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发表于 2009-4-21 13:41 |只看该作者 |倒序浏览 |打印

作者：Bruce C. Kone, Teresa Kuncewicz, Wenzheng Zhang,  Zhi-Yuan Yu作者单位：Departments of Internal Medicine and Integrative Biology, Pharmacology,and Physiology, The University of Texas Medical School at Houston, Houston,Texas 77030 + q3 ]) F3 |! G" ^/ ]



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      【摘要】# q! ~  {2 O8 U5 l, E# z
   Nitric oxide (NO) is a potent cell-signaling, effector, and vasodilatormolecule that plays important roles in diverse biological effects in thekidney, vasculature, and many other tissues. Because of its high biologicalreactivity and diffusibility, multiple tiers of regulation, ranging fromtranscriptional to posttranslational controls, tightly control NObiosynthesis. Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity,spatial distribution, and proximity of the NOS isoforms to regulatory proteinsand intended targets are governed. Dimerization of the NOS isozymes, requiredfor their activity, exhibits distinguishing features among these proteins andmay serve as a regulated process and target for therapeutic intervention. Anincreasingly wide array of proteins, ranging from scaffolding proteins tomembrane receptors, has been shown to function as NOS-binding partners.Neuronal NOS interacts via its PDZ domain with several PDZ-domain proteins.Several resident and recruited proteins of plasmalemmal caveolae, includingcaveolins, anchoring proteins, G protein-coupled receptors, kinases, and molecular chaperones, modulate the activity and trafficking of endothelial NOSin the endothelium. Inducible NOS (iNOS) interacts with the inhibitorymolecules kalirin and NOS-associated protein 110 kDa, as well as activatorproteins, the Rac GTPases. In addition, protein-protein interactions ofproteins governing iNOS transcription function to specify activation orsuppression of iNOS induction by cytokines. The calpain andubiquitin-proteasome pathways are the major proteolytic systems responsiblefor the regulated degradation of NOS isozymes. The experimental basis forthese protein-protein interactions, their functional importance, and potentialimplication for renal and vascular physiology and pathophysiology isreviewed.
      【关键词】 interactions synthases controlling
   calmodulin; caveolae; PDZ domains; Rac guanosine 3,5'-triphosphatase; heat shock protein 90( }3 X0 g6 m/ q2 f& M& {

NITRIC OXIDE ( NO ) IS A GASEOUS free radical that functions as anendogenous mediator in diverse biological effects in numerous tissues. In thekidney and vasculature, these processes include the control of systemic andmicrovascular tone, the glomerular microcirculation, renal sodium excretion,and inflammatory responses in the glomerulus and tubulointerstitium, amongmany others. NO also impacts the renin-angiotensin and eicosanoid systems,endothelin, cytokines, and other key regulators of inflammation. Because ofits potent chemical reactivity and high diffusibility, NO production by NOsynthases (NOS) is under complex, tight control to dictate specificity of its signaling and to limit toxicity to other cellular components. Indeed, NOproduction from each of the three major NOS isoforms, neuronal NOS (nNOS; alsotermed NOS1), inducible NOS (iNOS; also termed NOS2), and endothelial NOS(eNOS; also termed NOS3), is subject to a variety of transcriptional,translational, and posttranslational controls. The posttranslational controls, which include lipid modifications, phosphorylation events, and interactionswith protein partners, serve to govern the timing, magnitude, and spatialdistribution of NO release. In turn, these mechanisms specify the inputsignals that activate NO release and the effector functions of the molecule totarget specific proteins.' f' z4 U5 [2 E7 d' I, v

Studies in recent years have uncovered an increasingly important role ofphysical association of the NOS isoforms with a variety of regulatory andstructural proteins ( Table 1 ).These interactions may be categorized as constitutive interactions, such asthose between subunits of hemoglobin, and inducible or signal-dependent interactions, such as those between the subunits of GTP-binding proteins.Although protein-protein contacts involving each of the NOS isoforms and theirfunctional importance have been largely observed and deciphered in othertissues, their functional importance in the control of normal or perturbedrenal function has not been fully explored, and, in many cases, not yet been addressed at all. Thus there are tremendous opportunities for renal researchin this area. In addition to proteins interacting directly with the NOSproteins themselves, new evidence indicates that direct interactions betweentranscription factors or coregulatory proteins play an important role incontrolling the iNOS transcriptional response. In this review, we will firstdiscuss the known NOS-protein interactions, their influence on NO productionand intracellular locale, and the biological consequences of theseinteractions. We will then consider the known and potential impact of these interactions on renal and vascular function.
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Table 1. Protein partners of nitric oxide synthases

STRUCTURAL BIOLOGY OF NOS ISOFORMS

NO is generated from L -arginine, molecular oxygen, and NADPH byNOS enzymes. Three major NOS isoforms, which share a common basic structuralorganization and requirement for substrate cofactors for enzymatic activity,have been described. nNOS, principally expressed in neural tissues andskeletal muscle but also expressed in the macula densa segment( 104 ) as well as other tubulesegments, is typically viewed as aCa 2 /calmodulin-dependent enzyme, but it is also subjectto transcriptional and other posttranslational controls( 10 ). A mitochondrial variantof this enzyme, which, in contrast to the nNOS of the brain, is myristoylatedand phosphorylated at the COOH terminus, is widely distributed among tissues,including kidney, and may play a role in cellular energetics( 19 ). eNOS, expressedpredominantly in the endothelium, is also subject to rapid regulation by Ca 2 /calmodulin as well as transcriptional,posttranscriptional, and other post-translational controls( 60 ). iNOS is induced invirtually all tissues subjected to cytokines, endotoxin, or otherproinflammatory stimuli principally at the level of transcriptional control,and it is less responsive to intracellular Ca 2 transients owing to tight calmodulin binding at ambient intracellularCa 2 levels( 1 ).5 }- l6 T( b( ~' M  @

