Use of Surface Plasmon Resonance Coupled with Mass Spectrometry Reveals an Interaction between the Voltage-Gated Sodium Channel Type X r-Subunit and Caveolin-1 ¨ hman,† Anna Nilsson,‡ Alexandra Madeira,† Benita Sjo Elisabet O ¨ gren,† Per E. Andre´n,‡ and ,† Per Svenningsson* Center for Molecular Medicine, Section of Translational Neuropharmacology, Department of Physiology and Pharmacology, Karolinska Institute, 171 77 Stockholm, Sweden, and Department of Pharmaceutical Biosciences, Medical Mass Spectrometry, Uppsala University, Biomedical Centre, Uppsala, Sweden Received July 4, 2008
Abstract: The combination of surface plasmon resonance and mass spectrometry is emerging as a sensitive tool for the elucidation of protein-protein interactions. With the use of surface plasmon resonance-mass spectrometry, peptides, and brain extracts, we now report a novel interaction between the voltage-gated sodium channel type X R-subunit and caveolin-1, the central protein controlling caveolae formation. Surface plasmon resonance binding analyses show that this interaction involves amino acids 85-103 of voltage-gated sodium channel type X R-subunit and amino acids 81-100 of caveolin-1, a known scaffolding domain of caveolin-1. It is anticipated that the surface plasmon resonance-mass spectrometry approach utilized in this study will be important for the elucidation of protein-protein network analysis in native tissues including the brain. Keywords: surface plasmon resonance • protein-protein interactions • cavealoe • lipid rafts • ion channel
Introduction Physiological functions of proteins often depend on their ability to interact with other proteins, lipids, and DNA. An improved knowledge of protein-protein interaction networks is crucial for the understanding of many biological processes. Recent developments to combine surface plasmon resonance (SPR) technique and mass spectrometry (MS) have provided a powerful and sensitive tool dedicated to the identification and the characterization of protein-protein interactions.1 SPR is a phenomenon occurring when monochromatic p-polarized light is reflected on a gold-coated interface between two media.2 The intensity of the reflected light is reduced at a specific incident angle (SPR angle), which depends on the refractive index of the material near the surface on the non illuminated side. Molecules interacting close to the surface alter this refractive index. Consequently, the SPR angle is shifted, pro* To whom correspondence should be addressed. Dr. Per Svenningsson, Center for Molecular Medicine, Department of Physiology and Pharmacology, Karolinska Institute, Nanna Svartz va¨g 2, SE-17177 Stockholm, Sweden. E-mail:
[email protected]. Phone: +46-8-524 87914. † Karolinska Institute. ‡ Uppsala University. 10.1021/pr800498t CCC: $40.75
2008 American Chemical Society
ducing a signal measured in resonance units (RU) (1000 RU correspond to 1 ng bound protein/mm2).2 Results are presented as sensorgrams, which plot RU in real-time. P11, a member of the S100 family of calcium binding proteins, has been implicated in depression-like states and pain3,4 by virtue of its ability to bind to and modify the function of serotonin 5-HT1B receptors and voltage-gated sodium channels type X (Nav1.8), respectively. P11 has also been found to interact with multiple other ion channels and intracellular proteins,5 but no consensus motif for p11’s ability to interact with the other proteins has been found. However, in a previous study,6 it was found that amino acids 74-103 of Nav1.8 bind to p11 with two putative motifs for the interaction in amino acids 87-90 and 98-102. To further study the putative binding motifs in p11, we used a peptide consisting of amino acids 85-103 of Nav1.8 and immobilized it as a bait on a SPR sensor chips and allowed a solution from a brain extract to pass over the chip through the integrated microfluidics system of the SPR equipment. Bound ligands were eluted and analyzed with MS for identification. Surprisingly, no p11 was bound to the bait, but a novel interaction with caveolin-1 (Cav-1) was revealed and confirmed in several subsequent experiments including binding analyses using SPR technology. These data suggest that Nav1.8 may be found in caveolae.
