Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Abstract

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.

1. Introduction

Atherosclerosis is a complex inflammatory disease of the vessel wall [1]. The development of atherosclerotic plaque is caused, in part, by the progressive internalization and accumulation of modified lipids, particularly oxidized low-density lipoprotein (OxLDL) [2]. OxLDL is bound and internalized into cells by the C-type lectin-like OxLDL receptor (LOX-1) [3], which has been shown to play a significant role in atherogenesis [4]. LOX-1 is a type II transmembrane protein expressed on endothelial cells, monocytes, smooth muscle cells, and platelets. As a scavenger receptor, LOX-1 also binds to C-reactive protein [5], heat shock proteins [6], apoptotic cells [7], platelets [8], and bacteria [9]. LOX-1 has been associated with not only atherosclerotic disease [4] but also Alzheimer’s disease [10–11] and sepsis [12]. Because of the multifunctional properties of LOX-1, it has been considered an attractive therapeutic target for treating a variety of diseases [13–16].

Upon binding to OxLDL, LOX-1 can induce several cellular events [15,17–18]. In association with membrane type 1 matrix metalloproteinase, LOX-1 signaling can promote endothelial dysfunction through the activation of the small GTPases RhoA and Rac1 [19]. LOX-1 signaling can also promote pro-inflammatory changes in endothelial cells through the activation of NFκB [20], resulting in increased expression of adhesion molecules [21] and chemoattractants, such as monocyte chemoattractant protein-1 [22]. In addition, LOX-1 signaling can result in apoptosis due to the activation of protein kinase C and other protein tyrosine kinases [23]. Although there are numerous mechanisms through which LOX-1 signaling has been linked to cardiovascular disease, little is known regarding the membrane-proximal events in LOX-1 receptor signaling.

The cytoplasmic domain of LOX-1 has been shown to play a critical role in receptor trafficking and endocytosis [24] and in cell-surface sorting [25]. A recent study has also shown that intracellular proteins ROCK2 and ARHGEF1 can form a complex with LOX-1 in an OxLDL-dependent manner [26]. Moreover, a naturally occurring, alternatively spliced isoform of LOX-1, termed LOXIN, has been shown to act as a dominant negative regulator of LOX-1 [27]. Although LOXIN has defective ligand-binding abilities, it has an intact transmembrane and intracellular domain that can form a heterodimer with intact LOX-1 [28]. Together, these reports suggest that the cytoplasmic domain of LOX-1 is an important mediator of LOX-1 function. Because the cytoplasmic tail of LOX-1 has no known enzymatic or catalytic activity, intracellular signaling probably requires interaction between the LOX-1 cytoplasmic domain and intracellular proteins. To our knowledge, no intracellular proteins have been identified that directly interact with the LOX-1 cytoplasmic domain [29].

In this study, we performed affinity isolation experiments using a LOX-1 cytoplasmic domain peptide to identify intracellular proteins that interact with the LOX-1 cytoplasmic domain. The identification of such proteins may help to determine ways to regulate the function of LOX-1 in disease.

2. Materials and Methods

2.1 Cell culture

HUVECs were cultured in EGM-2 (Lonza, Houston, TX) supplemented with 10% (v/v) FBS (Gibco, Grand Island, NY), penicillin (100 units/ml), and streptomycin (100 µg/ml) (Gibco). Cells were grown at 37°C in humidified conditions with 5% CO₂. Before use, cells were trypsinized by using 0.05% Trypsin/EDTA (Gibco), collected, and washed in phosphate-buffered saline (PBS).

2.2 Peptides

A LOX-1 cytoplasmic domain peptide (amino acid residues 1–33) was synthesized with C-terminal modifications that included a dual glycine spacer, a penultimate lysine-long chain biotin (LC-Biotin), and a C-terminal glycine (sequence shown in Fig. 1). For a control, we used a scrambled LOX-1 cytoplasmic domain peptide generated with the same C-terminal modifications (sequence shown in Fig. 1). The peptides were synthesized, purified by using high-performance liquid chromatography (>90%), and verified by mass spectrometry (NeoBioscience, Cambridge, MA).