The NOS enzymes are bidomain proteins, in which a centralcalmodulin-binding motif separates an oxygenase (NH 2 -terminal) froma reductase (COOH-terminal) domain ( Fig.1 ). The oxidase domain contains a cytochrome P -450-typeheme active site and a binding site for tetrahydrobiopterin (BH 4 ),and the reductase domain contains an electron-transfer domain that bindsflavin mononucleotide (FMN) and FAD( 1 ). NOS activity requiresbinding of calmodulin and BH 4 and the formation of a homodimer.Calmodulin binding, triggered by transient elevations in intracellular freeCa 2 levels, serves as an allosteric modulator of thethree major NOS isoforms ( 2 ).nNOS and eNOS contain 40-50 amino acid inserts in the middle of theFMN-binding subdomain that serve as autoinhibitory loops( 87 ), destabilizing calmodulinbinding at low Ca 2 concentrations and inhibitingelectron transfer from FMN to the heme in the absence ofCa 2 /calmodulin( 69, 70 ). This insert is absentfrom the structure of iNOS.
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Fig. 1. Domain structure of nitric oxide synthases (NOS). Binding sites for L -arginine (ARG), heme (H), tetrahydrobiopterin (BH 4 ),and calmodulin (CaM) are indicated. The zinc tetrathiolate cluster is shown.Numbers indicate amino acid positions in murine inducible (i) NOS. FMN, flavinmononucleotide.

Crystallographic studies have brought the structural and biochemical basisof enzymatic activity and dimerization into clearer focus. Metal binding,disulfide and hydrogen bond formation, and three-dimensional domain swappingparticipate in the regulation of the assembly and activity of the three NOSisozymes. Structures of the dimeric rat eNOS oxygenase (eNOS ox )domain ( 76 ) and humaneNOS ox and iNOS ox domains( 30, 59 ) revealed a zinctetrathiolate center, involving two cysteines (human iNOS Cys 110 and Cys 115 ) from each subunit, positioned at the bottom of thedimer and arranged so that interaction of the NH 2 -terminal"hooks" from their own subunits is favored. Site-directedmutagenesis of human iNOS Cys 115 and the corresponding human eNOSCys 99 revealed that these residues are essential for dimerstability ( 15 ). Moreover, thehuman iNOS ox domain structure with bound zinc possesses a net gain ofeight hydrogen bonds, which favors dimer stability, compared with thezinc-free structure. The extensive dimer interface creates binding sites forBH 4, sequesters the heme from solvent, and helps to structure thesubstrate-binding site and the active site. The sequestered heme makesextensive van der Waals interactions with neighboring amino acids. The BH 4 -binding pocket is also buried within the protein near the dimerinterface. Arginine binds with the side-chain terminus nestled into the narrowpart of the active site, with the guanidino group situated near the heme( 30, 59, 76 )( Fig. 1 ).0 K$ p5 k" l& @- X$ c1 y" u
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Despite the structural similarity of the three NOS isozymes, homodimers ofthe isozymes differ markedly in the association strength of their monomers,their full interfaces, and the influence of L -arginine andBH 4 on their formation and stability. These differences indimerization represent distinguishing control points and therapeutic targetsto control NOS activity. Homodimerization is dependent on the binding of L -arginine, stoichiometric amounts of heme, and BH 4 ( 93 ). In turn, dimerization potentiates NOS activity by creating high-affinity binding sites for L -arginine and BH 4, removing heme from the solventphase, and facilitating electron flow from the reductase domain to theoxygenase domain heme ( 16, 30, 59 ). Studies of recombinant iNOS ox and nNOS ox domains indicate that L -arginine and BH 4 facilitate dimerization of themonomers and protect both dimers against trypsin proteolysis, whereas theeNOS ox dimer was resistant to proteolysis under all conditions( 73 ). For nNOS ox, L -arginine alone was more effective than BH 4 alone,whereas the opposite was true for iNOS ox ( 73 ). Both L -arginine and BH 4 participate in extensivehydrogen-bonding networks that stabilize the surrounding protein. Based onurea dissociation studies ( 73 )and studies of low-temperature SDS-PAGE( 99 ), dimeric interaction isstrongest in eNOS ox, followed by nNOS ox and theniNOS ox. Although the combination of L -arginine andBH 4 promotes the best dimer stability for all the NOS isozymes, theisozymes differ in the degree to which these two compounds individuallyfacilitate stability. Both the NH 2 -terminal hooks and zinc-bindingelements help to stabilize NOS dimers, and because these structures have the greatest sequence divergence among the NOS isozymes, they might conferisozyme-specific differences in dimer stabilities( 16, 37, 38, 73 ). The NOS reductase domainsmay also participate in the formation of a dimeric holoenzyme. Studies withisolated oxygenase and reductase domains in the yeast two-hybrid system indicate that only the oxygenase domain is involved in iNOS dimer formation,whereas interactions between the reductase domains and between the reductaseand oxygenase domains are critical for dimerization of nNOS and eNOS( 99 ).. j' B3 i6 l# U. C7 \) B