Experimental Section Peptides and Brain Extracts. The Nav1.8 (85-103) peptide corresponds to the sodium channel (type X) alpha subunit from the amino acid 85-103. The Cav-1 (81-100) peptide corresponds to Cav-1’s scaffolding domain. The Cav-1 (1-25) peptide, corresponding to the N-terminal domain of Cav-1 and a randomized peptide (Cav-1 RP) with the same amino acids as Cav-1 (81-100) peptide but in a different order were used as negative control. A peptide corresponding to a domain from the 5-hydroxytryptamine 1B receptor (5-HT1BR (252-271)) was also used as negative control. All these peptides were purchased from BACHEM, U.K. Brain extracts from mouse and rat were prepared by sonication in TBS buffer, followed by centrifugation at 2900g for 20 min at 4 °C. The pellets were discarded Journal of Proteome Research 2008, 7, 5333–5338 5333 Published on Web 11/07/2008
technical notes and the supernatants were centrifuged at 29 000g for 45 min at 4 °C. The obtained supernatant fractions were stored in TBS buffer. Ligand Fishing with Nav1.8 (85-103) or Cav-1 (81-100) Peptides. 1. Immobilization of the Peptides on a Sensor Chip. The immobilization experiments were performed using BIACORE 3000 SPR sensor with the corresponding control software version 4.1. The peptides were immobilized on CM5 sensor chips docked in the integrated µ-fluidic cartridge of the instrument (IFC) or in the surface prep unit (SPU), and HBSEP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20) was used as running buffer (BIACORE, Uppsala, Sweden). The immobilization experiments in the instrument were performed according to a standard protocol in which all four flow cells in the IFC were used and the flow rate was 10 µL/ min. Briefly, the surface of the sensor chip was activated with equal quantities of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (BIACORE, Uppsala, Sweden). Cav-1 (81-100) or Nav1.8 (85-103) peptides were injected at a concentration of 100 or 200 µg/mL, respectively diluted in 50 mM Tris-HCl buffer pH ) 5.0. Finally, the surface was inactivated with 1 M ethanolamine-HCl, and to remove noncovalently bound peptides, 1% acetic acid was injected in 10 aliquots of 5 µL. Immobilization in the surface preparatory unit (SPU) was performed according to an “Immobilization in the Surface Prep Wizard”, which means that the conditions of the experiment are already programmed by the software and every step is automatically controlled. This procedure is similar to the standard protocol for immobilization described above. However, noncovalently bound peptides were removed with 50 mM NaOH instead of 1% acetic acid. It is important to note that experiments done in the SPU cannot be monitored in real time but allow immobilization and recovering of increased amounts of proteins compared to experiments performed in the IFC. 2. Recovery of Proteins from Mouse and Rat Brain Extracts Interacting with Cav-1 (81-100) or Nav1.8 (85-103) Peptides. Recovery experiments in the IFC were performed in triplicates according to a user defined method with 12 cycles. HBS-N (0.01 M HEPES, pH 7.4, 0.15 M NaCl) (BIACORE, Upsala, Sweden)/10 mM octyl-β-D-glucopyranoside (OGP) (SigmaAldrich, Stockholm, Sweden) was used as the running buffer. The flow rate was 10 µL/min. In each cycle, brain extracts from mice or rats (0.25 mg/mL in TBS buffer, 10 mM OGP) were injected followed by MS-washes with 50 mM NaOH, 2% acetic acid/50 mM OGP and HBS-N/10 mM OGP. Subsequently, the flow cells were washed with H2O and HBS-N/10 mM OGP and incubated in recovery solution (0.25% TFA) for 30 s, in order to release proteins bound to the immobilized peptides. The recovery solution was then eluted into a protein LoBind tube (Eppendorf AG, Hamburg, Germany). To avoid accumulation of proteins from brain extracts, the flow area was flushed with recovery solution before an additional MS-wash. The recovery experiments in the SPU were also performed according to a user defined method with eight cycles, similar to the one described above. However, the running buffer used was HBS-N and the brain extracts contained 50 mM of OGP. The method starts with an injection of the brain extracts followed by a needle wash with 1.6% acetic acid/50 mM OGP. The flow area was incubated with 0.25% TFA and 50 mM NH4HCO3, for recovery of proteins bound to the immobilized peptide. The recovery solution was eluted into a protein LoBind 5334
