Article |
Address correspondence to Michel Bernier, Diabetes Section, National Institute on Aging, Gerontology Research Center, 5600 Nathan Shock Drive, Box 23, Baltimore, MD 21224-6825. Tel.: (410) 558-8199. Fax: (410) 558-8381. email: Bernierm{at}vax.grc.nia.nih.gov
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Abstract |
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Key Words: receptors; PLC; signal transduction; cultured cells; mass spectrometry
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Introduction |
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The cytoplasmic domain of the IR ß-subunit contains reactive cysteine thiol(s) that can modulate the receptor catalytic activity (Li et al., 1991; Bernier et al., 1995; Schmid et al., 1998). The importance of the IR cytoplasmic cysteines for the association between this receptor and intracellular effectors has been investigated in intact cells using 1,6-bismaleimidohexane (BMH), an irreversible thiol-specific homobifunctional cross-linking reagent (Garant et al., 2000). This approach has led to the identification of a complex between the human IR and a thiol-reactive membrane-associated protein (TRAP). The IRTRAP complex migrates as an 250 kD protein on SDS-PAGE under reducing conditions and does not contain the receptor
-subunit as assessed by immunoblot analysis. In the same report, point-mutation analyses have shown that cysteine 981 of the cytoplasmic domain of the human IR ß-subunit is the nucleophilic thiol responsible for the covalent binding to TRAP after BMH-induced cross-linking (Garant et al., 2000).
To further our understanding of the biological importance of TRAP in insulin signaling, we purified the IRTRAP complex and identified TRAP as PLC1 using matrix-assisted laser desorption/ionization (MALDI) analysis. Here, our coimmunoprecipitation assays demonstrated constitutive and insulin-inducible association of PLC
1 with the IR in a number of cultured cell lines and a primary culture of rat hepatocytes, which reflects the potential for physiological significance. Structurally, the catalytic region of PLC
1 contains an insert with two SH2 domains and an SH3 domain. It has been proposed that the two SH2 domains are essential for association of PLC
1 with activated growth factor receptor tyrosine kinases (Middlemas et al., 1994, Ji et al., 1999), whereas the SH3 domain directs PLC
1 to bind to the cytoskeleton (Park et al., 1999). Whether these and other motifs play an important function in the recruitment of PLC
1 to the IR remains unknown.
The dynamic association between PLC1 and the IR must depend on specific domains within both proteins. In an attempt to identify some of these motifs, we have expressed mutant forms of PLC
1 and analyzed the pattern of IRPLC
1 association in intact cells. Now, we report on the identification of a domain of PLC
1 containing the PH and EF-hand (PHEF) that is required for interaction with the IR. Overexpression of the PHEF fragment or reduction of PLC
1 expression using small interfering RNA (siRNA) abrogates MAPK regulation by insulin, strengthening the notion that PLC
1 plays an important role in insulin signaling (Kayali et al., 1998; Eichhorn et al., 2002).
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Results |
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Characterization of TRAP
The silver-stained gel of the anti-IR immunoprecipitates resolved four major bands that corresponded to the TRAP/ß-subunit complex, IR proreceptor (ß-dimer), and
- and ß-subunit, respectively, with apparent molecular masses ranging between
100 kD (ß-subunit) and
275 kD (TRAP/ß-subunit) (Fig. 2). The IR ß-subunit and TRAP/ß-subunit protein bands were subjected to in-gel digestion with trypsin, followed by peptide mass fingerprinting and MALDI analysis of the eluted peptides to provide tentative identification of each protein species. 17 and 15 peptide masses covering 17 and 9% of the IR ß-subunit, respectively, were found in both protein bands (estimated z value of 2.16 and 2.38, respectively), whereas 12 peptide masses within the TRAP/ß-subunit band matched the 155-kD PLC
1 (estimated z value of 2.39), corresponding to 10% of the molecule. These peptides covered various regions of PLC
1. Analysis of recombinant GST-tagged PLC
1 SH2/SH3 domain fusion protein by MALDI returned 18 peptide masses (estimated z value of 1.82), many of which were strong matches with those found in the TRAP/ß-subunit protein band. Subsequent immunoblot analyses revealed the presence of PLC
1 in the IRTRAP complex (see below).