Fig. 1.

Primary structure of the LOX-1 receptor. LOX-1 is a type II transmembrane protein with a short (33 amino acid) N-terminal intracellular domain, or cytoplasmic tail (CT), a single-pass transmembrane domain (TM), a neck region, and a C-terminal ligand-binding domain (LBD) (top image). The sequences of the LOX-1 cytoplasmic domain peptides and the scrambled LOX-1 peptides used in the affinity isolation experiments are shown with the penultimate lysine-long chain biotin (LC-Biotin) modifications (bottom image).

2.3 Cell lysate preparation

HUVECs were washed with PIPES buffer, which comprised 10 mM PIPES and 50 mM NaCl (pH 6.8), and resuspended (10 × 10⁶ cells/ml) in PIPES lysis buffer, which consisted of PIPES buffer containing 1% Triton X-100, 1 mM EDTA, 1 mM Na₃VO₄, 50 mM NaF, 150 mM sucrose, protease inhibitors (ie, mini-tablets containing inhibitors of chymotrypsin, thermolysin, papain, pronase, pancreatic extract, and trypsin; Roche, Indianapolis, IN), and 0.1 mM N-ethylmaleimide (Sigma, St Louis, MO). The cells were incubated overnight in the lysis buffer at 4°C with end-over-end rotation. Lysates were then passed through a 21-gauge syringe and centrifuged at 13,000g for 30 minutes at 4°C. Lysate protein concentrations were determined by using Quick Start Bradford Dye reagent (Bio-Rad, Hercules, CA).

2.4 Affinity isolation using LOX-1 cytoplasmic domain peptide

Affinity isolation assays were performed as previously described [30], with modifications. NeutrAvidin agarose beads (100 µl of 50% slurry; Thermo Scientific, Rockford, IL) were washed in PIPES buffer. Then, 150 µg of LOX-1 cytoplasmic domain peptide or control scrambled peptide was added to the beads in a final volume of 1 ml in PIPES buffer. After an overnight incubation at 4°C, FBS (20% v/v) was added to block nonspecific binding sites, and the beads were again incubated overnight at 4°C. The peptide-bound beads were then thoroughly washed in PIPES buffer for use in the affinity isolation assay. Between 500 and 700 µg (total protein) of HUVEC lysate was added to 100 µl of either unbound beads or peptidebound beads, and the binding volume was adjusted to 1 ml with PIPES lysis buffer. After an overnight incubation at 4°C, the beads were washed 5 times with PIPES lysis buffer and collected by centrifugation at 700g for 2 minutes. To elute the bound proteins, the collected beads were then heated at 95°C in 75 µl of 2× sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris HCl [pH 6.8], 20% glycerol, 4% SDS, and 0.005% bromophenol blue) for 5 minutes and centrifuged at 13,000g for 1 minute. The solubilized proteins were then separated by 4–20% Precise™ protein gels (Thermo Scientific).

2.5 Silver staining and LC/MS/MS analysis

The protein samples obtained by affinity isolation and run on Precise protein gels (4%–20%) were stained by using the Pierce Silver Staining kit (Thermo Scientific). Protein bands that were visible in the lanes with the LOX-1 affinity isolation products were excised, destained as per the manufacturer’s protocol, and sent to the University of Utah Mass Spectrometry and Proteomics core facility for analysis by liquid chromatography combined with tandem mass spectrometry (LC/MS/MS). In-gel digestion and LC/MS/MS analysis of these samples were performed as previously described [31].