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¨ hman et al. O tube (Eppendorf AG, Hamburg, Germany). The flow rate was 1 µL/min, and these experiments were also performed in triplicates. 3. Control Recovery Experiments of Proteins from Mouse and Rat Brain Extracts Interacting with Blank Chip or a Randomized Cav-1 Peptide. To control for unspecific interactions, the same SPR-MS set up as above was used with nonimmobilized CM5 sensor chips (blank chip). Control SPRMS experiments were also performed with a randomized peptide (Cav-1 RP), which has the same amino acid content as the Cav-1 (81-100) peptide but in a different order, immobilized on CM5 sensor chips. All control experiments were performed in duplicates. Trypsin Digestion and Resuspension. Sequencing grade modified trypsin (Promega Corp., Madison, WI) (0.02 µg/mL in 0.1 mM HCl), and 50 mM of NH4HCO3, pH 8.0, were added to the recovered protein solutions, which were left at room temperature for enzymatic cleavage overnight. During the optimization of the protocol, trypsin digestion was done at room temperature overnight or at 37 °C for 4 h. These conditions yielded no significant differences in sequence coverage and the number of detected proteins (data not shown). It should be noted that, under these conditions, hydrophobic domains are less accessible to the trypsin cleavage, which yields peptides mostly from hydrophilic domains.7 We did not study effects of reduction and alkylation of the samples on the efficacy of trypsin cleavage. The tryptic peptide solution was further lyophilized and resuspended in 8 µL of 0.25% acetic acid before MS-analysis. MS-Analysis of Peptide Solution from Recovery Experiments. The trypsinated protein solutions from the recovery experiments were analyzed on a capillary liquid chromatography (nanoLC) system (Ettan MDCL, GE Healthcare) coupled with an electrospray linear ion trap (LTQ) mass spectrometer (Thermo Scientific, San Jose, CA). The mobile phases used were solvent A (0.25% acetic acid in HPLC-grade water) and Solvent B (acetonitrile and 0.25% acetic acid in HPLC-grade water). The sample was loaded onto a precolumn (C18 PepMap100 5 µm, 300 µm (i.d.) × 5 mm, 100 Å; LC Packings, Amsterdam, The Netherlands) and then separated on an in-house packed analytical column, a 15-cm fused silica emitter with a 75 µm i.d. and a 375 µm outer diameter (Proxeon Biosystems; Odense, Denmark) packed with Reprosil-Pur C18-AQ 3-µm resin (Dr. Maisch GmbH; Ammerbuch-Entringen, Germany). The tryptic peptides were eluted during a 40 min gradient from 3% solvent B to 65% solvent B, followed by a wash phase of 20 min at 95% solvent B and a re-equilibration of 20 min at 3% solvent B. The flow rate of the sample loading pump was 10 µL/min and the splitted flow over the analytical pump was 200 nL/ min at 3% of solvent B. Mass spectra were acquired from 12 to 52 min in a data-dependent manner. Every full scan MS spectrum was followed by a zoom scan and a tandem MS spectrum of the peak with the highest intensity. Every peak was allowed to be selected twice before it was put on an exclusion list for 150 s. The MS/MS data were converted into a combined mgf-file. The mgf-files were searched against the database Sprot 48.8 (Mus musculus and Rattus norvegicus) using the search engine Mascot 2.1 with the following settings: trypsin as enzyme, no fixed modification, potential oxidation of methionine, error window of experimental peptide mass values as 1.5 Da, error window for MS/MS fragment ions as 0.7 Da, 2 missed cleavages, data file as MASCOT generic, and instrument as ESI-TRAP. The significance of the results is
technical notes
Interaction between NAV1.8 and Cav-1
Table 1. MS Analyses of the Solution from the Recovery Experiments with Mouse and Rat Brain Extracts on Immobilized Nav1.8 (85-103) Peptidea
species
accession number
name of the identified protein
found in an recovery experiments
MASCOT score
number of identified tryptic peptides
66 62
1 3
8 6
58 64 51 33 27 32 30 40
3 2 2 2 1 1 1 2
6 12 8 8 5 7 7 4
32 27 42 36
4 5 2 2
5 7 3 6
Mouse
P49817 O08553
Caveolin-1 Dihydropyrimidinaserelated protein 2
1 2
Rat
P41350
Caveolin-1
4
P45592
Cofilin-1
2
P47942
Dihydropyrimidinaserelated protein 2
3
P47819
GFAP
2
sequence coverage (%)
a Accession numbers refers to the number used to archive the protein in the Swiss-Prot database. Mascot scores above 27 are considered as significant. Furthermore, the criteria for a protein to be listed is that it, at least, was found in both species or in one species with more than one tryptic peptide or in several recovery experiments. The sequence coverage is expressed as the number of amino acids spanned by the assigned peptides divided by the sequence length. The total number of recovery experiments in each species was six.
