevance of dynamin S-nitrosylation. Initially, we treated HEK cell lysates with DETA-NO and verified that dynamin can be S-nitrosylated in situ (data not shown). We then exposed HEK cells to a sustained flux of NO (DETA-NO, half-life ≈56 h) and assayed for SNO-dynamin by performing immunoprecipitations with anti-SNO antibodies, followed by immunoblotting for dynamin. The ability to immunoprecipitate SNO-dynamin from intact cells established that the S-nitrosothiol in dynamin is stable in vivo (Fig. 2D). Moreover, the variations in amount of SNO-dynamin that were detected over 24 h of constant NO exposure indicated that dynamin may undergo cycles of S-nitrosylation and denitrosylation: S-nitrosylation peaked at 4 h, declined gradually through 12 h, and then increased after 16 h (Fig. 2D). Finally, treatment of HEK-eNOS cells with ISO (in the absence of exogenously added NO) rapidly induced the S-nitrosylation of endogenous dynamin (Fig. 2E). Hence, dynamin S-nitrosylation in situ is directly coupled to ligand binding. Steady-state levels of SNO-dynamin may reflect not only the activity of eNOS but also activities that subserve removal of NO groups (16).

As an initial test of the effects of S-nitrosylation on dynamin function, we assayed the GTPase activity of dynamin exposed to NO. Incubation of dynamin with either DEA-NO (20 min, 25°C, pH 7.0) or GSNO (20 min, 25°C, pH 7.0) produced a significant increase in GTP hydrolysis (P < 0.05). More detailed studies showed that GSNO (data not shown) and DEA-NO produced a sustained increase in dynamin GTPase activity (Fig. 3 A and B). Thus, S-nitrosylation of dynamin, like phosphorylation (6, 8, 17) of dynamin, increases its enzymatic activity. Because self-assembly of dynamin is a critical determinant of the rate of GTP hydrolysis (3, 5), we hypothesized that NO may promote dynamin assembly. Indeed, under conditions that produced an increase in GTPase activity, DEA-NO also induced the self-assembly of purified recombinant dynamin (Fig. 3C). In addition, pretreatment of recombinant dynamin with DEA-NO accelerated dynamin's ability to form helical tubes: Whereas native dynamin required 3 h to complete formation of spirals around the lipid tubes (control; 3h, 93 ± 8% vs. DEA-NO; 3h, 94 ± 6%; n = 3) (Fig. 3 D and E), NO-treated dynamin markedly accelerated the rate of spiral formation, yielding neat spirals within 30 min (DEA-NO; 30 min, 84% ± 7% vs. control; 30 min, 21% ± 3%; n = 3) (Fig. 3 F and G).

Fig. 3.
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Fig. 3.

Effect of S-nitrosylation on GTP hydrolysis and self-assembly. NO dose- (A) and time- (B) dependent effects on GTP hydrolysis by dynamin. Purified dynamin 1 was treated with the indicated concentration of DEA-NO (A) for 30 min or with DEA-NO (1 mM) for the indicated times (B), followed by measurement of GTP hydrolysis. Each point represents the mean ± SE of three independent experiments. Data are presented as percent increase in GTP hydrolysis in which the value 100 corresponds to spontaneous activity (≈20 pmol/μg protein per minute). *, P < 0.05 versus untreated samples. (C) NO induces self-assembly of dynamin. Purified dynamin 1 protein was treated with DEA-NO for 20 min at the indicated concentrations, separated on a native nonreducing gel, and stained with Coomassie blue. Numbers on the left are molecular mass markers (in kDa). (D–G) Purified dynamin proteins were mixed with preformed phosphatidylserine lipid and incubated (F and G), or not (D and E), with DEA-NO (1 mM) for 30 min (D and F) or 3 h (E and G). Representative electron microscopy images are shown. (Scale bar, 100 nm.)