DEGRADATION OF NOS PROTEINS. h) I3 S+ o, M. ]8 _& p
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Protein-protein interactions are controlled, at the most fundamental level,by the availability of the component proteins, which, in turn, is principallycontrolled by the net effects of synthesis and degradation of the proteins.NOS degradation is a regulated process influenced by diverse agents, includingglucocorticoids, caveolin-1, heat shock protein (Hsp) 90, neurotoxic molecules ( 45 ), and certain NOSinhibitors. The calpain and ubiquitin-proteasome pathways function as themajor proteolytic systems responsible for the regulated degradation of iNOS.Studies in IFN- -stimulated RAW 264.7 macrophage cells demonstrated thatdexamethasone promotes proteolytic degradation of iNOS and that the iNOS monomer is a direct substrate for cleavage byCa 2 -dependent neutral cysteine protease calpain I( 101 ). Access to thecalmodulin-binding domain appears to be critical for substrate cleavage,because purified calmodulin inhibits calpain I-mediated iNOS degradation invitro ( 102 ). Studies of iNOS,expressed by transfection in HEK-293 cells or induced in primary bronchialepithelial cells, A549 cells, or murine macrophages demonstrate that it isubiquitinated and that ubiquitination is required, to a large extent, fordegradation ( 56 ).Overexpression of caveolin-1 in human colon carcinoma cell lines, which havelow endogenous levels of this protein, results in cosegregation of a portion of cytokine-induced iNOS protein with caveolin-1 in detergent-insoluble membrane fractions and degradation there via the proteasome pathway( 22 ). Caveolin-1 and iNOS werefurther shown to bind to each other in vitro( 22 )( Table 1 ). These resultssuggest a model of tumor biology in which downregulation of caveolin-1 mayserve to promote uncontrolled iNOS activity, genotoxicity, and tumordevelopment. nNOS undergoes enhanced proteolytic degradation when exposed tocertain neurotoxins ( 45 ), when subjected to suicide inactivation with guanidine compounds ( 71 ), or when the Hsp90-basedchaperone system is inhibited with geldanamycin( 7 ). The fact that theheme-deficient monomeric form of nNOS is preferentially ubiquitinated overthat of the heme-bound homodimer suggests that ubiquitination of nNOSparticipates in the regulated proteolysis of the nonfunctional enzyme. The mechanisms of eNOS protein degradation have not been extensively studied. Theloss of endothelial function after cardiac ischemia is associated with a lossof eNOS activity due to a combination of intracellular acidosis-dependentdenaturation and proteolysis ( 40 )., @2 ?  O5 E& w1 F) O; N

HOW ARE PROTEIN-PROTEIN INTERACTIONS IDENTIFIED?( w) V+ j5 p( u1 G0 {. a' `

Several methods for identifying protein partners have been developed andsome applied to the study of NOS regulation. These include proteincross-linking ( 6, 66 ), green fluorescent protein ( 55, 74 ), phage display( 85 ), the yeast two-hybridsystem ( 27 ), chromatographictechniques ( 5 ), andfluorescence resonance energy transfer (FRET)( 54 ). One general limitationof these methods is that they generally can screen only one bait protein at atime. One of the most commonly used methods for detecting new interactions isthe yeast two-hybrid system( 27 ). This simple andinexpensive method requires no prior knowledge about the interacting proteins.The system exploits the ability to split the functional domains of atranscription factor into a DNA-binding domain and a transcriptionalactivation domain. Neither domain when expressed individually can activatetranscription, but they can be used to generate hybrid proteins to test for potential protein-protein interaction. Typically, investigators screen aprotein of interest ("bait") expressed as a fusion to aDNA-binding domain against a random library of DNA or cDNA fused to atranscriptional activation domain, expressed in yeast ("prey") bygenetic selection. Once the fusion proteins interact, the functionaltranscription factor is reconstituted and its activity can be readilymonitored using reporter gene assays or nutritional selection in yeast. Weused this method to identify an interaction of iNOS with Rac GTPases( 58 ). The yeast two-hybridsystem has a reputation for producing a significant number of false positives,and whereas the method can be used to map interactions within a complex, itcannot identify multiprotein complexes themselves, which are now routinelycharacterized by mass spectrometry (MS). In addition, because the system is based on a transcriptional readout, the protein to be studied must be largelydevoid of significant transcriptional activity. Several cytoplasmic proteinshave been shown to contain structural domains that exhibit transcriptionalactivity. Moreover, some proteins, such as cell-cycle regulators, exhibittoxicity when fused to a heterologous DNA-binding domain and expressed in yeast. Finally, the requirement of this system that the protein-protein interaction should occur at the yeast nucleus may not be appropriate forcytoplasmic or integral membrane proteins( 3 )." I0 C% s( m, ]  K$ x

An alternative method for the identification of protein-protein interactions, which is becoming increasingly popular, is to capture proteinsof interest and their interacting proteins by using high-affinity antibodiesin immunoprecipitation experiments followed by polyacrylamide gel separationof the protein mixtures. Individual or mixtures of protein bands in gels arethen subjected to in situ tryptic digestion, and the resultant peptidefragments are extracted and analyzed by MS. The unambiguous identification ofthe proteins in the mixture by MS is largely dependent on the amount, quality,and complexity of the digested peptides, the species of origin, and thestrength of the corresponding genomic and protein databases( 28 ). This methodologyrepresents an important component of the rapidly growing field of"functional genomics," and it was recently used to identify anassociation between porin and eNOS( 94 ).

With any method, additional tests are needed both to verify the interactionindependently and to establish its biological relevance. Commonly, proteinpairings identified by the yeast two-hybrid method are confirmed to occur inmammalian cells by immunoprecipitation with an antibody directed against one of the protein partners, followed by immunoblotting of the resultant immunoprecipitates with an antibody recognizing the other protein. Alternatively, glutathione S -transferase (GST) fusion constructs canbe generated for one of the proteins and used to retain the second proteinfrom cell or tissue lysates or generated as a radiolabeled in vitrotranslation product. The demonstration of colocalization by immunolabeling orFRET methods helps to solidify the findings. FRET detects the proximity offluorescence-labeled 100 A, and it can be used tomap protein-protein interactions in vivo( 54 ). Finally, the impact ofthe putative protein-protein interaction on target protein function,localization, or trafficking is tested in the presence and absence of theinteracting protein to determine the functional consequences.

PROTEIN-PROTEIN INTERACTIONS INVOLVING nNOS

PDZ Domain-Containing Proteins

The NH 2 terminus of nNOS contains a PDZ domain that participates in the formation of active nNOS dimers and interacts with a variety of otherproteins in specific regions of the cell. Among the NOS isoforms, the PDZdomain is unique to nNOS. Proteins containing PDZ domains typically localizeto specialized cell-cell contacts and often link components of signaltransduction pathways in ternary complexes. Direct interactions of severalproteins bearing PDZ domains with nNOS have been shown to influence the activity and/or the subcellular distribution of the enzyme in brain and muscle( 57 ). By anchoring nNOS tospecific targets in this manner, NO signaling is altered. In skeletal muscle, the nNOS PDZ domain scaffolds the enzyme to 1 -syntrophin,which independently binds both dystro-brevin and dystrophin, which binds toglycoproteins in the membrane( 11, 12 ). This arrangement associates nNOS with the sarcolemma, where it can generate NO to increaseperfusion of adjacent blood vessels of contracting muscle. Disruption of theseinteractions contributes to the pathophysiology of Duchenne musculardystrophy.

