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Originally published In Press as doi:10.1074/jbc.M611537200 on January 19, 2007

J. Biol. Chem., Vol. 282, Issue 11, 7973-7981, March 16, 2007
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The Microtubule-binding Protein Hook3 Interacts with a Cytoplasmic Domain of Scavenger Receptor A*

Hitomi Sano{ddagger}§1, Masaho Ishino, Helmut Krämer||2, Takeyuki Shimizu{ddagger}§, Hiroaki Mitsuzawa{ddagger}§, Chiaki Nishitani{ddagger}§, and Yoshio Kuroki{ddagger}§

From the Departments of {ddagger}Biochemistry and Hygiene, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan, the ||Center for Basic Neuroscience and Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and §CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Received for publication, December 18, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The class A scavenger receptor (SR-A) is a multifunctional transmembrane glycoprotein that is implicated in atherogenesis, innate immunity, and cell adhesion. Despite extensive structure-function studies of the receptor, intracellular molecules that directly interact with SR-A and regulate the receptor trafficking have not been determined. In the current study, we have identified a microtubule-binding protein, Hook3, as a novel interacting partner of SR-A. The association between a rat Hook3 isoform and SR-A was suggested by yeast two-hybrid screening and mass spectrometry analysis of SR-A-cytoplasmic domain-bound proteins in rat alveolar macrophages. The binding of overexpressed and endogenous human Hook3 to SR-A was demonstrated by pull-down assay and co-immunoprecipitations. Furthermore, endogenous murine SR-A and HK3 co-sedimented from cell lysates isolated from Raw264.7 murine macrophage cells. The interaction of Hook3 with SR-A was significantly stimulated after SR-A had recognized the extracellular ligand. Studies using truncations demonstrated that the positively charged C-terminal Val614–Ala717 region of human Hook3 was required for the interaction with the negatively charged residues, Glu12, Asp13, and Asp15 in the human SR-A cytoplasmic domain. By transfecting small interfering RNA targeting Hook3, total and surface expression, receptor-mediated ligand uptake and protein stability of SR-A were significantly promoted, whereas the protein synthesis and maturation were not altered. We propose for the first time that Hook3 may participate in the turnover of the endocytosed scavenger receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage scavenger receptor A (SR-A)3 is a type II transmembrane glycoprotein and acts as a prominent scavenger receptor of macrophages and dendritic cells (1). Scavenger receptors recognize a number of ligands including chemically modified or altered molecules (2, 3). The extracellular domain of SR-A is comprised of an {alpha}-helical coiled-coil region, a collagenous region, and a cystein-rich globular head at the C terminus (4). Four lysines in the most C-terminal portion of the receptor form a positively charged groove responsible for the binding to a variety of polyanionic ligands, including acetylated low density lipoproteins (AcLDL), oxidized LDL, lipopolysaccharides, lipoteichoic acids, fucoidan, poly(I), gp96, calreticulin, and so on (1, 5, 6). The uptake of modified lipoproteins by SR-A is considered to be involved in the process of atherosclerosis (7) but anti-atherogeneic effects have also been suggested (8). In innate immune systems, SR-A is responsible for phagocytosis of Gram-negative and -positive bacteria (9, 10), detoxification of microbial products (11, 12), and clearance of apoptotic cells (13). In addition to the ligand recognition for endocytosis, the extracellular domain of SR-A participates in cell adhesion, possibly through interactions with extracellular matrix proteins (14–16).

The N-terminal cytoplasmic tails of human, rabbit, and bovine SR-A consist of 50 amino acids, whereas the murine tail has 55 highly conserved amino acids (4, 17). Previous studies focusing on the cytoplasmic domain of SR-A identified several specific motifs and phosphorylation sites participating in receptor internalization or cell surface localization (18–21). We have reported that pulmonary surfactant protein A, which plays important roles in host defense in lungs, promotes SR-A-mediated phagocytosis by stimulating alveolar macrophages to induce increased surface expression of SR-A (22). However, the precise mechanism of intracellular trafficking of SR-A remains to be clarified. Cell surface SR-A proteins are activated upon ligand binding, enter cells via coated pits and traffic the endocytic pathway, recycling between early endosomal compartments and the cell surface (23). However, little information is available on the functional partners involved in the sorting and recycling pathways of SR-A. Although the interaction of SR-A with calnexin in the endoplasmic reticulum has recently been described (24), other intracellular molecules that interact with the cytoplasmic domain of SR-A and regulate the receptor trafficking have not been identified yet.