Dynamin has six cysteine residues. To identify the cysteine(s) that are targets of S-nitrosylation, each of the six candidate cysteines (C86, C169, C427, C442, C607, and C708) was changed to an alanine, and the mutant dynamins were expressed in both HEK and HEK-eNOS cells (Fig. 4A). Whereas none of the C-to-A mutants had a substantial effect on ISO-mediated β2AR internalization in native HEK cells, the C607A mutant markedly attenuated ISO-mediated β2AR internalization in HEK-eNOS cells (Fig. 4B), essentially recapitulating the effect of NOS inhibition, which was also confined to HEK-eNOS cells (Fig. 1B), and suggesting that the NOS effect is identified with the C607 residue. Notably, the attenuation of β2AR internalization by the C607A dynamin was comparable in magnitude with that seen with the GTPase-deficient dominant-negative K44A mutant (6, 18, 19). These data suggest that NO can regulate the function of dynamin in receptor-mediated endocytosis and that the effect of NO can be ascribed to S-nitrosylation of C607. Moreover, these results indicate that, in the presence of NO, the molecular machinery subserving receptor-mediated endocytosis critically depends on S-nitrosylation of dynamin. In other words, in the NO-governed mode, the internalization process depends on the S-nitrosylation of dynamin, whereas, in the absence of NO, dynamin and other components of the internalization machinery are alternatively regulated.

Fig. 4.
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Fig. 4.

Mapping the S-nitrosylated cysteine residue in dynamin 1. (A) Expression of wild-type and C-to-A dynamin mutant proteins in HEK-eNOS cells. An equal amount of total cell lysate was separated on SDS/PAGE, and dynamin expression was determined by immunoblotting using anti-dynamin 1/2(Top), anti-dynamin 1 (Middle), or anti-dynamin 2 (Bottom) antibodies. (B) Effect of the expression of individual C-to-A mutant forms of dynamin 1 on ISO-mediated β2AR internalization in native HEK and HEK-eNOS cells. Cells were transfected with cDNAs encoding the individual C-to-A dynamin 1 forms together with Flag-epitope-tagged β2AR and then subjected to ISO-mediated receptor internalization, as measured by flow cytometry. Data are presented as the fraction of internalized receptor, where 100% corresponds to the value of ISO-induced internalized receptor in cells expressing wild-type dynamin. Each point represents the mean ± SE of three independent experiments. *, P < 0.05 versus values obtained from corresponding native HEK and HEK-eNOS cells. (C) C607 is the principal site of S-nitrosylation in dynamin 1. Purified dynamin proteins were mixed with DEA-NO for 20 min and subjected to assay for SNO by using the biotin switch. The filter was stripped and reblotted with anti-dynamin 1 antibodies to demonstrate equivalent loading of the samples. (D) Dynamin 1 C607A exhibits impaired NO-mediated multimerization. Purified dynamin 1 proteins were treated as in C, separated on a native gel, and stained with Coomassie blue. (E) DEA-NO does not enhance dynamin 1 C607A-mediated GTP hydrolysis. Purified protein was treated with DEA-NO and subjected to GTP hydrolysis assays, as described in Fig. 3A. (F and G) In response to NO, dynamin C607A does not form spirals around lipid tubes. Dynamin 1 wild-type (F) and C607A (G) proteins were assessed for their ability to form spirals, as described in Fig. 3; DEA-NO (1 mM) for 30 min. (Scale bar, 100 nm.)


To provide further evidence that the dominant-negative-like effects of dynamin C607A can be identified with S-nitrosylation, we generated baculoviruses that encode for wild-type dynamin as well as C607A and C86A (control) mutant proteins, purified the recombinant proteins from Sf9 cells, and assessed their susceptibility to S-nitrosylation by using the biotin switch assay (18). Whereas dynamin C86A and wild-type proteins were S-nitrosylated by NO, the C607A mutant protein was not (Fig. 4C). Furthermore, in tests of self-assembly (Fig. 4D) and enzymatic activity (Fig. 4E), the dynamin C607A mutant could not be stimulated by DEA-NO. In contrast, NO induced an increase in GTPase activity of wild-type dynamin (Fig. 3A) and promoted the self-assembly of dynamin C86A (Fig. 4C). As an additional measure of dynamin function, we assessed its ability to form helical tubes when incubated with liposomes. Treatment with DEA-NO for 30 min induced the assembly of wild-type dynamin spirals (Fig. 4F) but had no effect on the C607A mutant protein (Fig. 4G). Collectively, these data demonstrate that S-nitrosylation of C607 mediates the stimulatory effects of NO on both the self-assembly and GTPase activities of dynamin.