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Immunodetection of the PLC1IR complex
The association of PLC1 with the IR was evaluated in CHO-IR cells that were left untreated or exposed to a saturating concentration of insulin (100 nM) for 330 min. Immunoblotting the anti-IR immunoprecipitates with anti-PLC
1 antibody showed a time-dependent increase in PLC
1 association with the IR in response to insulin that persisted throughout the 30 min of the experiment (Fig. 3 A). The interaction is stimulated by insulin in a dose-dependent manner, with detectable levels at 5 nM insulin (Fig. 3 B). Similar results were obtained by probing anti-PLC
1 immunoprecipitates with anti-IR antibody (unpublished data). When immunoprecipitation was performed with a control IgG, no cosedimentation of the IR with PLC
1 was detectable (unpublished data).
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To further evaluate the role of insulin in mediating tyrosine phosphorylation and association of PLC1 with the activated IR, anti-PLC
1 immunoprecipitates from vanadate-treated CHO-IR cells were probed with anti-IR. Insulin stimulation led to a significant increase (6.4 ± 1.6-fold; n = 6) in IR cosedimentation with PLC
1 and in phosphoPLC
1 levels (Fig. 3 D).
Physiological significance of the IRPLC1 association
Next, we investigated the role of insulin in the recruitment of PLC1 to the endogenous IR in insulin-responsive HepG2 cells. These cells were pretreated with orthovanadate and then left untreated or exposed to 100 nM insulin for 15 min. Fig. 4 A shows the results of a typical experiment analyzing PLC
1 immunoprecipitates that were blotted with the IR ß-subunit. In agreement with our previous results with CHO-IR cells from this report, a constitutive and insulin-inducible cosedimentation of the IR with PLC
1 was observed, suggesting that insulin could promote the recruitment of PLC
1 to the IR in a number of cell types. Higher tyrosine phosphorylation of PLC
1 was also noted in response to insulin when PLC
1 was immunoprecipitated and then visualized with either anti-phosphoPLC
1 (pTyr-783) or phosphotyrosine (clone RC20) antibody (Fig. 4 A). Next, we determined that endogenous PLC
1 interacted with the IR in primary culture of rat hepatocytes (Fig. 4 B). These results strongly support a physiological role for the PLC
1 association to the IR in insulin signaling.
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A number of IR-interacting proteins, including Gab-1 and IRS, contain a PH domain that allows their membrane association. To assess the importance of this domain in the recruitment of PLC1 to the IR in intact cells, various experiments were performed using HA-tagged PHEF domain (aa 1301) of rat PLC
1. This construct was readily detected as a 40-kD protein upon transient transfection in HEK-293 cells and upon immunoprecipitation using anti-HA or an antibody against the PLC
1 PH domain (Fig. 8 A). Expression of the HA-tagged PHEF construct led to a 60 ± 11% decrease (P < 0.01; n = 4) in the ability of insulin to stimulate recruitment of cellular PLC
1 to the activated IR (Fig. 8 B, top left). To determine if a PLC
1 mutant lacking the PHEF motif could also interfere with this interaction, an NH2-terminal truncation of 301 amino acids was performed to generate the
PHEF PLC
1 mutant. HEK293 cells expressing HA-tagged
PHEF displayed no reduction in the binding of endogenous PLC
1 with the IR (Fig. 8 B, top right), but markedly abrogated the PLC
1EGF receptor interaction (Fig. 8 C, middle right). Importantly, expression of the PHEF construct did not block PLC
1 association with the activated EGF receptor in HEK293 cells (Fig. 8 C, middle left) or HepG2 cells (unpublished data). Ligand-mediated phosphorylation of the EGF receptors was normal in all conditions tested (Fig. 8 C, top panels). These results are consistent with the PHEF domain being required for PLC
1 interaction with the IR.