To get a better understanding of the mechanism underlying SR-A recycling, we sought to find SR-A-interacting molecules participating in the receptor trafficking. In this report, we demonstrate for the first time that a microtubule-binding protein, Hook3 (HK3), is one of the molecules interacting with the cytoplasmic tail of SR-A. Microtubules directly interact with a variety of endocytic compartments and are required for organelle movement (25, 26) and maturation (27). We suggest that HK3 may be involved in the mechanism of the intracellular trafficking and clearance of the endocytosed receptor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Anti-V5 polyclonal antibody (Ab) for immunoblotting was purchased from Medical & Biological Laboratories (Nagoya, Japan). Anti-FLAG M2 monoclonal antibody (mAb), anti-V5 polyclonal Ab for flow cytometric analysis, and anti-V5 mAb-agarose (V5–10) were obtained from Sigma. The rat anti-mouse scavenger receptor A mAb (2F8) was purchased from Serotech (Oxford, United Kingdom). Anti-GAPDH mAb was purchased from Ambion (Austin, TX). Anti-human HK3 (hHK3) polyclonal antibody was generated as described previously (28). A polyclonal antibody against a rat HK3 isoform-{Delta}N was raised in rabbits against a keyhole limpet hemocyanin-conjugated synthetic peptide (QLIMSSIKHLPEG) corresponding to the C-terminal sequence of rat Ac1288 mRNA.

Cell Culture—Human embryonic kidney (HEK) 293 cells were cultured in DMEM containing 10% FCS, and Raw264.7 murine macrophage cells (from ATCC) were maintained in RPMI 1640 medium containing 10% FCS.

Plasmids and Transfection—The full-length human SR-AI cDNA was isolated from THP-1 cells by reverse transcriptase-PCR, and human SR-A (hSR-A) lacking the first 24 amino acids of the cytoplasmic domain (SR-A{Delta}1–24) was amplified by PCR from the full-length hSR-AI cDNA using the 5'-primer containing a methionine start codon and a spacer alanine codon. The mutant SR-A, SR-A12,13,15A, in which Glu12, Asp13, and Asp15 were replaced by alanine, was generated by site-directed mutagenesis (QuikChange Mutagenesis kit, Stratagene) using the full-length hSR-AI cDNA as a template. These SR-A cDNAs were subcloned into pcDNA3.1D/V5/His-TOPO vector (Invitrogen) containing the C-terminal fusion V5 tag. Human HK3 cDNA was obtained from HEK293 cells by reverse transcriptase-PCR and inserted into p3xFLAG-CMV-14 vector (Sigma) containing the C-terminal fusion FLAG tag. The C-terminal truncation of hHK3, {Delta}C-hHK31–555, and the N-terminal truncation, {Delta}N-hHK3168–718, were described previously (28). HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen), and stable cell lines were maintained in DMEM containing 10% heat-inactivated FCS and 0.5 mg/ml G418 sulfate (BioLinks, Tokyo, Japan).

Yeast Two-hybrid Screening—The yeast two-hybrid screening was performed using BD Matchmaker Library Construction & Screening Kits (Clontech, Palo Alto, CA) according to the manufacturer's instruction. As a bait plasmid, hSR-AI cDNA was subcloned into pGBKT7 vector including TRP1, and transformed into yeast strain Y187. Rat alveolar macrophages were obtained by lavaging the lungs with PBS containing 0.5 mM EDTA, and total RNA was isolated using TRIzol reagent (Invitrogen). As a prey, the double strand cDNA library of rat alveolar macrophages was then constructed and cotransformed into yeast strain AH109 together with pGADT7-Rec vector including Leu2. AH109 contains ADE2, HIS3, MEL1, and lacZ under the control of heterologous GAL4 upstream activating sequences. After mating the bait and prey culture, the mating mixture was spread onto master plates lacking adenine, histidine, leucine, and tryptophan to select for colonies containing both plasmids. The positive clones were finally isolated, sequenced, and analyzed using BLAST search.

Analysis of GST-hSR-A-CD-associated Proteins—The cDNA sequence encoding the hSR-AI cytoplasmic domain (CD) (MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK) was amplified by PCR from the full-length hSR-AI cDNA using 3'-primer containing stop codon (TGA). The hSR-A-CD cDNA was inserted into pGEX-4T-1 vector (Amersham Biosciences) and the plasmid was inoculated to Escherichia coli JM109. Protein expression was induced by amplifying the bacteria in the presence of 1 mM isopropyl beta-D-thiogalactoside (Amersham Biosciences) for 4 h, and bacterial extract was obtained by probe sonication on ice in PBS containing 1% (v/v) Triton X-100. The fusion protein was then isolated on a glutathione-Sepharose 4B (Amersham Biosciences), and cell lysates isolated from rat alveolar macrophages were applied to a GST-hSR-A-CD-conjugated Sepharose column. After the extensive washing, bound proteins were eluted with PBS (pH 7.4) containing 1 M NaCl, precipitated with 20% trichloroacetic acid on ice for 2 h, and collected by centrifugation at 20,000 x g for 10 min. The pellet was dissolved in SDS sample buffer and pH adjusted to an alkaline range with 2 M Tris for resolution on SDS-PAGE. Coomassie Blue-stained bands were excised from gels, destained in 50% acetonitrile, and analyzed using mass spectrometry.