2.6 Coimmunoprecipitation assay

HUVECs were treated with or without 10 µg/ml of OxLDL (770252-7, Kalen Biomedical, Montgomery Village, MD) for 1 hour, lysed by using the previously described method in section 2.3, and then incubated overnight at 4°C with 1 µg of anti-LOX-1 monoclonal antibody (sc66155, Santa Cruz Biotechnology, Dallas, TX) or isotype-specific (IgG1) control antibody (0102-01, SouthernBiotech, Birmingham, AL). Next, 20 µl Protein-G beads (GE Healthcare, Pittsburgh, PA) prewashed in PIPES buffer was added to the cell lysate and rotated for 2 h at 4°C. The beads were then washed 5 times with PIPES lysis buffer, and the proteins were eluted by adding 2× SDS sample buffer (40 µl) and heating the beads at 95°C for 5 minutes. Eluted proteins were separated by 4–20% Precise™ protein gels (Thermo Scientific), and western blot analysis was performed by using anti-CCT1 (sc53454, Santa Cruz Biotechnology) or anti-CCT4 (sc137094, Santa Cruz Biotechnology) antibodies.

2.7 Indirect immunofluorescence confocal microscopy

HUVECs (2 × 10⁵ cells) were cultured in supplemented EGM-2 medium on sterile 12-mm glass cover slips in a 12-well plate overnight at 37°C in humidified conditions with 5% CO₂. The following day, cells were incubated with or without 10 µg/ml of DiI-OxLDL (Kalen Biomedical) for 1 hour and washed in PBS twice before undergoing fixation with 3.7% paraformaldehyde for 15 minutes at room temperature. To permeabilize the cells, we placed them in PBS containing 10% horse serum and 0.2% Triton X-100 for 10 minutes on ice. The cells were blocked overnight at 4°C with SuperBlock blocking buffer (Thermo Scientific) supplemented with 5% horse serum and 5% goat serum. For immunostaining, 100 µl of primary antibody (diluted to 1 µg/ml in blocking buffer supplemented with 0.05% Tween 20 and 0.001% Triton X-100) was pipetted onto flattened parafilm, and a cover slip containing the cells was placed over it. After a 45-minute incubation at room temperature under humidified conditions, the cover slips were removed, placed in wells of a 6-well plate, and washed 3 times for 10 minutes each with PBS containing 10% horse serum, 0.05% Tween 20, and 0.001% Triton X-100. The cells were then immunostained with isotype-specific secondary antibodies (2–4 µg/ml) in a fashion similar to that used for the primary antibodies. Primary antibodies included anti-LOX-1 (ab60178, Abcam, Cambridge, MA) and anti-CCT1 (sc53454, Santa Cruz Biotechnology), and their respective secondary antibodies were Alexa Fluor 488 goat anti-rabbit (Invitrogen, Carlsbad, CA) and Alexa Fluor 555 goat anti-rat (Invitrogen). Isotype-specific IgG and the respective secondary antibody were used as a control. Immunostaining for multiple proteins was performed in series, with the primary antibody or control isotype-specific IgG being followed by the respective secondary antibodies. Lastly, to aid in the visualization of the nuclei, Hoechst 33258 (Sigma) was diluted to 2 µg/ml in blocking buffer with 0.05% Tween 20 and 0.001% Triton X-100 and added to the fixed cells. After incubating the cells for 10 minutes at room temperature, the cells were washed extensively. The cover slip with the cells was then placed over a drop of fluorescence mounting media (Dako, Carpinteria, CA) on a superfrost slide and left to dry at room temperature for 15 minutes. The slides were stored in a cold room until confocal microscopy was performed. Similar methods were used to label early and late endosomes with anti-EEA1 (Early Endosome Antigen-1 antibody, sc-365652; Santa Cruz Biotechnology) and anti-M6PR1 (Mannose 6-Phosphate Receptor antibody [2G11], MA1-066; Thermo Scientific), respectively. All images were obtained by using a Leica TCS SP5 II confocal microscope. Colocalization imaging was performed by scanning an XY plane at a single Z position. Identical settings were used for both control and test slides.