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To further test the requirement of PLC1 for insulin signaling, we used PLC
1-/- fibroblasts reconstituted with the IR alone or together with wild-type PLC
1. After serum withdrawal, cells were stimulated in the absence or presence of insulin, then in the phosphorylation of endogenous ERK, and AKT phosphorylation was measured in total cell lysates using phosphospecific antibodies. Insulin-stimulated ERK phosphorylation was activated to a greater extent in cells reconstituted with wild-type PLC
1, whereas there was only an
20% increase in AKT phosphorylation levels by insulin (Fig. 9 A). Lastly, the role of PLC
1 in insulin action was determined using siRNA methodology. HepG2 cells transfected with a control siRNA duplex had no reduction in PLC
1 expression (Fig. 9 B, second panel). However, with a PLC
1-specific siRNA duplex targeting to the 29792999 region of the human PLC
1 mRNA-coding sequence, the expression of PLC
1 was dropped to
30% of the levels of siRNA controls. Exposure of these cells to insulin activated the phosphorylation of IRSs and AKT to levels equivalent to those in insulin-stimulated cells transfected with control siRNA (Fig. 9 B). More significantly, incubation with PLC
1 siRNA attenuated ERK phosphorylation elicited by insulin (Fig. 9 B, fifth panel). These results demonstrate the efficiency of the siRNA template and indicate the pathway of insulin signaling that PLC
1 may relate to.
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Discussion |
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Increase in PLC1-mediated PI(4,5)-bisphosphate hydrolysis has been reported in anti-IR immunoprecipitates from insulin-stimulated 3T3-L1 adipocytes (Eichhorn et al., 2001). However, whether the binding of PLC
1 to the IR was direct or through an accessory protein remains unclear. It should be noted that c-Cbl tyrosine phosphorylation by insulin requires the adaptor protein APS, which coordinates interaction between c-Cbl and the activated IR (Liu et al., 2002). Our data show the direct interaction between PLC
1 and the IR using cross-linking methodology in intact cells. A significant conformational change of the cytoplasmic region of the receptor ß-subunit occurs as the result of IR autophosphorylation (Baron et al., 1992; Lee et al., 1997). Hence, the mechanism by which PLC
1 is recruited to the IR in response to insulin may involve change in conformational flexibility at the interface between the two proteins, which brings the pair of reactive thiols (Cys 981 of the IR [Garant et al., 2000] and that of PLC
1) in close proximity. The inter-thiol distance could be separated by as much as 8 Å, as the BMH analogue (BMOE) was efficient at promoting the formation of a covalent IRPLC
1 complex.
Our results show insulin-stimulated phosphorylation of a positive regulatory residue (Tyr-783) on PLC1 both in CHO-IR and HepG2 cells, as well as in HEK293 cells and PLC
1-/- fibroblasts transiently expressing wild-type PLC
1. A commercially available phosphoPLC
1 antibody (pTyr-783) was used, and the results were confirmed with anti-phosphotyrosine. By contrast, no PLC
1 tyrosine phosphorylation was detected upon addition of insulin in 3T3-L1 adipocytes (Eichhorn et al., 2001). It has been suggested that kinases of the Src family have the ability to phosphorylate and activate PLC
1 (Nakanishi et al., 1993). Src-related kinases are abundant in caveolin-rich raft preparations of adipocytes (Mastick and Saltiel, 1997; Müller et al., 2001) and CHO-IR cells (unpublished data), and are believed to play a role during insulin signaling (Sun et al., 1996). Because the IR appears to be incapable of directly phosphorylating PLC
1 (Nishibe et al., 1990), it is possible that upon insulin stimulation, PLC
1 is repositioned for phosphorylation by raft-associated Src-family kinases. PLC
1 contains several tyrosine residues that are targets of receptor and nonreceptor tyrosine kinases and whose phosphorylation may contribute to positive or negative regulation of PLC
1 (Kim et al., 1991; Plattner et al., 2003). However, a subset of these phosphotyrosine moieties may function as a docking site for SH2 domaincontaining proteins during signal transduction (Pei et al., 1997) rather than participating directly in the regulation of PLC
1.