Pull-down Assay—In addition to hSR-AI-CD (Met1–Lys50), GST-conjugated CD11 (Met1–Gln11), CD24 (Met1–Asp24), and CD44 (Met1–Glu44) were purified as described above. HEK293 cells expressing FLAG-tagged hHK3 were lysed in lysis buffer as described previously (22), and equal amounts of the lysates (150 µg) were incubated in 0.5 ml of lysis buffer with GST- or GST-hSR-A-CD-Sepharose at room temperature for 3 h. After washing the Sepharose, bound proteins were eluted in SDS sample buffer by boiling for 5 min. The eluted proteins were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membranes, and probed with anti-FLAG mAb in PBS (pH 7.4) containing 0.1% (v/v) Triton X-100 and 3% (w/v) skim milk. The membrane was further incubated with horseradish peroxidase-labeled anti-mouse IgG, and the proteins on the membrane were visualized using a chemiluminescence detection kit (SuperSignal, Pierce).

Immunoprecipitation Assay—For cross-linking, cells were treated with 0.5 mM dithiobis(succinimidylpropionate) (Pierce) at 37 °C for 30 min. Equal amounts of cell lysates from HEK293 cells stably expressing hSR-A were incubated in 0.5 ml of lysis buffer (22) with anti-V5-agarose beads at room temperature for 2 h. Cell lysates from Raw264.7 murine macrophage cells were incubated with anti-mouse SR-A mAb (2F8) (10 µg/ml) at 4 °C overnight, and immune complexes were recovered by incubation at 4 °C for 2 h with protein G-Sepharose beads (Amersham Biosciences). After washing the Sepharose, the immunoprecipitated proteins were analyzed by Western blot as described above using anti-HK3 polyclonal antibody (1:4000).

To examine the effect of the presence of an SR-A ligand, HEK293 cells in a 12-well plate were washed with DMEM and further incubated with either fucoidan or mannose (500 µg/ml) for the indicated periods at 37 °C in DMEM containing 10% FCS. After the incubation, cells were collected and lysed, and the immunoprecipitation assay was performed as described above.

siRNA—Synthesis of small interfering RNA (siRNA) was performed using the Silencer siRNA Construction Kit (Ambion, Austin, TX) following the manufacturer's instructions. The selected sequence for siRNA targeting HK3 was 5'-TTGATCGTCAGCTGAAGAAAA. siRNA targeting GAPDH and negative control siRNA were purchased from Ambion. Cells were transfected with siRNA by the reverse transfection method. The mixture of Lipofectamine 2000 and purified siRNA (30 nM for HEK293 cells or 15 nM for Raw264.7 cells) was plated onto a dish, and then trypsinized cells were added and incubated for 48–72 h. Cell lysates isolated from siRNA-transfected cells were analyzed by Western blot using anti-HK3 polyclonal antibody (1:4000), anti-GAPDH mAb (1:4000), and either anti-mouse SR-A mAb 2F8 (1:50) or anti-V5 polyclonal antibody (1:1000).

Surface Expression of hSR-A—Adherent cells in a 6-well plate were collected and incubated for 30 min on ice in PBS containing 1% (w/v) bovine serum albumin. The cells were further incubated in the same buffer with anti-V5 polyclonal Ab (8 µg/ml) for 1 h on ice followed by incubation with Alexa 488-labeled anti-rabbit IgG (Molecular Probes, Eugene, OR) for 45 min. After washing, the cell-associated fluorescence was assessed using FACSCalibur and CellQuest software (BD Biosciences).

AcLDL Internalization—Adherent cells were incubated with Alexa Fluor 488-AcLDL (3.75 µg/ml, Molecular Probes) at 37 °C for 1 h. The cells were then trypsinized, washed, and resuspended in PBS. AcLDL internalization was assessed by quantifying the fluorescence associated with ~50,000 cells using flow cytometry. To determine nonspecific binding, fucoidan (500 µg/ml) (Sigma) was added 5 min before addition of AcLDL.

hSR-A Protein Synthesis—HEK293 cells stably expressing hSR-A (1.5 x 105) were transfected with or without hHK3 siRNA or negative control siRNA (30 nM) using a 12-well plate. After incubation for 72 h, the cells were washed and incubated for 15 min in methionine-free DMEM containing 10% dialyzed FCS. The cells were then labeled with 100 µCi/ml RedivueTM L-[35S]methionine (Amersham Biosciences) for 3 h, washed with PBS, and lysed in lysis buffer. V5-tagged hSR-A was sedimented from the cell lysates using anti-V5-agarose and subjected to SDS-PAGE followed by autoradiography.