Dynamin's role in vesicle trafficking is not restricted to receptor internalization (1, 24). To explore the possibility that the effects of S-nitrosylation may be generalized to other dynamin-regulated functions, we examined the role of NO in internalization of uropathogenic Escherichia coli into bladder epithelial cells, which express endogenous dynamin 2 and eNOS (data not shown). Treatment with DETA-NO (at doses that do not affect bacterial viability) or with l-NAME (Fig. 5B) caused significant increases and decreases, respectively, in E. coli invasion (Fig. 5A). In addition, forced expression of C607A dynamin, like the dominant-negative K44A mutant, greatly decreased E. coli invasion, whereas overexpression of the C86A mutant (which served as a control) did not affect bacterial entry (Fig. 5C).

Fig. 5.
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Fig. 5.

Effect of dynamin S-nitrosylation on bacterial uptake in epithelial bladder cells. (A) NO regulates bacterial invasion. Cells were pretreated with l-NAME (10 mM) for 16 h or DETA-NO (1 mM) for 4 h and mixed with E. coli at a multiplicity of infection 100 for 1 h at 37°C. Extracellular bacteria were killed with gentamicin, and cell lysates were used to quantify the invading bacteria by counting colonies grown on LB agar plates. Typically, control cells yielded 80–200 bacterial colonies. (B) l-NAME and DETA-NO do not affect bacterial growth. E. coli were directly mixed with l-NAME (10 mM) or DETA-NO (1 mM) for 3 h at 37°C and their growth determined. (C) Effect of dynamin C607A expression on E. coli invasion. Cells were transfected with cDNAs encoding the indicated forms of dynamin 1, and bacterial invasion was quantified. CN, control. Data in A–C represent the mean ± SE of three experiments done in triplicate. *, P < 0.05 versus control.


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Discussion

Dynamin proteins are master regulators of vesicle trafficking, including receptor endocytosis (1) and pathogen invasion (19, 24). Self-assembly of dynamin, hydrolysis of GTP, and movement of dynamin to the membrane are obligatory events in endocytotic vesicle budding. Our data indicate that NO plays a critical role in regulating these fundamental aspects of dynamin function. NO that is derived from eNOS activates dynamin by S-nitrosylating a single cysteine residue that is conserved among dynamin proteins (25, 26). eNOS, which is present in caveolae (27), may serve as an anchor for dynamin, thereby stabilizing dynamin at the plasma membrane. The NO-dependent interactions between dynamin and eNOS may regulate both the subcellular localization and multimerization of dynamin as well as its assembly-linked activities.

Our findings demonstrate that agonist-mediated internalization of the β2 adrenergic and EGF receptors can be regulated by a NO-dependent mechanism and that the target of NO is C607 of dynamin, which is confined to a locus within the pleckstrin homology (PH) domain at which a cysteine residue and SNO motif (28) is found in numerous PH domain-containing proteins. Although cells can still support vesicle fission in the absence of NO, the magnitude of receptor internalization is lower in this situation, a finding that is well rationalized by the stimulatory effects of S-nitrosylation on dynamin self-assembly and GTPase activity. The observation that the role of C607 is conserved across dynamin functions (i.e., both receptor internalization and bacterial invasion) increases further the likelihood that NO regulates membrane trafficking in many physiological situations. Our results also indicate that the mechanism used for internalization may depend critically on the availability of NO, and it is noteworthy that disease states are often characterized by NO deficiency. The dynamin C607A mutant should provide a unique means to assess the role of S-nitrosylation in the various cellular functions of dynamin in health and disease, and points to a new therapeutic target in the fight against viral and microbial infection.

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Materials and Methods

Cell Culture and Transfection. HEK293 and bladder epithelial 5637 cells were obtained from American Type Culture Collection, and HEK293 cells stably expressing eNOS (HEK-eNOS) were obtained from W. Sessa (Yale University, New Haven, CT). HEK and HEK-eNOS cells were maintained in DMEM, and bladder epithelial cells were maintained in RPMI medium 1640. The culture media contained 10% FBS. Cells were transiently transfected by using the appropriate cDNAs (see Supporting Materials and Methods, which is published as supporting information on the PNAS web site) and Lipofectamine (Invitrogen). Experiments were performed 2 days after transfection, and cells were serum-starved overnight in culture medium containing 10 mM Hepes, pH 7.5, and 0.1% BSA.