2.8 Chaperonin-containing TCP-1 (CCT) complex purification

CCT was purified from bovine testes according to previously established procedures, and the integrity of the oligomeric CCT complex was confirmed by using single-particle cryoelectron microscopy [32–33]. The final purified protein concentration was determined by using the Bradford assay with BSA standards (Pierce), and substrate folding activity of the CCT complex was assessed with a luciferase refolding assay, as previously described [34].

2.9 Direct binding assay

NeutrAvidin agarose beads (100 µl of 50% slurry, Thermo Scientific) were washed in CCT lysis buffer, which comprised 25 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgCl₂, 10% glycerol, 0.1% Triton X-100, 20 mM EDTA, 0.1% v/v Tween-20, and protease inhibitors (Roche). Then, 100 µg of the biotinylated LOX-1 peptide or the control scrambled peptide was added to 100 µl of the resuspended beads, and the CCT lysis buffer was used to bring the final volume to 1 ml. After an overnight incubation at 4°C, the peptide-bound beads were washed once in CCT lysis buffer, and the nonspecific binding sites were blocked with 1 ml of FBS during an overnight incubation at 4°C. The blocked beads were then washed and resuspended in 1 ml of CCT lysis buffer. Purified endogenous CCT (100 µg) from bovine testis was then combined with the peptide-bound beads and incubated overnight at 4°C in the presence or absence of ATP (0.1 mM). After the incubation, the beads were washed 5 times with CCT lysis buffer and collected by centrifugation at 700g for 2 minutes. Collected beads were heated at 95°C in 50 µl of 2× SDS sample buffer for 5 minutes and centrifuged at 13,000g for 1 minute. The eluted proteins (20 µl) were then separated by 4–20% Precise™ protein gels (Thermo Scientific), and western blot analysis was performed to detect CCT1. Purified CCT was run on the same gel as a control.

3. Results

3.1 CCT complex proteins identified as novel LOX-1 cytoplasmic domain-interacting proteins

To identify the intracellular molecules that interact with the LOX-1 cytoplasmic domain, we synthesized the cytoplasmic tail of LOX-1 as a biotinylated peptide (Fig. 1), conjugated this peptide to NeutrAvidin agarose beads, and used these beads in affinity isolation experiments with lysate from HUVECs (Fig. 2A). We used beads alone and beads conjugated to a scrambled sequence of the LOX-1 peptide as controls. The proteins eluted from the beads after affinity isolation were separated on 4–20% protein gels and silver stained. The bands for each of the proteins enriched on the LOX-1 peptide beads (Fig. 2B, asterisks) were excised, destained, and subjected to LC/MS/MS analysis. The proteins identified included 6 of the 8 subunits in the CCT complex: subunits 1, 3, 4, 5, 6A, and 7 (Supplementary Table S1).

Fig. 2.

Identification of proteins that interact with the LOX-1 cytoplasmic domain. (A) A biotinylated LOX-1 cytoplasmic domain peptide was used as bait in affinity isolation experiments to identify proteins that interact with the LOX-1 cytoplasmic domain. A scrambled biotinylated LOX-1 peptide was used as a control. Biotinylated peptides were bound to NeutrAvidin agarose beads for the assay. After blocking, the beads were incubated with HUVEC lysate at 4°C. The beads were then washed, and bound protein was eluted off the beads with SDS sample buffer. (B) A gel showing the eluted proteins separated by SDS-PAGE and silver stained. Proteins that were bound to LOX-1 (shown by asterisks) were excised and analyzed by liquid chromatography with tandem mass spectrometry. B, beads alone; L, LOX-1 cytoplasmic domain peptide; sL, scrambled LOX-1 cytoplasmic domain peptide.