Biotinylated hSR-A Degradation—HEK293 cells expressing hSR-A were transfected with or without HK3 siRNA as described above. The cells were washed with cold PBS (pH 7.4) and cell-surface proteins were labeled with EZ-link Sulfo-NHS-Biotin (1 mg/ml) (Pierce) in cold PBS at 4 °C for 30 min. The reaction was stopped by washing and incubating the cells in PBS (pH 7.4) containing 100 mM glycine at 4 °C for 10 min. The cells were then incubated at 37 °C in DMEM containing 10% FCS in the presence of 100 µg/ml fucoidan for the indicated periods. Biotinylated proteins were precipitated from the cell lysates by using streptavidin-agarose (Invitrogen) and analyzed by Western blot. The amount of biotinylated hSR-A was quantified using Luminous Imager (Aisin Seiki, Kariya, Japan).


Figure 1
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  		FIGURE 1.
Analysis of hSR-A-associated protein in rat alveolar macrophages. A, yeast two-hybrid screening identified the interaction between hSR-A and the rat Hook3 isoform sequenced as rat Ac1288 mRNA (rat HK3 isoform-{Delta}N). This isoform and the related sequence (XM_224952) (rat HK3 isoform-{Delta}C) are splice variants of the predicted rat homolog of human HK3. The amino acid regions that are dark gray are identical among hHK3 and rat HK3 isoforms, the regions that are black are identical between the rat HK3 isoform-{Delta}N and hHK3, and the regions that are gray are identical between hHK3 and the rat HK3 isoform-{Delta}C. The regions that are unfilled are either missing or extra exons. B, cell lysates from rat alveolar macrophages were passed through the GST-conjugated hSR-A-CD-bound glutathione-agarose affinity matrix, and bound proteins were eluted with PBS containing 1.0 M NaCl for resolution on SDS-PAGE. A protein band identified with Coomassie Blue staining (lane1) was analyzed by mass spectrometer. The cell lysates were probed with the {alpha}-rat HK3 isoform-{Delta}N Ab (lane 2) by Western blot analysis.

 
Other Methods—Stripping for re-probing of Western blot was performed using Re-Blot Plus mild antibody stripping solution (Chemicon International, Temecula, CA). The significance of differences was determined using the Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Rat HK3 Isoform Is a Ligand of hSR-A—Because SR-A is expressed primarily by macrophages, we first performed a yeast two-hybrid screening assay between hSR-A cDNA and the rat alveolar macrophage cDNA library. As a bait, the Y187 yeast strain was transformed with hSR-AI cDNA in pGBKT7 vector. For a prey, we constructed a double strand cDNA library of rat alveolar macrophages and cotransformed it into the AH109 strain with pGADT7-Rec vector. The Ade, His, Leu, Trp, and LacZ positive clones were sequenced and analyzed using BLAST search. The results indicated that one of the positive clones encoded a sequence identical to rat Ac1288 mRNA (accession number AY310154 [GenBank] ). This sequence is a splice variant of the predicted rat homolog of a human microtubule-binding protein Hook3 (XM_224952 [GenBank] ) that is derived from the same genomic region in rat chromosome 16q12.4 (gi:62750816). When compared with the full-length sequence of human HK3, the AY310154 [GenBank] sequence lacks the region corresponding to the N terminus of hHK3, whereas the XM_224952 [GenBank] sequence does not involve the C-terminal region of hHK3. Thus, we here designate the AY310154 [GenBank] -encoded protein as a rat HK3 isoform-{Delta}N, and the XM_224952 [GenBank] -encoded protein as a rat HK3 isoform-{Delta}C (Fig. 1A). Although the interaction of hSR-A with a rat HK3 isoform-{Delta}N was suggested, we could not find positive clones encoding a rat HK3 isoform-{Delta}C in the yeast two-hybrid screening.

To screen the ligands of hSR-A by another method, we next constructed a GST fusion protein with the full-length CD of hSR-A (Fig. 2A) and prepared an affinity matrix using glutathione-agarose beads. Cell lysates from rat alveolar macrophages were passed through the matrix, and bound proteins were eluted with 1.0 M NaCl for resolution on SDS-PAGE. As shown in Fig. 1B, lane 1, we could detect a protein band with a molecular mass of ~65 kDa. Interestingly, mass spectrometry suggested that the protein contained sequences identical to hHK3. To confirm the molecular mass of this rat HK3 isoform, we purified a rabbit polyclonal antibody raised against the keyhole limpet hemocyanin-conjugated synthetic peptide (QLMSSIKHLPEG) corresponding to the C-terminal sequences of the rat HK3 isoform-{Delta}N. When the cell lysates from rat alveolar macrophages were analyzed by Western blot using the purified antibody against the rat HK3 isoform-{Delta}N, a protein band of ~65 kDa was detected (Fig. 1B, lane 2). The result indicated that the 65-kDa protein eluted from the hSR-A-CD column may be the rat HK3 isoform-{Delta}N. Thus, the results of two different approaches: yeast two-hybrid assay and mass spectrometry analysis of hSR-A-CD-bound proteins, suggested that there was an interaction between hSR-A and a homologeous protein of hHK3 in rat alveolar macrophages.