PLC1 accumulates preferentially to cortical actin structures in EGF-stimulated A431 cells (Diakonova et al., 1995), where it binds to actin-binding proteins via its SH3 domain (Park et al., 1999). Furthermore, interaction between the COOH-terminal SH2 domain of PLC
1 and the actin cytoskeleton has been demonstrated in an in vitro binding assay (Pei et al., 1996). Our data show that upon insulin stimulation, the IR and tyrosine-phosphorylated PLC
1 colocalize with the actin clusters that ringed the plasma membrane. These results are consistent with the important role played by PLC
1 in cytoskeletal reorganization and membrane ruffling after cell activation (Yu et al., 1998). Similarly, PI 3-kinase is linked to cytoskeletal reorganization (Vanhaesebroeck et al., 2001) and for full activation of PLC
1 in some models (Rhee, 2001). Inhibition of PI 3-kinase activity by wortmannin has provided an opportunity to assess the mechanism of PLC
1 binding to membrane-associated IR in response to insulin. We found that the insulin-stimulated formation of PI (3,4,5)-trisphosphate does not act as a targeting signal for PLC
1 interaction with the IR.
A principal conclusion of this report is that SH2 domains have little role, if any, in promoting PLC1 recruitment to the IR. In contrast, disabling both SH2 domains was found to prevent the N-C- PLC
1 mutant to associate with ligand-activated receptors for PDGF (Ji et al., 1999) and EGF (this paper). In this regard, Grb14 has been proposed to interact with the IR in an SH2-independent manner, with the BPS domain being the main interacting region (Kasus-Jacobi et al., 2000). It is noteworthy that the binding of the N-C- PLC
1 mutant to the IR occurs even though the mutant is not phosphorylated at Tyr-783 in response to insulin, indicating that efficient PLC
1 association with the IR may not require this phosphorylation event. We established that the NH2-terminal region of PLC
1 encompassing the PHEF domain is able to bind to the IR, as is the full-length protein, thereby selectively blocking recruitment of endogenous PLC
1 to the activated IR, but not EGF receptors. Importantly, our data show that a truncated PLC
1 mutant lacking the PHEF region fails to bind to the IR, which is consistent with the notion that the PHEF-hand domain is necessary for PLC
1 association with the IR. Mutations in the PH domain of PLC
1 did not affect recruitment of PLC
1 to the EGF receptor (Matsuda et al., 2001). It is now believed that PH domains can interact specifically with a subset of signaling molecules rather than exerting promiscuous effects. For example, the IRS-1 PH domain has recently been shown to bind to a protein ligand referred to as PHIP (Farhang-Fallah et al., 2000), and interaction of F-actin with proteins that contain PH domains directs them to sites of cytoskeletal rearrangement at the plasma membrane (Yao et al., 1999). On the other hand, the ß-adrenergic receptor kinase PH domain must bind to heterotrimeric G-protein ß
subunits and with PI (4,5)-bisphosphate to promote effective membrane targeting (Pitcher et al., 1995). The importance that EF-hand alone has in modulating IRPLC
1 association will be the subject of future investigations.