PDZ domain interactions also link nNOS to the muscle isoform ofphosphofructokinase in skeletal muscle and neurons( 29 ) and function at neuronalsynapses to bring nNOS in proximity to the N -methyl-D-aspartate(NMDA) receptor, allowing glutamate-stimulated Ca 2 influx to specifically activate nNOS( 97 ). For example, nNOSinteracts with postsynaptic density (PSD)-95 and PSD-93 proteins in neurons( 11 ) via direct PDZ-PDZ domaininteractions. In the rat kidney, PSD-93 is expressed in the basolateralmembranes of the thick ascending limb of the loop of Henle, macula densa cells, distal convoluted tubules, cortical collecting ducts, outer and innermedullary collecting duct, glomerular epithelium, and Bowman's capsule( 83, 96 ). In the macula densa, asite of abundant nNOS expression, a subpopulation of nNOS colocalizes withPSD-93 adjacent to cytoplasmic vesicles and the basolateral membrane( 83, 96 ). The functional importanceof this apparent association to macula densa signaling is unknown.+ _9 f6 r" i0 V7 W# D

Another protein, the COOH-terminal PDZ ligand of nNOS (CAPON), which ishighly enriched in the brain, competes with PSD-95 for interaction with nNOSin the brain ( 49, 50 ). Overexpression of CAPONresults in dissociation of PSD-95-nNOS complexes in transfected cells,disrupts the proximity of nNOS to NMDA-mediated Ca 2 influx, and thereby may restrict NO generation. CAPON contains both aCOOH-terminal domain for binding to the nNOS PDZ domain and anNH 2 -terminal phosphotyrosine-binding domain that binds the smallmonomeric G protein Dexras1( 21 ). Thus CAPON serves as anadaptor protein in a ternary complex that enhances the ability of nNOS toactivate Dexras1 ( 21 )( Fig. 2 ). Similarly, CAPONinteracts with synapsin I, II, and III through an NH 2 -terminalphosphotyrosine-binding domain interaction on the synapsins to form a ternarycomplex comprising nNOS, CAPON, and synapsin I( 49 ). Using affinitychromatography of brain extract with the nNOS PDZ domain, theCOOH-terminal-binding protein (CtBP), a phosphoprotein first identified as abinding partner to adenovirus E1A, was identified as an nNOS-binding partner.Immunoprecipitation studies show that CtBP and nNOS associate in the brain.When CtBP is expressed in Madin-Darby canine kidney cells, its distribution isprimarily nuclear; however, when wild-type but not PDZ motif-mutated CtBP iscoexpressed with nNOS, its localization becomes more cytosolic. These results suggest a new function for nNOS as a regulator of CtBP nuclear localization( 80 ).

Fig. 2. Interaction of neuronal (n) NOS with the N -methyl-D-aspartate(NMDA) receptor (NMDAR) and PDZ domain-containing proteins at neuronalsynapses. nNOS binds via PDZ-PDZ domain interactions to postsynaptic density(PSD)-95, and PSD-95 associates with the NMDAR. The resultant ternary signalcomplex efficiently couples Ca 2 entry through the NMDARto activation of nNOS. The nNOS PDZ domain also binds to the adaptor proteinCOOH-terminal PDZ ligand of nNOS (CAPON), which binds the small monomeric Gprotein Dexras1 and links it to nNOS. The proximity of these proteinsfacilitates the ability of nNOS-derived nitric oxide (NO) to S -nitrosylate and thereby activate Dexras1.
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In a yeast two-hybrid assay, Ort and co-workers( 72 ) found that thecytoplasmic domain of islet cell autoantigen 512 of type 1 diabetes, areceptor tyrosine phosphatase-like protein associated with neuronal andendocrine secretory granules, binds the PDZ domain of nNOS. Whereas theexpression of the majority of these nNOS-interacting proteins has not beenreported in the kidney, it remains possible that they influence the renal nerves or that they are influenced by the metabolic consequences of uremia andcontribute to neurological dysfunction in that setting.: n7 F' T$ F; _

Finally, the plasma membraneCa 2 /calmodulin-dependentCa 2 -ATPase serves as a regulator ofCa 2 homeostasis and signal transduction networks of thecell. The COOH-terminal region of plasma membrane Ca 2 /calmodulin-dependentCa 2 -ATPase isoform 4b binds to the PDZ domain of nNOS,negatively regulating NO production in HEK-293 embryonic kidney cells andneuro-2a neuroblastoma cell models( 88 ). Mutational analysisproved that the PDZ domains of both proteins were involved in theinteractions. These results suggest an intriguing integration betweenCa 2 and NO signaling pathways( 88 ) that may impact renalCa 2 transport.& N0 }6 G# X" s& B/ U8 l
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Protein Inhibitor of NOS

The protein inhibitor of NOS (PIN) or dynein light chain, is an 89-aminoacid polypeptide isolated from a rat hippocampal cDNA library using the yeasttwo-hybrid system and various fragments of nNOS as bait( 51 ). PIN was originallyreported to inhibit only the nNOS isoforms by dissociating the active nNOShomodimer ( 51 ). However, thesefunctional results have recently been challenged. Hemmens et al.( 48 ), using recombinant proteins, demonstrated that all NOS isoenzymes form contacts with and areinhibited by PIN. This result is in keeping with the fact that the PINrecognition sequence of nNOS (residues Met 228 to His 244 of rat nNOS) ( 20 ) is notincluded in eNOS or iNOS, lies outside the catalytic core of nNOS, and is not part of the dimerization region of nNOS. Subsequent studies byRodriguez-Crespo et al. ( 84 )suggested that PIN neither inhibits nNOS activity nor dissociates the nNOSdimer as originally proposed. These authors suggested that PIN might functionas a dynein light chain involved in nNOS axonal transport rather than as annNOS inhibitor. Within the rat kidney, PIN immunoreactivity is evident inglomerular and vasa rectae endothelial cells and apical membranes of innermedullary collecting ducts( 83 ). Rats subjected to nephrectomy exhibited threefold greater levels of inner medullary PINlevels compared with controls, suggesting that PIN inhibition of nNOS mightserve to differentially regulate NO synthesis( 83 ).