Figure 2
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  		FIGURE 2.
Human SR-A interacts with human HK3. A, amino acid sequence of the N-terminal cytoplasmic tail of hSR-A. Underline, negatively charged residues. B, cell lysates from HEK293 cells expressing FLAG-hHK3 were incubated with GST- or GST-conjugated hSR-A-CD-bound glutathione-Sepharose. The pulled down hHK3 was detected by Western blot analysis using {alpha}-FLAG Ab. GST-conjugated CD44, CD24, or CD11 includes amino acids 1–44, 1–24, or 1–11 of the hSR-A cytoplasmic tail, respectively. C, HEK293 cells were transfected with SR-A, SR-A12,13,15A, or SR-A{Delta}1–24, and SR-A proteins were precipitated by {alpha}-V5-agarose. Co-precipitated hHK3 was detected by Western blot analysis using {alpha}-HK3 Ab. D, HEK293 cells were transfected with {Delta}N-hHK3168–718 or {Delta}C-hHK31–555 together with or without V5-hSR-A, and V5-tagged proteins were precipitated. Co-precipitated hHK3 was probed with {alpha}-HK3 Ab.

 
Human HK3 Interacts with the Negatively Charged Residues of hSR-A Cytoplasmic Domain—Because our data indicated that the rat HK3 isoform-{Delta}N binds to the cytoplasmic tail of hSR-A, we next investigated whether human HK3 interacted with hSR-A. To determine the hSR-A region required for interaction with hHK3, the glutathione-agarose affinity matrix was prepared using GST-conjugated CD44, CD24, or CD11, which was composed of 1–44, 1–24, or 1–11 amino acids of the hSR-A cytoplasmic tail, respectively. We designed CD44 because 1–49 amino acids of murine SR-A, which correspond to 1–44 amino acids in hSR-A, have been demonstrated to be necessary for receptor internalization (18). CD11 was prepared because the region Glu12–Lys50 is highly conserved between human and murine, whereas the first 11 amino acids have no similarity. CD24 was designed to focus on the negatively charged assembly (Fig. 2A, underline). Cell lysates from FLAG-tagged hHK3-expressing cells were prepared, and pull-down assays were performed by using GST, GST-CD, -CD44, -CD24, or -CD11-bound glutathione-Sepharose. As shown in Fig. 2B, hHK3 was pulled down by CD44 and CD24 as well as by GST-CD, but not by CD11. These results indicate that overexpressed hHK3 interacts with the hSR-A cytoplasmic domain, and further suggest that the Glu12–Asp24 region in the hSR-A cytoplasmic domain was involved in the binding to hHK3.

The binding between endogenous hHK3 and hSR-A was examined by immunoprecipitations. We constructed full-length hSR-A, deletion mutant SR-A{Delta}1–24, which lacks the first 24 amino acids of hSR-A, and SR-A12,13,15A in which negatively charged Glu12, Asp13, and Asp15 are replaced by alanine. All constructs possess the V5-tag at the C terminus. When these hSR-A constructs were expressed in HEK293 cells and immunoprecipitated by anti-V5-agarose, endogenous hHK3 was co-sedimented with full-length hSR-A but not with either SR-A{Delta}1–24 or SR-A12,13,15A (Fig. 2C). The results are consistent with those of the GST-CD pull-down assay and demonstrate that the Glu12–Asp24 region, and in particular the negatively charged residues Glu12, Asp13, and Asp15 in the hSR-A cytoplasmic domain, may be critical for hHK3 binding.

Human SR-A Interacts with the C-terminal Region of hHK3—For the purpose of identifying the hHK3 region required for interaction with hSR-A, immunoprecipitations were performed using HEK293 cells transfected with hHK3 truncations. As shown in Fig. 2D, hSR-A clearly interacted with the N-terminal truncated form of hHK3 ({Delta}N-hHK3168–718), which lacks 1–167 amino acids of hHK3, consistent with the absence of these sequences in the rat HK3 isoform-{Delta}N (Fig. 1A). In contrast, the C-terminal truncation ({Delta}C-hHK31–555) in which the C-terminal 556–718 amino acids are deleted failed to co-precipitate with hSR-A. These results indicate that the C-terminal region His556–Arg718 of hHK3 is required for the interaction with hSR-A. In this region, only amino acids Val614–Ala717 are shared between human HK3 and the rat HK3 isoform-{Delta}N. Taking into account the interaction of hSR-A with the rat HK3 isoform-{Delta}N, we concluded that the Val614–Ala717 region of hHK3 is the critical region for the binding to hSR-A. This conclusion seems to be consistent with the results obtained by yeast two-hybrid screening in which we could not find positive clones encoding the sequence of the rat HK3 isoform-{Delta}C. In the rat HK3 isoform-{Delta}C, the region corresponding to the Val614–Ala717 of hHK3 is absolutely absent. Furthermore, because the C terminus of hHK3 is positively charged, the binding of this region to the negatively charged residues of hSR-A is considered to be reasonable.