3.2 CCT constitutively interacts with LOX-1

To verify the interactions between the LOX-1 cytoplasmic domain and CCT complex proteins, we performed a western blot analysis of proteins obtained by either LOX-1 affinity isolation or by immunoprecipitation. For this analysis, antibodies against 2 of the 8 subunits of the CCT complex, CCT1 (TCP1α) and CCT4 (TCP1δ), were used to confirm the presence of the whole CCT complex [33,35]. Western blot analysis of the affinity isolation products showed CCT1 and CCT4 bound to the LOX-1 cytoplasmic domain peptide but not to the scrambled peptide or to the beads alone (Fig. 3A, top panels), suggesting a specific interaction between LOX-1 and the CCT complex. Western blot analysis of the products obtained by LOX-1 immunoprecipitation in HUVECs showed that CCT1 and CCT4 coimmunoprecipitated with endogenous LOX-1 but not with protein bound by the isotype-matched control antibody, further demonstrating the specificity of the interaction between LOX-1 and the CCT complex (Fig. 3B). Furthermore, indirect immunofluorescence staining of fixed HUVECs showed that LOX-1 and CCT1 colocalized in small, vesicular-like structures (Fig. 3C). Interestingly, these vesicles were found to be partially associated with early and late endosomes, which were identified by using markers EEA1 and M6PR1, respectively (Supplementary Fig. S1). These results supported the LC/MS/MS data, although they did not indicate whether the interaction between the LOX-1 cytoplasmic domain and the CCT complex is direct or mediated indirectly by an adaptor molecule.

Fig. 3.

Interaction between the CCT complex and the LOX-1 cytoplasmic domain. (A) Western blot analysis of the affinity isolation products showing that both CCT1 and CCT4 specifically bound to the LOX-1 peptide but not to the beads alone or to the scrambled LOX-1 peptide (top panel). Peptide loading was visualized with Ponceau staining of blots (lower panel, just below the 10 kDa molecular weight marker). CL, cell lysate of HUVECs; B, beads alone; L, LOX-1 cytoplasmic domain peptide; sL, scrambled LOX-1 cytoplasmic domain peptide; IB, immunoblotting. (B) Western blot analysis of the proteins immunoprecipitated from HUVECs showing that the CCT complex subunits CCT1 (top) and CCT4 (bottom) coimmunoprecipitated with the proteins captured by the LOX-1–specific mouse monoclonal antibody (LOX) but not with those captured by the IgG1 isotype control antibody (Iso). Blots shown are representative of at least 4 independent experiments. In the top panel, the light chain (IgG-L) of the LOX-1 mAb and the isotype-matched control mAb can be seen at approximately 25 kDa in both lanes. In the bottom panel, both the IgG heavy chain (IgG-H) and IgG-L of the LOX-1 monoclonal antibody and the isotype control antibody are visible at approximately 55 kDa and 25 kDa, respectively. CL, cell lysate of HUVECs. IP, immunoprecipitation; IB, immunoblotting. (C) Immunofluorescence staining of HUVECs shows the colocalization of LOX-1 and CCT-1 in small, vesicular-like structures (white vesicles shown by arrows in merged image of LOX-1 and CCT1). Nuclei were stained with Hoechst 33258 (blue), and all images were acquired by using a Leica TCS SP5 II confocal microscope. Colocalization imaging was performed by scanning an XY plane at a single Z position.

3.3 CCT directly interacts with the LOX-1 cytoplasmic domain

CCT requires ATP for its activity [33,36] and to bind to intracellular proteins [37–40]. Therefore, to determine whether CCT directly interacts with LOX-1, we performed a direct binding assay in which purified CCT was incubated with LOX-1 cytoplasmic domain peptides in the presence or absence of ATP. As a control, we incubated scrambled peptide with CCT in the presence of ATP. Any bound CCT was then eluted off the beads, and western blot analysis was performed to detect CCT1. Figure 4 shows that CCT1 associated with the LOX-1 peptide in the presence of ATP but not in the absence of ATP. In contrast, CCT1 did not bind to the control scrambled peptide in the presence of ATP (Fig. 4). These findings demonstrate that CCT can directly bind to the LOX-1 cytoplasmic domain and that this interaction is ATP-dependent. Because CCT is known to affect the folding of some proteins [41], we also examined whether knocking down the expression of CCT1 would affect LOX-1 expression. In HUVECs treated with CCT1 siRNA, LOX-1 expression was unchanged compared to that in HUVECs treated with control siRNA (Supplementary methods and Supplementary Fig. S2).