Isolation of Plasma Membrane. Cells incubated with or without 10 μM isoproterenol were washed with ice-cold PBS, scraped into buffer A (0.25 M sucrose, 10 mM Tris, pH 7.4, and 1 mM EDTA) and disrupted by Dounce homogenization. Nuclei and unbroken cells were removed by centrifugation at 1,000 × g for 10 min, and a plasma membrane fraction was precipitated by centrifugation of the supernatant at 3,000 × g for 15 min. Crude plasma membranes were washed three times with buffer A and resuspended in RIPA buffer (50 mM Tris·HCl, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM Na2HPO4, protease inhibitor mixture set II, 1 mM phenylmethylsulfonyl fluoride, and 100 μM Na3VO4). Protein concentration was determined by using the Bradford assay, and equal amounts of proteins were fractionated on SDS/PAGE gel.

Purification of Recombinant Dynamin. Protein purification was carried out as described in ref. 29; see also Supporting Materials and Methods.

Determination of Protein S-Nitrosylation. Measurement of S-nitrosylated dynamin in cultured cells was performed by immunoprecipitation with anti-nitrosocysteine antibody, followed by immunoblotting with dynamin antibody. Measurement of S-nitrosylated cysteine residues in vitro was performed by using the biotin switch method (30). Briefly, dynamin was diluted in HEN buffer (250 mM Hepes, 1 mM EDTA, and 0.1 mM neocuprine, pH 7.7) to a concentration of 0.2–0.5 mg/ml and mixed in the dark with DEA-NO (10 μM, 100 μM) for 20 min at ambient temperature. Proteins were desalted by using Micro Bio-Spin 6 chromatography column (Bio-Rad) preequilibrated with HEN buffer, mixed with SDS and methyl methanethiosulfonate (Sigma), briefly vortexed, and incubated at 50°C for 20 min. Proteins were desalted again by using the Micro Bio-Spin 6 columns and mixed with 0.2 mM biotin-HPDP (Pierce) and 2.5 mM ascorbate at ambient temperature for 1 h. The proteins were separated on SDS/PAGE, transferred to a nitrocellulose filter, blotted with avidin-conjugated horseradish peroxidase and visualized by chemiluminescence.

GTP Hydrolysis Assay. GTPase activity was determined by measuring the release of 32Pi from [γ-32P]GTP-dynamin as described in ref. 29. Purified recombinant dynamin 1 (2 μg) was added to a final volume of 75 μl of GTPase assay buffer (20 mM Hepes, pH 7.0, and 10 mM MgCl2) on ice. Reactions were initiated by the addition of 25 μl of 1 mM [γ-32P]GTP mixture (≈200 cpm/pmol) in GTPase assay buffer, followed by incubation at 30°C for the indicated times. The reactions were terminated by the addition of 1 ml of isobutyl alcohol/benzene (vol/vol, 1:1) and 0.25 ml of 4% tungstosilic acid in 3 N H2SO4, followed by brief mixing. Ammonium molybdate (10%) was added, followed by vigorous vortexing and brief centrifugation, and the aqueous phase solution, containing released 32Pi from [γ-32P]GTP, was counted by using a β-counter (Packard).

Dynamin Lipid Tube Assay. A dynamin lipid-tube-formation assay was done essentially according to the protocol of Hinshaw (3). Dry lipid mixture was prepared by evaporation under a stream of nitrogen and subsequently hydrated with preheated buffer (20 mM Hepes, pH 7.2, 1 mM MgCl2, 150 mM NaCl, 2 mM EGTA, 1 mM DTT, 1 mM PMSF, and complete protease inhibitors) for at least 30 min. Lipid mixture was extruded 15 times through a 1-μm polycarbonate membrane (Avanti Polar Lipids) to form unilamellar vesicles. Dynamin was treated, or not, with DEA-NO and mixed with the synthetic phosphatidylserine 18:1 lipid vesicles for 2 h at 25°C. The dynamin lipid tubes were adsorbed to carbon-coated electron microscope grids, washed with HCB 150, and stained with 1% uranyl acetate. Images were selected randomly and were obtained by using a Philips 301 electron microscope at 80 kV. The number of tubes with neatly assembled, or not, dynamin was counted from three independent experiments.