Interaction between HK3 and SR-A Was Promoted by the Presence of an SR-A Ligand—We next examined whether the interaction between SR-A and HK3 was stimulated after SR-A had recognized extracellular ligands. HEK293 cells expressing hSR-A were incubated for the indicated periods with an SR-A ligand, fucoidan, and hSR-A was immunoprecipitated from cell lysates by anti-V5-agarose. As shown in Fig. 3A, the amount of hHK3 co-sedimented with hSR-A was apparently increased after fucoidan was added to the cells. In contrast, the amount of hHK3 co-precipitated with hSR-A was not significantly changed after the cells were incubated with mannose (Fig. 3B). Re-probing the membrane with anti-V5 polyclonal antibody revealed that the amount of sedimented hSR-A was not significantly altered between the incubation periods. These results suggest that the association between hHK3 and hSR-A is significantly stimulated by ligand binding to hSR-A.


Figure 3
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  		FIGURE 3.
The interaction between HK3 and SR-A was promoted by the presence of an SR-A ligand. A, HEK293 cells (SR-A –) or V5-hSR-A-expressing HEK293 cells (SR-A +) were incubated with or without fucoidan (500 µg/ml) for the indicated periods. V5-tagged proteins were precipitated from the cell lysates and co-sedimented proteins were probed with {alpha}-hHK3 Ab. The membrane was stripped and re-probed with {alpha}-V5 polyclonal Ab. B, HEK293 cells expressing hSR-A were incubated with fucoidan or mannose (500 µg/ml) for the indicated periods, and the immunoprecipitation assay was performed as described above. IB, immunoblot.

 


Figure 4
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  		FIGURE 4.
Endogenous murine SR-A interacts with murine HK3, and HK3 knock-down may affect SR-A expression. A, murine SR-A was precipitated from Raw264.7 macrophage cells by rat anti-mouse SR-A mAb 2F8 or control rat IgG (10 µg/ml), and the immune complex was probed with anti-HK3 antibody. B, Raw264.7 cells were transfected with or without HK3 siRNA or negative control siRNA. Cell lysates were analyzed by Western blot using mAb 2F8, {alpha}-HK3, or {alpha}-GAPDH Ab. IB, immunoblot.

 
Endogenous HK3 and SR-A Co-sediment from Murine Macrophage Cells—To confirm whether endogenous SR-A interacts with endogenous HK3, an immunoprecipitation assay was also performed using Raw264.7 murine macrophage cells. When endogenous murine SR-A was precipitated by rat anti-mouse SR-A monoclonal antibody 2F8 and probed with anti-HK3 antibody, endogenous murine HK3 was clearly detected (Fig. 4A). In contrast, control rat IgG did not precipitate HK3. These results suggest the interaction between endogenous HK3 and SR-A in macrophage cells, and indicate that SR-A binds to murine HK3 in addition to the rat HK3 isoform and human HK3.

HK3 May Down-regulate the Expression of SR-A—To explore the physiological meaning of HK3-SR-A interaction, we sought to knockdown the expression of HK3 by using specific siRNA. The HK3 protein expression in Raw264.7 cells, HEK293 cells, or hSR-A-expressing HEK293 cells was significantly decreased by siRNA transfection in a manner dependent on the siRNA concentration. Because overexpression of hHK3 results in the disruption of the Golgi morphology (28), we tested the effect of HK3 knockdown on the Golgi complex. However, we found that transfection with functional molecular concentrations of HK3 siRNA did not induce significant disruption of the Golgi morphology, when HEK293 cells were stained for a cis-Golgi marker, GM130, and examined by confocal microscopy (data not shown).

Raw264.7 murine macrophage cells were transfected with or without 15 nM HK3 siRNA or negative control siRNA. Western blot analysis demonstrated specific inhibition of HK3 expression (Fig. 4B). However, unexpectedly, total expression of murine SR-A appeared to be increased by HK3 siRNA transfection compared with negative siRNA or mock transfection. When HEK293 cells stably expressing V5-tagged hSR-A was transfected with 30 nM HK3 siRNA or GAPDH siRNA, the amount of hSR-A also seemed to be promoted by HK3 siRNA transfection (Fig. 5A). We then examined whether cell surface localization of SR-A was also increased in HK3-deficient cells. Cell surface localization of hSR-A was assessed by flow cytometry, which demonstrated that the surface expression of hSR-A was significantly increased in cells transfected with HK3 siRNA (Fig. 5B). These results suggest that HK3 knock-down induces positive effects on both total and surface levels of SR-A. Furthermore, consistent results were observed by using hSR-A truncations, which failed to interact with hHK3. Expression levels of SR-A12,13,15A or SR-A{Delta}1–24 were greater than that of full-length hSR-A in transfected HEK293 cells (Fig. 5C). The cell surface localization of the receptor as assessed by flow cytometry was also increased more in SR-A12,13,15A-expressing cells or SR-A{Delta}1–24-expressing cells than in hSR-A-expressing cells (Fig. 5D). When the cells expressing SR-A12,13,15A or SR-A{Delta}1–24 were transfected with HK3 siRNA, the surface localization of the mutants was 97 ± 13 or 100 ± 5.5% of that in untransfected cells, respectively, indicating that HK3 siRNA did not affect the surface levels of these SR-A mutants, which failed to interact with HK3. The results demonstrate that the HK3 siRNA molecule by itself did not induce unspecific effects on surface levels of SR-A. Therefore, all these results suggest that HK3 may play a role in down-regulation of SR-A expression.