Fig. 4.

Direct binding of CCT to the LOX-1 cytoplasmic domain. Purified CCT protein from bovine testes was used in direct binding assays either in the presence (+) or absence (−) of ATP (0.1 mM). The western blot shows that CCT1 specifically bound to the LOX-1 peptide in the presence of ATP (L+) but not to the LOX-1 peptide in the absence of ATP (L−) or to the scrambled LOX-1 peptide in presence of ATP (sL+). Peptide loading was visualized by using Streptavidin-HRP (lower panel, just below the 10 kDa molecular weight marker). Blots shown are representative of 3 independent experiments. IB, immunblotting.

3.4 OxLDL suppresses the LOX-1/CCT interaction

Oxidized low-density lipoprotein is one of the LOX-1 receptor ligands that play an important role in atherosclerosis development [17]. To determine whether OxLDL regulates the interaction between LOX-1 and CCT, we treated HUVECs with DiI-labeled OxLDL (DiI-OxLDL) and immunostained the cells for LOX-1 and CCT1. When HUVECs were cultured without OxLDL, CCT1 colocalized with LOX-1 in small, vesicle-like structures (Fig. 3C). However, in HUVECs treated with DiI-OxLDL, the colocalization of LOX-1 and CCT1 within these vesicles decreased (Fig. 5A). To quantify the colocalization of LOX-1 and CCT1 in the cells before and after the OxLDL treatment, we calculated Manders’ colocalization coefficients M1 and M2 (Supplementary methods). Before the OxLDL treatment, the M1 and M2 coefficients were 85.3 ± 8.8% and 71.4 ± 4.3%, respectively. After the OxLDL treatment, the M1 and M2 coefficients were significantly reduced to 19.2 ± 3.2% and 17.2 ± 1.7%, respectively (Supplementary Fig. S3). This suggests that OxLDL may suppress the interaction between LOX-1 and CCT. To further examine this possibility, we assessed whether CCT complex proteins coimmunoprecipitate with LOX-1 in HUVECs treated with or without OxLDL. Western blot analysis showed that CCT1 coimmunoprecipitated with LOX-1 in untreated cells but not in OxLDL-treated cells (Fig. 5B). Thus, our findings indicate that OxLDL suppressed the CCT interaction with LOX-1.

Fig. 5.

OxLDL disassociates the CCT1/LOX-1 interaction. (A) Immunofluorescence staining of HUVECs treated with DiI-OxLDL (10 µg/ml) for 1 hour showed that OxLDL decreased the colocalization of LOX-1 and CCT1, as compared to that seen in untreated cells (shown in Fig. 3C). (B) Western blot analysis showing the proteins that coimmunoprecipitated with LOX-1 in HUVECs treated with (+) or without (−) OxLDL. CCT1 coimmunoprecipitated with LOX-1 in the untreated cells but did not in the OxLDL-treated cells. The 2 bands found below CCT1 at ~50 and ~25 kDa are the heavy chain (IgG-H) and light chain (IgG-L) of the LOX-1 monoclonal antibody, respectively. CL, cell lysate of HUVECs; IP, antibody used for immunoprecipitation; IB, antibody used for immunoblotting.

4. Discussion

LOX-1 signaling is thought to play an essential role in the initiation and progression of atherosclerosis[17]. However, little is known about the intracellular proteins that can directly bind to the cytoplasmic domain of LOX-1. In this study, affinity isolation experiments showed that the LOX-1 cytoplasmic domain interacts with subunits of the CCT complex in HUVECs. This LOX-1/CCT interaction was verified by using biochemical and cell biology methods. Furthermore, we showed that CCT1 requires ATP to directly bind to the cytoplasmic domain of LOX-1. To our knowledge, this is the first report to show that an intracellular protein, CCT, can interact directly with the LOX-1 cytoplasmic domain.