Receptor Sequestration. Agonist-induced internalization of Flag-β2AR was determined by flow cytometry (6, 29). HEK and HEK-eNOS cells transiently expressing the Flag-β2AR in six-well dishes were stimulated with freshly prepared 10 μM ISO for the indicated times. After washing with ice-cold PBS, the cells were incubated with M2 anti-Flag antibody (1:625 dilution) for 1 h, washed, and incubated with FITC-conjugated anti-mouse antibody for another 1 h. Receptor sequestration was defined as the fraction of total cell surface receptors removed from the plasma membrane after agonist treatment and, thus, were not accessible to antibody from outside the cell.

Immunoprecipitation and Immunoblotting. Overnight serum-starved dynamin-expressing cells in 100-mm plates were pretreated with the appropriate concentrations of chemicals as indicated in the figure legends. Cells were stimulated with 10 μM ISO at 37°C for the indicated times, washed twice with ice-cold PBS, lysed in 1 ml RIPA buffer (29) on ice, and clarified by centrifugation. Cell lysates were mixed with anti-dynamin or anti-nitrosocysteine antibody and protein G plus/protein A-agarose beads and rotated for 4 h at 4°C. For the dynamin immunoprecipitation experiment described in Fig. 2, we used limiting amounts (1 μg/1 mg cell lysate) of the anti-dynamin antibody. Immunoprecipitated proteins were resolved on 4–20% SDS-polyacrylamide gels (Invitrogen), transferred to nitrocel-lulose membranes, and immunoblotted with dynamin antibodies. Proteins were visualized by using chemiluminescence and quantified by using Fluor-S MultiImager (Bio-Rad).

In Vitro Bacterial Invasion. Transiently transfected human bladder epithelial cells were equally seeded into 96-well plates and allowed to grow to confluence. E. coli strain ORN103 transformed with the pSH2 plasmid encoding the type 1 fimbrial gene cluster (chloramphenicol-resistant) were obtained from S. N. Abraham (Duke University, Durham, NC) and grown overnight in LB broth with chloramphenicol (31). Cells were exposed to bacteria (at a multiplicity of infection 100) in serum-free RPMI medium 1640 supplemented with 1% BSA for 1 h at 37°C. Cell culture medium was replaced with fresh medium containing 100 μg/ml membrane-impermeable gentamycin for 1 h to kill noninvading bacteria. Cells were washed three times with PBS and lysed with 0.1% Triton X-100 in PBS, followed by plating at 1:10 and 1:100 dilutions onto LB agar plates containing 80 μg/ml chloramphenicol. Colonies were counted the following day and quantified against the number of colonies present in untransfected control cells.

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Acknowledgments

We thank R. J. Lefkowitz and S. N. Abraham for helpful comments and suggestions; C. Sample, S. Ahn, M. Delahunty, X. Q. Zhang, X. R. Jiang, M. J. Duncan, M. Foster, and A. Matsumoto for technical help; and J. House for secretarial assistance. This work was supported in part by National Institutes of Health Grant GM 62231 (to Y.D.) and the Sandler Program for Asthma Research (J.S.S.).

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Footnotes

  • ↵ ** To whom correspondence should be addressed at: Department of Pathology, CA2016, Medical College of Georgia, Augusta, GA 30912. E-mail: ydaaka{at}mcg.edu.

  • ↵ † N.H.M. and K.O. contributed equally to this work.

  • Author contributions: G.W., N.H.M., K.O., J.S.S., and Y.D. designed research; G.W., N.H.M., and K.O. performed research; G.W., N.H.M., K.O., J.S.S., and Y.D. analyzed data; and G.W., N.H.M., J.S.S., and Y.D. wrote the paper.

  • Conflict of interest statement: No conflicts declared.

  • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviations: β2AR, β2 adrenergic receptor; DEA-NO, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; DETA-NO, (Z)-1-[N-(2-aminoethyl)-N-(2-amonioethyl)-amino]diazen-1-ium-1,2-diolate; HEK, human embryonic kidney; ISO, isoproterenol; l-NAME, N G-nitro-l-arginine methyl ester; NOS, NO synthase; eNOS, endothelial NOS; SNO, S-nitrosothiol.

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  1. Published online before print January 23, 2006, doi: 10.1073/pnas.0508354103 PNAS January 31, 2006 vol. 103 no. 5 1295-1300
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