Our findings suggest that PHEF overexpression may exert selective effects in insulin action through alteration in PLC1 signaling. Expression of PHEF has been found to inhibit endogenous PLC
1 association with the IR with concomitant reduction in ERK (but not AKT) phosphorylation in response to insulin. Similarly, increase in ERK phosphorylation by insulin was markedly reduced after blocking PLC
1 expression in HepG2 cells using siRNA methodology. Additionally, reconstitution of PLC
1 in PLC
1-/- fibroblasts significantly elevates the ability of insulin to promote ERK activation. PLC
1 has been implicated in the regulation of MAPK activation in some systems (Zhang et al., 2000; Jacob et al., 2002). Together, our results support the hypothesis that PLC
1 association with the IR is necessary for ERK regulation in response to insulin. This may be of physiological significance, as the unique structure of PLC
1 with its PH, SH2, and SH3 domains may allow scaffolding of effector proteins harboring phosphotyrosine residues or proline-rich domains near the activated IR. The SH3 domain of PLC
1 has been shown to be involved in SOS-mediated Ras activation (Kim et al., 2000) and to interact with c-Cbl (Tvorogov and Carpenter, 2002). The finding that the activated hybrid receptor encompassing the tyrosine kinase domain of the IR requires PLC
1 for efficient calcium mobilization is potentially important (Telting et al., 1999). On the other hand, a PLC
1 mutant lacking the lipase activity can induce DNA synthesis (Smith et al., 1994), indicating that the products of PLC
1 activation and its associated mobilization of intracellular calcium may not be required for all aspects of PLC
1 signaling. In view of the fact that PLC
1 can fulfill functions that are not necessarily dependent on its enzymatic activity, this raises the possibility of a unique activation mechanism whereby PLC
1 acts as an adaptor protein. To what extent the findings reported here relate to the role of PLC
1 in insulin action remains to be elucidated.
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Materials and methods |
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Plasmids and mutagenesis
The pRK5 vector containing cDNA for the HA-tagged rat PLC1 wild-type and the PLC
1 SH2 domain double mutant (N-C-) were obtained from Graham Carpenter (Vanderbilt University, Nashville, TN). The plasmid encoding the human EGF receptor (pXER) was provided by Alexander Sorkin (University Colorado Health Science Center, Denver, CO). The HA-tagged PHEF domain of rat PLC
1 (1301) was amplified from the pRK5/HA-PLC
1 plasmid using PCR-based site-directed mutagenesis with primers to introduce a HindIII site between EF-hand and catalytic domain "X" of PLC
1. A 2,961-bp HindIIIHindIII fragment was excised, and the linearized pRK5/HA-tagged PHEF plasmid was then self-ligated. An HA-tagged truncated PLC
1 mutant lacking the PHEF domain (
PHEF) was created using PCR-based site-directed mutagenesis with primers to introduce EcoRI sites both at the junction between HA epitope and PH domain and between EF-hand and catalytic domain "X". A 903-bp EcoRIEcoRI fragment was excised and the linearized pRK5/HA-tagged
PHEF plasmid was then self-ligated. The constructs were verified by DNA sequence analysis.
Cell culture and metabolic labeling
CHO cells stably expressing wild-type human IR or both the IR and EGF receptors (CHO-EI cells) have been described previously (Kole et al., 1996). HEK293 and liver-derived HepG2 cells were purchased from American Type Culture Collection (Manassas, VA), and PLC1-/- mouse embryonic fibroblasts were gifts from Dr. G. Carpenter (Ji et al., 1998). All CHO cell lines were expanded and maintained in Ham's F12 supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, whereas HepG2 and HEK293 cells were maintained in DME and McCoy's 5A medium containing 10% FBS and antibiotics. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C.
For metabolic labeling experiments, confluent monolayers of CHO-IR were incubated for 16 h with 60 µCi/ml Trans 35S-label (ICN Biomedicals) in methionine- and cysteine-free RPMI 1640 medium containing 3% FCS. After a series of PBS washes, cells were serum starved for 3 h and were then subjected to treatments as described below.