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Interactions with several receptors involved in signal transduction havebeen reported. The COOH-terminal four amino acids (GEEV) of the human 1A -adrenergic receptors have been reported to interact withthe PDZ domain of nNOS ( 75 ).Moreover, nNOS coimmunoprecipitated with epitope-tagged A -, 1B -, and 1D -adrenergic receptors, but thefunctional significance is unknown( 75 ). The serotonin 5-HT2Breceptor appears to interact via its COOH-terminal PDZ domain with both nNOSand eNOS to trigger their activation ( 63 ). The bradykininB 2 receptor and nNOS were coimmunoprecipitated in human embryonickidney cells stably transfected with human nNOS, suggesting that thebradykinin B 2 receptor may functionally interact with nNOS in vivo( 43 ). In agreement with thiswork, a synthetic peptide derived from the known inhibitory sequence of thebradykinin B 2 receptor (residues 310-329) was found tointeract with both eNOS and nNOS. Binding of this peptide blockedflavin-to-heme electron transfer of nNOS( 43, 98 ).
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Hsp90 and Caveolin-3: ?  T- o' [* Z5 C
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Hsp90 is a highly abundant cytosolic protein known to serve as a molecularchaperone in protein folding and maturation events, but it has receivedincreasing recognition as an integral component of signaling networks.Formation of a nNOS-Hsp90 heterocomplex that results in enhanced NO formationhas also been reported ( 8 ).Mechanistic studies showed that Hsp90 increases the activity of recombinantrat nNOS, increases the binding of calmodulin to nNOS to shift thecalmodulin-nNOS dose-response curve markedly to the left, and augments themaximal activity of nNOS ( 91 ).In the aggregate, these results indicate that Hsp90 directly enhancescalmodulin binding and thereby increases nNOS catalytic function. Caveolin-3has been shown to interact with nNOS in skeletal muscle, where it appears tocomprise a component of the dystrophin complex( 100 ).

PROTEIN-PROTEIN INTERACTIONS INVOLVING eNOS0 E0 o6 b3 x  k3 w7 \& g3 n
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Calmodulin, Caveolin, and Hsp90% Y8 J! l. Q' R4 t2 F% u4 h

It has long been recognized that the Ca 2 -dependentactivation of eNOS occurs through calmodulin binding when intracellular Ca 2 concentrations rise. Binding of calmodulin to itsspecific motif on eNOS displaces an adjoining( 87 ) loop on eNOS and nNOS,thereby promoting NADPH-dependent electron flow from the reductase domain tothe oxygenase domain of the protein. eNOS and calmodulin have beencoimmunoprecipitated from human endothelial cells( 86 ), and inhibitors ofcalmodulin have shown a requirement of calmodulin for eNOS activity( 87 ). As intracellularCa 2 concentrations fall, calmodulin dissociates fromeNOS. This bimodal conceptualization ofCa 2 /calmodulin-dependent regulation of eNOS has beengreatly refined to include a complex array of protein-protein interactions( Fig. 3 ) that was the subjectof a recent, intensive review( 42 ).( [/ I6 N" y: i+ o' s/ i
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Fig. 3. Protein interactions regulating endothelial (e) NOS in caveolae.Myristoylation and palmitoylation of eNOS target it to the plasma membrane ofcaveolae. Interaction with caveolin inhibits eNOS activity ( A ),whereas stimuli such as shear stress promote recruitment of heat shock protein(Hsp) 90 and Ca 2 /CaM to eNOS, increasing its activity. B : Akt, activated by shear stress, VEGF, or histamine, phosphorylatesand activates eNOS. eNOS interacting protein (NOSIP) and eNOS traffic inducer(NOSTRIN) participate in translocation of eNOS away from the caveolae tointracellular targets, which results in diminished eNOS activity.3 c# v' ?- o7 |/ v

The majority of functional eNOS in quiescent endothelial cells resides incaveolae, the result of dual acylation. Myristoylation of eNOS targets theprotein to the Golgi apparatus, where it undergoes palmitoylation. Themyristoylated and palmitoylated eNOS is then targeted to the caveolae membrane( 36, 61, 82, 89, 92 ). Several groupsindependently reported that caveolin-1, the resident integral membrane proteinof caveolae, directly interacts with and inhibits in a dynamic fashion eNOS invitro and in endothelial cells in vivo( 35, 36, 53, 67 ). Similarly, caveolin-3 wasshown to interact with eNOS in ventricular myocytes ( 23 ). The binding regionswithin caveolin-1 for bovine eNOS reside on amino acids 60-101 and, to alesser degree, amino acids 135-178( 35, 47, 53 ). Synthetic peptides corresponding to the caveolin scaffolding region (amino acids 82-101)disrupt immunocomplexes containing eNOS and caveolin-1 ( 67 ). Conversely, amino acids350-358 of eNOS comprise a consensus caveolin-binding motif( 90 ). In the resting state, eNOS appears to be tethered to caveolin-1 and inactive. However, severalagonists that raise intracellular Ca 2 concentrations, such as bradykinin, promote calmodulin binding to eNOS and caveolin dissociation from the enzyme, resulting in an activated eNOS-calmodulin complex ( Fig. 3 ). Whenintracellular Ca 2 levels return to resting levels, thecycle is reversed, with calmodulin dissociating and caveolin reassociatingwith now-inactive eNOS ( 26, 67 ). This work is supported byseveral experimental observations in vitro and in caveolin-1 knockout mice.First, the activity of purified eNOS is suppressed by incubation with peptides derived from the scaffolding domains of caveolin-1 and -3( 35 ). Second, heterologousoverexpression of wild-type caveolin-1 in COS-7 cells limited eNOS activity,whereas eNOS harboring of a mutated caveolin-binding motif was not inhibited( 67 ). Finally, mice withtargeted disruption of caveolin-1 exhibit enhanced eNOS activity, as judged bythe NO-dependent relaxation of preconstricted aortic rings( 18, 79 ).& y5 u$ y  q) D7 _( m' w6 G

Mechanistically, Ghosh and co-workers( 39 ) reported that the bindingof caveolin-1 to the reductase domain of eNOS inhibits NO synthesis bylimiting the enzyme's ability to bind calmodulin and to donate electrons tothe eNOS heme moiety. However, others have distinguished calmodulin-dependentand -independent effects of caveolin-1 on NOS function. A calmodulin-dependentmechanism, characterized by an increase in the affinity of eNOS forcalmodulin, may be operative at lower Ca 2 concentrations, whereas a calmodulin-independent mechanism apparent athigh-Ca 2 concentrations may stimulate eNOS reductaseactivity without a further change in calmodulin binding( 95 ).