The CCT complex is a large (approximately 1 mDa), ATP-dependent, protein-folding apparatus that consists of 2 hetero-oligomeric rings stacked upon each other. Each ring is made up of 8 subunits (CCT1-CCT8) [33,36]. In our study, we used coimmunoprecipitation and immunofluorescence experiments to verify that components of the CCT complex colocalize and interact with LOX-1 in intact HUVECs. Thus, it is unlikely that the interaction identified by our affinity isolation experiments resulted merely from the in vitro recognition of the LOX-1 cytoplasmic domain peptide by CCT after cell lysis.

The CCT complex is mainly involved in the folding of cytoskeletal proteins [41], and many such CCT substrates have recently been identified [37–40,42]. If CCT also affects the folding of the LOX-1 protein, we would predict that reducing the expression of CCT may also affect LOX-1 expression. However, our experiments showed that the siRNA-mediated knockdown of CCT1 does not significantly change the total expression of LOX-1 protein, suggesting that CCT does not regulate LOX-1 folding or expression. Our results also showed that CCT1 and LOX-1 colocalize in small, vesicle-like structures and that OxLDL decreases this association. Further investigation showed that the vesicles in which CCT1 and LOX-1 colocalize are early and late endosomes. Murphy and colleagues [15,24] have previously shown that the LOX-1 receptor is constitutively internalized and recycled to the cell surface, independent of OxLDL, possibly by the endosomal pathway. Although knockdown of CCT1 did not change overall expression levels of LOX-1 protein, future work will determine whether membrane turnover and trafficking is dependent on CCT complex function. CCT has also been reported to participate in the uptake of exogenous antigens and the presentation of these antigens on both class I and class II major histocompatibility complex molecules [6,43–44], suggesting a potential role for the LOX-1/CCT interaction in this process. Understanding the physiological relevance of the LOX-1/CCT interaction requires further analysis and may provide insight into the regulation of LOX-1 function.

An alternatively spliced isoform of LOX-1, termed LOXIN, has been identified that lacks exon 5, which is a primary component of the LOX-1 ligand-binding domain. In LOXIN, the extracellular dimerization domain and the cytoplasmic domain are intact [29]. LOXIN has been identified as both a homodimer and as a heterodimer with LOX-1 [28]. The formation of a heterodimer between LOXIN and LOX-1 reduces OxLDL binding and decreases the cell surface expression of LOX-1 [28]. It is possible that LOXIN homodimers or LOXIN/LOX-1 heterodimers negatively regulate LOX-1 function by interacting with CCT and competing with or sequestering CCT from LOX-1. Future studies may help determine whether differential CCT interactions occur with LOXIN homodimers or LOXIN/LOX-1 heterodimers in intact cells.

In conclusion, we have demonstrated for the first time that the intracellular CCT complex can directly and constitutively interact with the LOX-1 cytoplasmic domain. A better understanding of this receptor-proximal molecular association may lead to the identification of novel targets for the pharmaceutical intervention of cardiovascular diseases in which LOX-1 has been implicated, such as atherosclerosis [4]. Future studies will delineate these interactions in greater detail and increase our understanding of the role these interactions play in regulating LOX-1 function under physiologic and pathologic conditions.

Highlights.

• -Affinity isolation was used to identify proteins that bind LOX-1 cytoplasmic domain.
• -CCT complex subunits were found to constitutively bind the LOX-1 cytoplasmic domain.
• -LOX-1/CCT binding was verified in cells via immunoprecipitation and immunostaining.
• -Purified native CCT could directly bind to the LOX-1 cytoplasmic domain peptide.
• -Oxidized low-density lipoprotein suppressed the LOX-1/CCT interaction.