determined by probability based scoring. The probability that the observed matches between the MS/MS data obtained and the sequences in the Sprot 48.8 database is a chance event is calculated. The probabilities are converted into scores according to an algorithm. On the basis of the size of the Sprot 48.8 database and p < 0.05, scores around 27 and 28 and above implicate significant results. The results are presented according to the guidelines for the analysis and documentation of peptide and protein identifications published on the Molecular and Cellular Proteomics Web site.8 Specifically, the criteria for a protein to be presented is that it, at least, was found in both species or in one species with more than one tryptic peptide or in several recovery experiments. Binding Analyses of Nav1.8 (85-103), Cav-1 (81-100), Cav-1 (1-25) and 5-HT1BR (252-271) against Cav-1 (81-100), and Cav-1 RP. 1. Immobilization. Cav-1 (81-100) and the Cav-1 RP were diluted to a concentration of 25 µg/mL in 10 mM acetic acid and immobilized on a CM4 chip, in one flow cell each. The immobilization was performed with the wizard “Aim for immobilized level” included in the control software. This wizard is similar to the standard protocol used for immobilization in the instrument, except washes with 1% acetic acid, but allows for immobilization of different compounds in separate flow cells. 2. Binding Analyses. Nav1.8 (85-103) (100 µg/mL in TBS buffer) was injected on the CM4 sensor chips with immobilized Cav-1 (81-100) and Cav-1 RP, with a flow rate of 10 µL/min. This was replicated three times. Cav-1 (1-25) and 5-HT1BR (252-271) were also injected as negative controls. The chip was washed with either 10 mM HEPES buffer, 25 mM NaOH or 50 mM NaOH, depending on the amount of peptide bound to the chip. Since Cav-1 (81-100) binds to itself, injection of this peptide (10 µg/mL in TBS buffer) with a flow rate of 10 µL/ min was used as a positive control to verify the condition of the chip before and after the injections of Nav1.8 (85-103), Cav-1 (1-25) and 5-HT1BR (252-271). Statistical Analyses. Statistical analyses were done using either two-tailed Student’s t test or one-way ANOVA followed
by Dunnett’s for pairwise comparisons, and the data were presented as means ( SEM (Standard Error of Mean). Twotailed Student’s t test was chosen when two groups were compared. One-way ANOVA was chosen when three groups with one parameter were compared and Dunnett’s test for pairwise comparisons was selected as the posthoc test as it is recommended when there are three groups and where two groups are separately compared with one control group.
Results Ligand Fishing with Nav1.8 (85-103) Peptide. The immobilization of Nav1.8 (85-103) in the IFC gave immobilization levels corresponding to 2 to 3 ng/mm2 of immobilized peptide. The amount of bound proteins in the recovery experiments varied between 0.5 and 1.5 ng/flow cell of which approximately 0.5 ng per flow cell could be recovered. These recovered proteins were then digested and analyzed by MS. The results from the MS-analyses are presented as lists of proteins, containing amino acid sequences that correlate to peptides found in the protein solution from the recovery experiments. The MASCOT scores and the number of peptides found from the identified proteins were also validated. Furthermore, the sequence coverage represents the length of the tryptic peptides compared to the total length of the protein identified. Proteins found in several recovery experiments are also considered as relevant. A compilation of the proteins found in solutions from the recovery experiments in ligand fishing with Nav1.8 (85-103) peptide is presented in Table 1. It is noteworthy that no interaction with p11 was found. Proteins identified from peptide solution in control experiments have been excluded. Binding Properties of Cav-1 (81-100) and the Randomized Peptide (Cav-1 RP). According to the results described above, Cav-1 protein is often retrieved in the recovery experiments using immobilized Nav1.8 (85-103) peptide. Thus, a peptide corresponding to the scaffolding domain of Cav-1 (Cav-1 (81-100)) was used to further study the interacting properties Journal of Proteome Research • Vol. 7, No. 12, 2008 5335
technical notes
¨ hman et al. O recovery experiments, and/or in recovery experiments with immobilized Cav-1 RP have been excluded. Binding Analyses of Nav1.8 (85-103), Cav-1 (81-100), Cav-1 (1-25) and 5-HT1B (252-271) against Immobilized Cav-1 (81-100) and Cav-1 RP. Injection of Nav1.8 (85-103) resulted in an increase of RU significantly higher with immobilized Cav-1 (81-100) peptide than with Cav-1 RP and a blank flow cell (Figure 2). RU levels did not increase when Cav-1 (1-25) and 5-HT1BR (252-271) were injected as negative controls. Thus, these data indicate specific interaction between Nav1.8 (85-103) and Cav-1 (81-100), since Cav-1 (1-25) and 5-HT1BR (252-271) did not bind to Cav-1 (81-100). The injection of the Cav-1 (81-100) peptide before and after the injections of Nav1.8 (85-103), Cav-1 (1-25) and 5-HT1BR (252-271), resulted in a higher increase of RU in the flow cell in which Cav-1 (81-100) was immobilized than in the flow cell with immobilized Cav-1 RP and the blank flow cell. This was expected since Cav-1 is known to bind to itself and also confirms that the conditions of the sensor chip remained satisfactory throughout the experiment.