Isolation and culture of rat hepatocytes
Hepatocytes were isolated from 5-mo-old male Fischer 344 rats by the collagenase perfusion method of Seglen (Ikeyama et al., 2002). The isolated cells were seeded onto Biocoat Collagen I cellware (BD Discovery Labware) in William's E medium supplemented with 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin for 2 h in 5% CO2 at 37°C to allow attachment to the dishes. The medium was then replaced with serum-free William's E medium plus the above supplements, and cells were cultured for an additional 16 h before treatment. This procedure results in <5% contamination with nonhepatocyte cells.
Transient transfection assays
HEK293 cells were cultured for 24 h until 6080% confluence was reached. Transient transfection was performed according to the manufacturer's protocol for the use of FuGENETM 6. In brief, empty expression vector (pcDNA3.1) and expression plasmids encoding HA-tagged PLC1 [wild type or N-C-] together with recombinant human IR or human EGF receptor were mixed with the transfection reagent and directly added into the culture plates at a ratio of 1.5 µg of each plasmid per 60-mm dish. Both CHO-EI cells and PLC
1-/- mouse embryonic fibroblasts were transfected using LipofectAMINETM 2000 according to the manufacturer's protocol. 24 h after transfection, cells were serum starved for 18 h and then subjected to a 30-min treatment with 200 µM orthovanadate followed by stimulation with 100 nM insulin or 20 nM EGF for 510 min at 37°C. Transfection efficiency was monitored using a plasmid DNA encoding eGFP.
siRNA preparation and cell transfection
The siRNA sequence targeting human PLC1 (GenBank/EMBL/DDBJ accession no. NM_002660) was from position 29792999 relative to the start codon. This PLC
1 sequence was reversed and used as unspecific siRNA control. 21-nt RNAs were purchased from Dharmacon in deprotected and desalted form, and the formation of siRNA duplex (annealing) was performed according to the manufacturer (Dharmacon). Subconfluent HepG2 cells were transiently transfected with siRNAs using OligofectamineTM according to the manufacturer's protocol (Life Technologies). In brief, 100 µl Opti-MEM® I medium and 10 µl OligofectamineTM per 60-mm dish were preincubated for 5 min at RT. During the time for this incubation, 100 µl Opti-MEM® I medium was mixed with 20 µl of 20 µM siRNA. The two mixtures were combined and incubated for 20 min at RT for complex formation. The entire mixture was then added to the cells in one dish resulting in a final concentration of 100 nM for the siRNAs. Cells were usually assayed 4872 h after transfection. Specific silencing was confirmed by at least three independent experiments.
Immunofluorescence and confocal microscopy
Cells grown on coverslips were fixed in fresh 4% PFA in PBS for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min at RT. The cells were incubated with blocking buffer (8% BSA in PBS) for 20 min at RT, washed in PBS supplemented with 0.5% BSA and 0.05% Tween 20, and incubated with anti-IR ß-subunit (1:100; 06492, Upstate Biotechnology), phosphoPLC1(1:50; Tyr-783), or PLC
1 (1:200; Upstate Biotechnology) antibody for 16 h at 4°C. After washing, cells were stained with Alexa Fluor® secondary antibody (1:1,000). For immunolocalization of F-actin, fixed cells were incubated with Alexa Fluor® 568conjugated phalloidin. Nuclear counterstaining was performed by incubating coverslips with TO-PRO®-3 in PBS for 5 min before mounting slides with Vectashield® (Vector Laboratories). Images were acquired using an inverted confocal microscope (LSM-410; Carl Zeiss MicroImaging, Inc.) with a 63x oil-immersed objective, and processed using the MetaMorph® software (Universal Imaging Corp.). No fluorescent staining was observed when the primary antibody was omitted.
IRTRAP cross-linking in intact cells
Serum-starved cells were washed twice in PBS, and were then incubated in Krebs Ringer phosphate buffer for 5 min at 37°C. 100 nM insulin was added for 5 min and cells were then transferred to thermoregulated aluminum cooling plates set at 6°C. The cross-linking reaction was initiated by the addition of 100 µM BMH or vehicle (DMSO) and quenched 10 min later with 4 mM L-cysteine. In some instances, cross-linking was performed in the presence of 100 µM BMOE or BMB. For wortmannin treatment, 100 nM wortmannin was added to the cells 30 min before insulin stimulation.