HK3 May Alter the Ligand Uptake by SR-A—The next question was whether the receptor-mediated ligand uptake was affected by interfering with the association between SR-A and HK3. As shown in Fig. 6A, AcLDL uptake was increased significantly in cells expressing SR-A12,13,15A, which failed to interact with HK3, compared with cells expressing wild type hSR-A. The results correlate well with the increased cell surface expression of the mutant (Fig. 5D). Likewise, HK3 siRNA-transfected cells internalized more AcLDL than control cells (Fig. 6B), consistently with the increased surface expression of the receptor in HK3-deficient cells (Fig. 5B). The ligand-induced internalization of the receptor was therefore not impaired in the absence of HK3. Taken together, these results clearly indicate that neither cell surface sorting nor early endocytic trafficking of SR-A require HK3 interaction.

In contrast, the mutant SR-A{Delta}1–24, which did not bind HK3, failed to internalize AcLDL (Fig. 6A) despite the increased surface expression (Fig. 5D). The inability of the SR-A{Delta}1–24 to internalize AcLDL seems to be independent of HK3 function, because a previous study has demonstrated that deleting amino acids 1–49 of murine SR-A, which correspond to amino acids 1–44 in hSR-A, greatly reduced AcLDL uptake despite increased surface expression (18). Our results appear to be consistent with their results and further indicate that receptor internalization requires amino acid region 1–24 of hSR-A.


Figure 5
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  		FIGURE 5.
Total expression and cell surface localization of SR-A is increased by inhibiting the association with HK3. A, HEK293 cells stably expressing V5-SR-A were transiently transfected with or without GAPDH siRNA or HK3 siRNA. Cell lysates were analyzed by Western blot using {alpha}-V5, {alpha}-HK3, or {alpha}-GAPDH Ab. B, the flow cytometric analysis of cell surface expression of SR-A in HEK293 cells (gray zone), HEK293/SR-A cells (dashed line), HEK293/SR-A/GAPDH-siRNA cells (gray line), or HEK293/SR-A/HK3-siRNA cells (black line). Relative fluorescence is shown in the right panel. C, Western blot analysis of cell lysates from HEK293 cells stably expressing SR-A, SR-A12,13,15A, or SR-A{Delta}1–24 was performed using {alpha}-V5 or {alpha}-GAPDH Ab. D, cell surface expression of SR-A in HEK293 cells expressing SR-A, SR-A12,13,15A, or SR-A{Delta}1–24 was assessed by flow cytometry and expressed as relative fluorescence. *, p < 0.05; **, p < 0.01, compared with the intensity of SR-A-transfected cells (data expressed as mean ± S.E. of three experiments).

 
SR-A Degradation but Not Synthesis Is Affected by HK3—Because the total amount of SR-A appeared to be increased after HK3 siRNA transfection, we next sought to determine whether HK3 altered protein synthesis or degradation. After the cells were incubated with [35S]methionine for 3 h, hSR-A in cell lysates was immunoprecipitated and subjected to SDS-PAGE. The autoradiography showed that the HK3 siRNA transfection did not alter the synthesis of 35S-labeled hSR-A when compared with negative control siRNA transfection or mock transfection (Fig. 7A). In addition, no significant change was observed in the molecular mass of the synthesized hSR-A. These results indicate that HK3 may not participate in protein synthesis or the maturation process of hSR-A.