Discussion Figure 1. Interactions between proteins from rodent’s brain extract and immobilized Cav-1 (81-100) or Cav-1 RP. The amount of protein from mouse brain extracts (A) or rat brain extracts (B) interacting with immobilized Cav-1 (81-100) or Cav-1 RP during the recovery experiments was evaluated. These results are from 3 independent experiments (12 cycles and 4 flow cells each) and are expressed as percentage of maximal amount of proteins from brain extracts bound to Cav-1 (81-100) peptide (Two-tailed Student’s t test was used to compare Cav-1 RP with Cav-1 (81-100), *p < 0.001).
of Cav-1 protein. As a negative control for the interaction studies with Cav-1 (81-100), a randomized peptide (Cav-1 RP) with the same amino acid composition as Cav-1 (81-100) but in a different order was designed. To verify the disruption of scaffolding properties of the Cav-1 RP, the binding capacity of the Cav-1 RP was compared to the binding capacity of the Cav-1 (81-100) in recovery experiments with brain extracts. The amount of proteins from brain extracts interacting with the Cav-1 RP was significantly lower than the amount of proteins from brain extracts interacting with Cav-1 (81-100) (Figure 1). The Cav-1 RP was further used as a negative control in the ligand fishing experiments with Cav-1 (81-100) and in the binding analyses. Ligand Fishing with Cav-1 (81-100) Peptide. The immobilization of the Cav-1 (81-100) peptide both in the IFC and the SPU gave levels corresponding to 10 ng/mm2 of immobilized peptide. The amount of proteins bound in each cycle in the recovery experiments varied between 0.5 and 2.0 ng/mm2, of which approximately 0.5 ng/mm2 could be recovered. As described above, the results from the MS-analyses are presented as lists of proteins taking into account different criteria as MASCOT score, the sequence coverage, the number of tryptic peptides retrieved and the number of recovery experiments where the protein is identified. A compilation of the proteins found in solutions from the recovery experiments in ligand fishing with Cav-1 (81-100) peptide is presented in Table 2. Proteins identified from peptide solution in blank chips 5336
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A better knowledge concerning protein-protein interactions in biological systems and an exploration of the nature of these interactions can be useful to define the function and the meaning of protein networks. Such knowledge can also explain the emergence of pathological conditions and the elaboration of new therapeutic strategies. For this purpose, novel technologies are being developed. With the use of SPR combined with MS, the present study has found a new interaction between Nav1.8 and Cav-1, which could be confirmed by SPR binding analysis. The original purpose of this study was to use SPR-MS technology to fish out p11 from brain extracts using peptides directed toward an amino acid sequence in the Nav1.8 reported to bind to p11. However, no p11 was recovered in SPR-MS analysis using a synthesized peptide consisting of amino acids 85-103 of Nav1.8. It should be noted that the region defined by Poon et al.6 actually consisted of amino acids 74-103, so additional experiments using a peptide spanning all these amino acids may recover