Immunoprecipitation and immunoblotting
Cells were lysed in immune precipitation buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1 mM orthovanadate, 100 mM NaF, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 0.02% sodium azide, 0.25 mM Pefabloc-SC [Boehringer], 1 mM benzamidine, 8 µg/ml aprotinin, and 2 µg/ml leupeptin) for 20 min on ice, and then centrifuged at 10,000 g for 20 min at 4°C to sediment insoluble materials. The clarified lysates were incubated with the indicated antibodies for 16 h at 4°C with rocking. Then, protein A/G-agarose (Oncogene Research Products) beads were added and the incubation was continued at 4°C for 2 h. The beads were pelleted by centrifugation and washed twice in the same buffer and twice in 50 mM Hepes, pH 7.4, and 0.1% Triton X-100 before solubilization in Laemmli sample buffer supplemented with 5% 2-mercaptoethanol. In some experiments, cells were lysed directly in Laemmli sample buffer containing 5% 2-mercaptoethanol and 1 mM orthovanadate. After heating at 70°C for 10 min, proteins were separated by SDS-PAGE and were electrotransferred onto polyvinylidene difluoride membranes. Detection of individual proteins was performed by immunoblotting with specific primary antibodies and visualized by ECL. Signals were quantitated by densitometry coupled with the ImageQuant software (Molecular Dynamics). Where indicated, membranes from 35S-labeling experiments were dried and autoradiography was performed.
Purification of the IRTRAP complex
10 x 150-mm dishes of CHO-IR cells were incubated with 100 nM insulin for 5 min and were then subjected to cross-linking reaction with BMH as shown above. After immunoprecipitation of the cell lysates with anti-IR antibodies prebound to protein G-agarose, the immune pellets were washed extensively and then incubated with 1 ml 1.5x Laemmli sample buffer without 2-mercaptoethanol for 60 min at RT. The eluted proteins were then concentrated down to 50 µl using an Ultrafree® centrifugal filter (molecular weight cut-off of 100 kD, Millipore). The concentrated material was incubated with 2-mercaptoethanol (7.5% final concentration) for 10 min at 70°C, and was then resolved by SDS-PAGE.
TRAP identification by MALDI mass spectrometry
Colloidal bluestained bands were cut out of the gels for in-gel digestion as follows. The gel pieces were equilibrated for 20 min in 200 µl 25 mM ammonium bicarbonate, 50% acetonitrile. The supernatant was decanted and the same procedure was repeated until full decoloration of the gel. The gel pieces were dried, rehydrated for digestion with 5 µg/ml porcine trypsin (Roche) in 25 mM ammonium bicarbonate, and incubated at 37°C overnight. The reaction was stopped by adding 1 vol of 50% acetonitrile, 0.5% trifluoroacetic acid. The peptides were extracted from the gel matrix by sonication for 0.51 h. Peptide mass fingerprinting was performed using a mass spectrometer (Voyager-DE STR; PerkinElmer) operating in delayed reflector mode at an accelerating voltage of 20 kV. The peptide samples were cocrystallized with matrix on a gold-coated sample plate using 1 µl matrix (-cyano-4-hydroxy-transcinnamic acid) and 1 µl sample. After internal calibration with protein standards (renin, angiotensin, and adrenocorticotropic hormone), the monoisotope peptide masses were assigned and then used in database searches with ProFound (http://prowl.rockefeller.edu/profound_bin/webProFound.exe). Cysteines were modified by acrylamide, and methionine was considered to be oxidized. One missed cleavage was allowed.
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Acknowledgments |
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Submitted: 29 January 2003
Accepted: 2 September 2003
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