Increased vascular flow( 81 ) and fluid shear stress( 33 ) also promote eNOSdissociation from caveolin and association with calmodulin to activate theenzyme in endothelial cells. Conversely, serum or its LDL fraction fromhypercholesterolemic patients increases the expression of caveolin-1 and theformation of caveolin-eNOS heterocomplexes in endothelial cells and thereby limits basal and agonist-stimulated NO release( 24, 25 ). This pathologicalresponse may represent in part the basis for endothelial dysfunction inhypercholesterolemia. Similarly, estrogen promotes increased caveolin-3 anddecreased eNOS in the heart( 103 ).

Hsp90 has been found to serve as an allosteric activator of eNOS.Garcia-Cardena and colleagues( 33 ) demonstrated that vascular endothelial growth factor, histamine, and fluid shear stress promoterapid association of Hsp90 with eNOS in endothelial cells and augment eNOSactivity. Blockade of Hsp90-mediated signaling limits both agonist-induced NOproduction and vasorelaxation. Hsp90 and eNOS appear to form a ternary complexthat includes the kinase Akt. Fontana et al.( 31 ) found that the M region of Hsp90 interacts with the NH 2 terminus of eNOS and with Akt. Moreover, stimulation of endothelial cells with vascular endothelial growthfactor promoted recruitment of eNOS and Akt to this same domain of Hsp90,facilitating Akt-driven phosphorylation of eNOS and promoting NO release.Hsp90 appears to enhance calmodulin binding to nNOS( 91 ), but this propertyremains to be firmly established for eNOS. Similarly, it remains to beestablished whether Hsp90 also serves as a scaffolding protein thatparticipates in the ushering of other regulatory molecules.. ~) ?2 q$ j+ s6 J. G

Recent evidence builds on this model by suggesting that calmodulin, caveolin, Hsp90, and eNOS form part of a dynamic, integrated signaling complex( Fig. 3 ). Immunoprecipitationof radiolabeled protein from endothelial cells with an anti-eNOS antibodyfollowed by immunoblotting of these immunoprecipitates revealed the presence of caveolin-1, Hsp90, and eNOS in the same complex( 44 ). In vitro reconstitutionexperiments showed that eNOS interacted with the other two proteins, but Hsp90and caveolin-1 did not interact with each other. Neither calmodulin nor Hsp90could directly disrupt the eNOS-caveolin interaction, but Hsp90 facilitated the ability of calmodulin to displace caveolin from eNOS( 44 ). Whether the threeproteins remain bound to eNOS in the caveolar membrane and eNOS conformationaltransitions stimulate more efficient NO production in response to a stimulusor whether recruitment of calmodulin and Hsp90 results in weak dissociation ofcaveolin from eNOS but the complex is maintained in the caveolus remainsunclear.

G Protein-Coupled Receptors

Multiple G protein-coupled receptors resident in caveolae appear tocontribute to the eNOS-membrane complex and regulate eNOS activity. Thecapacity to bind and inhibit eNOS appears to be a common feature ofmembrane-proximal regions of intracellular domain (ID) 4 of the bradykininB 2, angiotensin AT 1 receptor ( 52 ), and the ET-1 ETBreceptors, but not of the ATP P2Y2 receptor ( 64 ). Ju and colleagues( 52 ) usedcoimmunoprecipitation and in vitro binding assays to demonstrate that thebradykinin B 2 receptor, via its COOH-terminal ID4 (amino acids310-329), interacts with eNOS in a ligand- andCa 2 -dependent manner. In the resting state, thereceptor docks with eNOS in the caveolar membrane and participates in itsinactivation. Activation of endothelial cells with bradykinin orCa 2 ionophore triggers dissociation of theeNOS-B 2 receptor complex and activates the eNOS enzyme( 52 ). The sites of binding toeNOS and the mechanisms by which the bradykinin B 2 receptor andcaveolin-1 inhibit eNOS appear to be distinct( 52 ). Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the bradykininB 2 receptor decreases the binding affinity of the receptor domainfor the eNOS enzyme and relieves eNOS inhibition. Furthermore,bradykinin-induced tyrosine phosphorylation of the bradykinin B 2 receptor in cultured endothelial cells appears to promote a transientdissociation of eNOS from the receptor, accompanied by a transient increase inNO production. At a mechanistic level, Golser and colleagues( 43 ) found that binding of theID4 peptide disrupts electron transfer from flavin to heme in eNOS. Takentogether, these data suggest that reversible and inhibitory interactions withG protein-coupled receptors participate in the complex regulation of eNOSactivity in endothelial cells and that these interactions are regulated byreceptor phosphorylation( 64 ).
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Transporters" X0 R; B% l/ ]. e/ u  [, F