p11. However, Cav-1, a member of the caveolin family, was repeatedly retrieved in recovery experiments with brain extracts injected on immobilized Nav1.8 (85-103) peptide. Cav-1 is the central protein that controls caveolae formation. It is expressed in various cell types, such as astrocytes, oligodentrocytes, Schwann cells, endothelial cells, dorsal root ganglia (DRG) and hippocampal neurons.9,10 Although the Cav-1 structure has not been resolved by crystallography, there is evidence that it has a central hydrophobic segment inserted in the membrane. This transmembrane domain splits the molecule into two cytoplasmic domains, the N- and C-terminals. This arrangement of the protein makes it resemble an unusual hairpin structure. In addition, Cav-1 assembles into oligomers, containing about 15 individual molecules. This oligomerization seems to be important for the formation of an interlocking network of Cav-1 that makes up the striated caveolar coat.11 The interacting regions of Cav-1 are supposed to be the N-terminal region and the scaffolding domain. The N-terminal region has been shown to associate with the actin cytoskeleton via a dimeric protein called filamin, and the scaffolding domain is crucial for caveolin oligomerization as well as for protein interactions.12 Since Cav1’s scaffolding properties have not been fully explored, we
technical notes
Interaction between NAV1.8 and Cav-1
Table 2. MS-Analyses of the Peptide Solution from the Recovery Experiments with Mouse and Rat Brain Extracts on Immobilized Cav-1 (81-100) Peptidea
found in an experiments
MASCOT score
number of identified tryptic peptides
109 60 59 55 44 111 96 59 111 87 74 50
1 1 1 1 1 3 1 1 2 3 2 1
8 8 8 8 8 21 8 8 5 10 4 1
3 1 1 2 3 4 5 5 1 2 2 2 2 2 2 4 2 1
5 1 1 8 9 11 12 29 16 10 2 5 11 11 11 29 20 1
3
1
species
accession number
Mouse
P49817
Caveolin-1
5
P18160
Cofilin-1
3
P17183
Gamma-enolase
3
Q64018
Glycine receptor alpha-1 chain precursor
4
Rat
name
O55131
Septin-7
4
P54227
Stathmin
3
Q64332
Synapsin-2
2
P41350
Caveolin-1
3
P45592
Cofilin-1
2
P07727
Glycine receptor alpha-1 chain precursor Nav1.8
1
38 37 35 52 43 39 38 50 44 43 36 34 73 73 64 139 84 34
1
28
Q62968
sequence coverage (%)
a
Accession numbers refers to the number used to archive the protein in the Swiss-Prot Database. Mascot scores above 27 are considered as significant. Furthermore, the criteria for a protein to be listed is that it, at least, was found in both species or in one species with more than one tryptic peptide or in several recovery experiments. The sequence coverage is expressed as the number of amino acids spanned by the assigned peptides divided by the sequence length. The total number of recovery experiments in each species was six.
Figure 2. Binding of Nav1.8 (85-103) on immobilized Cav-1 (81-100), immobilized Cav-1 RP and on a blank chip. Bound Nav1.8 (85-103) peptide is significantly higher with immobilized Cav-1 (81-100) peptide than with immobilized Cav-1 RP or on the blank flow cell. These results are based on 3 independent experiments, and are expressed in percentage of the maximal amount of Nav1.8 (85-103) bound to Cav-1 (81-100) peptide (One way ANOVA followed by Dunnett’s for pair wise comparisons was used to compare Cav-1 RP or blank with Cav-1 (81-100), *p < 0.01).