The cation arginine transporter CAT1 represents another component of theeNOS-membrane complex in caveolae of endothelial cells. CAT1, eNOS, andcaveolin-1 colocalize to plasmalemmal caveolae in pulmonary artery endothelialcells, and an eNOS-specific antibody immunoprecipitates CAT1-mediated argininetransport from solubilized plasma membrane proteins( 65 ). The proximity of CAT1and eNOS proteins may serve to direct arginine delivery to eNOS and therebyoptimize NO release. Using coimmunoprecipitation experiments followed by MSanalyses, Sun and Liao ( 94 )identified a human voltage-dependent anion/cation channel or porin as an eNOS-binding partner. In vitro studies showed that a GST-porin fusionconstruct specifically retained eNOS and that this interaction augmented eNOSactivity. Increasing intracellular Ca 2 concentrations with Ca 2 ionophore or bradykinin to activate eNOSmarkedly increased porin-eNOS interaction, suggesting that intracellular Ca 2 transients may regulate this interaction.
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eNOS Traffic Inducer
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Zimmermann and colleagues( 105 ) used the yeasttwo-hybrid system with the eNOS ox domain as bait to isolate a novel506-amino acid eNOS-interacting, eNOS traffic inducer protein (NOSTRIN). NOSTRIN contains an NH 2 -terminal cdc15 domain and a COOH-terminal SH3 domain, and its transcripts are abundantly expressed in highlyvascularized tissues, including kidney, as well as vascular endothelial cells.Coimmunoprecipitation experiments confirmed eNOS-NOSTRIN interaction in vitroand in vivo and established that NOSTRIN's SH3 domain was necessary andsufficient for eNOS binding. NOSTRIN colocalized with eNOS at the plasmamembrane of human umbilical venous endothelial cells. When overexpressed, NOSTRIN triggered redistribution of eNOS from the plasma membrane tovesicle-like structures containing NOSTRIN. This redistribution correlatedwith a coincident inhibition of NO release. These results suggested thatNOSTRIN participates in the complex regulation of eNOS trafficking, targeting,and activity ( 105 ) ( Fig. 3 ).
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eNOS-Interacting Protein

eNOS-interacting protein (NOSIP) is a 34-kDa protein that avidly binds tothe COOH-terminal region (amino acids 366-486) of the eNOS ox domain. NOSIP-eNOS complex formation was inhibited by a synthetic peptide ofthe caveolin-1 scaffolding domain, suggesting competition between NOSIP andcaveolin-1 for eNOS binding, but it was not regulated by stimulation of cellswith Ca 2 ionophore. Although NOSIP did not alter eNOSactivity in vitro, it promoted translocation of eNOS from the plasma membraneto intracellular sites when coexpressed in cells, resulting in diminishedionomycin-stimulated NO release. Thus NOSIP appears to uncouple eNOS fromplasma membrane caveolae, thereby suppressing NO synthesis( 17 )( Fig. 3 ). The functional roleof NOSIP in vivo and its specificity for eNOS over the other NOS isoforms isunknown.

GTPases and Kinases

The GTPase dynamin-2 is known to participate in internalization events incaveolae, vesicle formation and trafficking, and receptor-mediatedendocytosis. Dynamin-2 coimmunoprecipitated with eNOS from lysates of bovineaortic endothelial cells and colocalized in these cells predominantly in aGolgi membrane distribution( 13 ). Treatment of endothelialcells with a Ca 2 ionophore enhanced thecoimmunoprecipitation of dynamin, suggesting that the interaction between theproteins can be triggered by intracellular Ca 2 transients ( 13 ). Theproline-rich domain of dynamin-2 was found to interact with the FAD-bindingregion of the eNOS reductase domains and positively regulate eNOS activity, atleast in part, by potentiating electron transfer between FAD and FMN of theeNOS reductase domain( 14 ).
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Several kinases, including RAF, Erk, and AKT, can be coimmunoprecipitated with eNOS from endothelial cells, and the association of these proteins can beprovoked by agonists, suggesting that they may contribute somewhat to the eNOSsignal ( 9, 68 ). Because these kinasesaffect eNOS activity through phosphorylation rather than binding per se, thephysical coupling appears to be important for specifying the phosphorylationevent. Bradykinin ( 46 ), VEGF,and shear stress trigger Akt-mediated phosphorylation ofSer 1177/1179 eNOS (bovine eNOS numbering), leading to eNOS activation in vitro and in vivo( 32, 62, 68 ).: ^5 w# c' l! \0 S; M

For the eNOS regulatory system in endothelial cells, the only obvious factis that it is extremely complex, with multiple potential interactions, many ofwhich can be independently regulated. Given the role of eNOS-generated NO insystemic and renal hemodynamics, however, understanding how these networks operate in different vascular beds in vivo will be extremely important.

PROTEIN-PROTEIN INTERACTIONS INVOLVING iNOS
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Inhibitory Proteins

Three proteins that bind iNOS and exert inhibitory actions on it have beenidentified. Murine macrophages express a 110-kDa protein, termedNOS-associated protein-110 kDa (NAP110), that interacts with theNH 2 terminus of iNOS and inhibits iNOS catalytic activity bypreventing dimerization ( 77 ).Expression of NAP110 may serve as a defense mechanism by which macrophagesexpressing iNOS protect themselves from cytotoxic levels of NO. A yeast two-hybrid screen of a hippocampal cDNA library showed that the first 70 aminoacids of iNOS interact with kalirin, a neural-specific protein known tofunction as an interactor with a secretory granule peptide biosynthetic enzyme( 78 ). Kalirin interacts withiNOS in vitro and in vivo and prevents iNOS dimerization, and therefore iNOSactivity. Kalirin may play a neuroprotective role during inflammation of thecentral nervous system by inhibiting iNOS activity. As noted earlier,caveolin-1 complexes with iNOS in human colon carcinoma cells to promote iNOSproteolysis ( 22 ).5 v4 t* x) m, o, |4 [
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Activating and Trafficking Proteins
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Using a yeast two-hybrid screen of a mouse kidney cDNA library with murineiNOS as bait, our laboratory recently defined a regulatory interaction of iNOSwith the Rac1 and Rac2, members of the Rac family of Rho-like GTPases( 58 ). Racs function as molecular switches in regulating a variety of important biological responsepathways. The interaction was evident in coimmunoprecipitation assays inextracts harvested from activated RAW 264.7 cells and in GST pull-down assaysusing GST-Rac1 and GST-Rac2 fusion constructs to immobilize in vitrotranslated iNOS. Deletion analysis revealed that the point of interaction withRac2 resides in the iNOS ox domain( Fig. 4 ). iNOS and Rac1colocalized in activated RAW 264.7 macrophages( Fig. 4 ).[ 35 S]methionine-labeled iNOS was found to interact directly withGST-Rac2 in the absence of calmodulin or iNOS substrates or cofactors. Stableoverexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitritegeneration ( 60%) and iNOS activity ( 45%) without measurablyaffecting iNOS protein abundance and led to a redistribution of iNOS to ahigh-speed, Triton X-100-insoluble fraction and enhanced appearance of iNOS inRac2-containing vesicles, suggesting a role of Rac in redistributing iNOS inthe cell. In addition to increased NO production, the Rac2-overexpressingcells generated greater levels of superoxide anion, and thus greater amounts of the chemical byproduct of NO and superoxide anion, peroxynitrite, than thecontrol cells after exposure to LPS IFN-. In the aggregate, theseresults indicated that iNOS and Rac2 are functionally coupled through theirjoint effects on both iNOS-mediated NO generation and Rac2-driven NADPHoxidase activity ( 58 ).% s# z' Q& b0 [

Fig. 4. Interaction of inducible (i) NOS and Rac2. Left : deconvolutionimmunofluorescence microscopy with anti-iNOS and anti-Rac2 antibodies togetherwith secondary antibodies conjugated to different fluors (green for iNOS, redfor Rac2) revealed colocalization (orange) of a population of iNOS and Rac2 inthis merged image of LPS-treated RAW 264.7 macrophage cells. Right :glutathione S -transferase (GST) pull-down assays with GST fusionconstructs of murine iNOS comprising the isolated oxygenase (OXY; amino acids1-498), reductase (RED; 499-1144), and full-length proteins (FL;amino acids 1-1144) showed that only the oxygenase domain andfull-length GST-iNOS constructs could specifically retain 35 S-labeled, in vitro translated Rac2. Xa, eluate from factorXa-treated samples (factor Xa liberates the iNOS construct from the GSTplasmid); W, samples simply washed without factor Xa cleavage. Data are fromRef. 58.3 @( M& d: G! }2 v* \

NO is also known to modulate solute transport processes in polarized epithelial processes, suggesting that local NO delivery to its moleculartargets is achieved. In cultured human proximal tubule epithelial cells, iNOSwas found to localize to the apical domain within a submembranous proteincomplex tightly bound to cortical actin and physically interacting with theapical PDZ protein ezrin-radixin-moesin-binding phosphoprotein (EBP50). Mutational analysis indicated that this interaction was dependent on the lastthree COOH-terminal amino acids of iNOS, SAL, but also required the presenceof additional unknown cellular proteins. The EBP50-iNOS interaction at theapical membrane apparently serves to direct vectorial NO production at thissurface, facilitating NO delivery to its targets, which might include apicaltransporters or other proteins known to be complexed with EBP50, such as type3 Na /H exchanger( 41 ).! S8 v& L9 P$ Z7 u' w% J' o
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HOMOCELLULAR INTEGRATION OF PROTEIN-PROTEIN INTERACTIONS: LINKING NOSISOFORMS TO DIFFERENT SPATIAL COMPARTMENTS AND FUNCTIONS IN THE SAME CELL

Differential spatial arrangements and interactions with effector proteinsof NOS isoforms in cardiomyocytes mediate independent, and at times opposite,effects on cardiac structure and function. NO is known to inhibit L-typeCa 2 channels in the heart but also to stimulateCa 2 release from the sarcoplasmic reticulum via theryanodine receptor. The molecular basis for these opposing responses appearsto involve selective interactions of eNOS and nNOS with specific proteins andsubcellular compartments within the same cell( 4 ). In cardiomyocytes,caveolin-3 selectively interacts with eNOS and brings the enzyme intoproximity with -adrenergic receptors and L-typeCa 2 channels, an arrangement that permits NO to inhibit -adrenergic-stimulated inotropy ( 4 ). In contrast, nNOSselectively complexes with the ryanodine receptor in the sarcoplasmicreticulum, facilitating the ability of NO to promoteCa 2 release and cardiac contractility( 4 ). The balance of theseindependent and opposite roles of nNOS and eNOS serves to regulate cardiaccontractility. Because several cell types and tissues express more than oneNOS isoform, it will be interesting to determine whether such coordinate ordiscoordinate actions of the NOS isoforms contribute to other biological orpathobiological responses.
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CONCLUSIONS9 P: C8 M7 h2 C

Protein-protein interactions represent an important and increasingly complex mechanism in the control of NOS activity and NO-dependent effects inthe kidney and vasculature. The challenging work for the future concerns theintegration of the various protein-NOS interactions into coherent models ofsignaling and stimulus-response coupling in normal physiology and disease. Themajority of these protein-protein interactions have not yet been explored inthe kidney, where important insights not only into their impact on renalfunction but also into the cell specificity of different interactions andtheir associated responses could be gained. Obviously, because all NOSisoforms are represented in the kidney and are known to contribute to renalhemodynamics, innervation, solute and water transport, inflammation, and disease progression, NOS-associated proteins in the control of NO productionshould be considered in attempts to understand these functions and conditionsmechanistically. As the binding motifs for NOS-associated proteins are definedand functional proteomics gains more widespread use, new candidate proteins for NOS interaction and regulation will be identified and tested. The pointsof contact between heterologous proteins and the NOS isoforms could serve asexperimental and potentially therapeutic targets to modulate NOS activity.$ W; C3 s4 A1 H# j: b1 O6 B9 G
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DISCLOSURES" I4 e! s6 G2 n( I- f5 h
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This work was supported in part by National Institutes of Health GrantsDK-47981, DK-50745, and GM-20529, a Department of Defense DREAMS grant, andendowment funds from The James T. and Nancy B. Willerson Chair./ k# r8 L- J* {2 ^: X/ Y- k/ N

The authors regret that because of space limitations, many important contributions could not be referenced.
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Address for reprint requests and other correspondence: B. C. Kone, Div. ofRenal Diseases and Hypertension, The Univ. of Texas Medical School at Houston,6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: Bruce.C.Kone{at}uth.tmc.edu ' u '@' d ' ).
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