decided to use the Cav-1’s scaffolding domain (Cav-1 (81-100)) as a bait in experiments coupling SPR and MS technology using brain extracts as described above. Nav1.8 was retrieved in one experiment, providing additional evidence for an interaction between Nav1.8 and Cav-1. The fact that the interaction between Cav-1 and Nav1.8 was less obvious when Cav-1 was
used as a bait may be due to the multiple hydrophobic regions of native Nav1.8 which are not optimally cleaved by our trypsin protocol. Our results also suggest several additional interactions with immobilized Cav-1 (81-100) peptide, including ligand gated receptors, cytoskeletal proteins and various kinds of enzymes. Some of these interactions have already been published. For example, oligomerization of Cav-1, suggested in the recovery experiments with mouse and rat brain extracts, has already been shown.12 Furthermore, an interaction between Cav-1 and stathmin was found in the recovery experiments with brain extracts from mouse. Cav-1 and stathmin have previously been suggested to interact in vascular smooth muscle cells.13 The confirmation of these previously known interactions indicates that the method is reliable. However, the number of identified peptides was relatively small and the sequence coverage was relatively low. This can, at least partly, be due to that hydrophobic domains are less accessible to the used trypsin cleavage protocol.7 Furthermore, some proteins with high abundance were considered as nonspecific interactors as they appear in peptide solutions from all recovery experiments, even if blank sensor chips are used. These proteins, such as keratin, myelin basic protein, actin, hemoglobin, pyruvate kinase and tubulin, are not only excluded but could also interfere with binding of specific interactors. Similarly, proteins found in recovery experiments with immobilized Cav-1 RP have also been excluded, since changing the order of the amino acids of the Cav-1 scaffolding domain Journal of Proteome Research • Vol. 7, No. 12, 2008 5337
technical notes appears to effectively disrupt its interacting properties. Indeed, we compared the amount of proteins from brain extracts that interact with Cav-1 (81-100) and a randomized peptide (Cav-1 RP) that shows the same composition in amino acids than Cav-1 (81-100) but in a different order and observed a significant decrease of interaction with the randomized peptide. The possible interaction between Nav1.8 and Cav-1 was further investigated with binding analyses with peptides from these proteins, with Cav-1 RP as a negative control. The stronger binding between Nav1.8 (85-103) and Cav-1 (81-100) compared to Nav1.8 (85-103) and Cav-1 RP provides evidence for an interaction between Nav1.8 and Cav-1. The fact that neither Cav-1 (1-25) nor 5-HT1B (252-271) interacted with Cav-1 (81-100) in the binding analysis further confirms the specificity of the interaction between Cav-1 (81-100) and Nav1.8 (85-103). It should also be pointed out that the amino acid sequences of the Cav-1 (81-100) and Nav1.8 (85-103) peptides contain putative β-strands which are known to interact.11,6,14 Somewhat surprisingly, there appears to be no putative β-strands or consensus motif among the other interactors to Cav-1 presented in Table 2. The physiological importance of the possible interaction between Nav1.8 and Cav-1 is difficult to predict. There appears to be at least some colocalization of Nav1.8 and Cav-1 in the nervous system. As mentioned above, Cav-1 is expressed in the hippocampus, cortex, cerebellum and DRG.9,15 Nav1.8 is enriched and highly expressed in the DRG and trigeminal ganglion nerves, but is found in other regions of the central nervous system.16,10 An interaction between Nav1.8 and Cav-1 has not previously been described in the brain, but, interestingly, studies have shown that cardiac sodium channels are localized in caveolin-rich membrane domains.17 Cav-1 is a scaffolding protein and can recruit signaling proteins to lipid rafts.9 For example, studies have shown that caveolins interact with several G-protein coupled receptors (GPCR), and/or regulate signaling from them. The functions of these interactions can be to localize and assemble these receptors in lipid rafts.18 Perhaps Cav-1 recruits and assembles voltage gated sodium channels containing the Nav1.8, in order to increase their role in action potentials regulation. As indicated above, Nav1.8 has been implicated in pain transmission.19,4 In addition, overexpression of Nav1.8 has been seen in some pathological conditions, like in experimental autoimmune encephalomyelitis (EAE), via a mechanism that has been suggested to involve the p75 nerve growth factor (NGF) receptor.16 Interestingly, NGF receptors have been shown to coimmunoprecipitate with Cav-1 in caveolae20 and Cav-1 expression is increased in the spinal cord of rats with EAE.21 The possible functional relationship between Nav1.8, Cav-1 and the NGF receptors would be interesting to study in future work.
Conclusion This study has used a SPR-MS approach to show that Nav1.8 interacts with Cav-1 and that this interaction involves amino acids 85-103 of Nav1.8 and 81-100 of Cav-1. The approach utilized in this study with a combination of surface plasmon resonance and mass spectrometry holds much promise for the elucidation of protein-protein networks and may turn out to be more widely used in the proteomics field.
Acknowledgment. This study was supported by Swedish Research Council (Vetenskapsrådet) and Torgny
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Journal of Proteome Research • Vol. 7, No. 12, 2008
¨ hman et al. O and Ragnar So¨derbergs Foundation och Swedish Brain Fund (Hja¨rnfonden). Dr Alexandra Madeira was supported by a postdoctoral fellowship from the French Medical Research Foundation (Fondation pour la Recherche Medicale).
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