However, the stability of hSR-A seemed to be significantly altered by HK3. Cell surface proteins were labeled with biotin, followed by chase incubation in the presence of fucoidan to induce hSR-A internalization and stimulate the interaction with HK3. After the incubation, the biotinylated proteins were sedimented by streptavidin-agarose and the amount of biotinylated hSR-A was detected by Western blot analysis (Fig. 7B). Consistently with the result shown in Fig. 5A, the basal surface SR-A expression before chase incubation was increased in HK3 siRNA-transfected cells compared with untransfected cells. When the amount of biotinylated receptors retained in the cells was quantified and expressed as a percentage of the initial value of the corresponding cells, biotinylated hSR-A in control cells was degraded more rapidly than in HK3 siRNA-transfected cells. The level of biotinylated hSR-A in control cells was decreased to 35 or 9% of the initial value, after incubating for 9 or 22 h, respectively. In contrast, as much as 76 or 49% of biotinylated hSR-A was retained in HK3 siRNA-transfected cells for as long as 9 or 22 h, respectively. The increased amount of total proteins and cell surface expression of hSR-A in HK3-deficient cells (Fig. 5, A and B) was therefore considered to be due to the increased protein stability rather than the up-regulation of protein synthesis. In addition, the reduced proteolysis of surface hSR-A was not a consequence of the decreased amount of receptor endocytosis, because AcLDL uptake by hSR-A was increased in HK3-deficient cells compared with control cells (Fig. 6B). We therefore concluded that HK3 may be involved in transferring the endocytosed receptor to the lysosomal pathway, or in inhibiting receptor recycling. These interpretations seem to be consistent with the data indicating that HK3 preferred to target ligand-induced endocytosed SR-A (Fig. 3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The current study demonstrates the interaction between SR-A and HK3 by yeast two-hybrid screening, mass spectrometry of hSR-A cytoplasmic domain-bound proteins, GST pull-down assay, and co-immunoprecipitations. We found that hSR-A associates with the rat HK3 isoform-{Delta}N and human HK3. The interaction between endogenous murine SR-A and HK3 was also suggested in Raw264.7 murine macrophage cells. The positively charged C-terminal region of hHK3 is important for interaction with the negatively charged residues in the N terminus of the hSR-A cytoplamic tail. The association with HK3 was stimulated by ligand binding to SR-A, which results in its internalization. Conversely, AcLDL uptake, total and cell surface expression of SR-A, and its stability were promoted by down-regulation of HK3 expression. From these results, we consider that HK3 may function as a positive regulator of the SR-A degradation process (Fig. 8). Because protein synthesis and maturation were not affected by transfecting HK3 siRNA, we consider it unlikely that HK3 participates in the processing of SR-A, although HK3 is abundant in the cis-Golgi (28).


Figure 6
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  		FIGURE 6.
AcLDL uptake by SR-A is promoted by inhibiting the association with HK3. A, HEK293 cells (gray zone) or HEK293 cells transfected with SR-A (black line), SR-A12,13,15A (gray line), or SR-A{Delta}1–24 (dotted line) were incubated with Alexa 488-labeled AcLDL (3.75 µg/ml) for 1 h at 37°C. After treatment with trypsin-EDTA, cell-associated fluorescence was assessed by flow cytometry. To determine nonspecific AcLDL uptake in SR-A-transfected cells, fucoidan was added 5 min before incubation with AcLDL (dashed line). The right panel shows relative fluorescence intensity. B, Alexa 488-labeled AcLDL uptake by SR-A-expressing HEK293 cells transfected with or without GAPDH siRNA or HK3 siRNA. *, p < 0.05; **, p < 0.01, compared with the intensity obtained by SR-A-expressing cells (data expressed as mean ± S.E. of three experiments).

 
Because immunoelectron microscopy study has detected HK3 also on unidentified small vesicles (28), we speculate that HK3 may interact with SR-A outside the Golgi. However, at present, we have not been able to identify the intracellular compartments in which HK3 and SR-A colocalize. Under the confocal microscopic analysis using anti-SR-A antibodies, we observed that intracellular SR-A was stained diffusely as shown in previous studies (18, 29), presumably because of the nature of the antibodies we used. It was difficult to determine the colocalization of SR-A and HK3 under this condition. Further studies are necessary to clarify the precise mechanism by which HK3 interacts with SR-A and regulates the intracellular trafficking of SR-A.

Hook proteins constitute a family of microtubule-binding proteins that bind to organelles and microtubules. In secretory and endocytic pathways, vesicular transport such as trafficking from early to late endosomes or lysosomes, and trafficking through the Golgi complex is dependent on microtubules (30, 31). The Drosophila Hook protein (dHK) is required for normal trafficking of endocytosed ligands to early endosomes and accumulation into multivesicular bodies (32–34). It has been suggested that dHK negatively regulates the fusion between mature multivesicular bodies and late endosomes or lysosomes (33). hHK3 has been considered to participate in defining the architecture and localization of the Golgi complex (28), and its function in the trafficking of endocytosed compartments has not been clarified. However, a role of hHK3 in the endocytic pathway was suggested in the recent study indicating the interaction of hHK3 with the Salmonella SpiC protein (35), which inhibits endosome-lysosome fusion in vitro and interferes with recycling of the transferrin receptor in vivo (36). Because lysosome distribution in Raw264.7 macrophages was disrupted by expression of either SpiC or {Delta}C-hHK31–555, it has been speculated that the SpiC protein interacts and inactivates hHK3 to prevent phagosome-lysosome fusion (35). Taken together with our data, this suggests that hHK3 may participate in the endosome-lysosome pathway and regulate clearance of endocytosed ligands and receptors. Because phagosome-lysosome fusion was significantly impaired in SR-A-deficient macrophages infected with Listeria monocytogenes (37), HK3 may contribute to SR-A-mediated bactericidal mechanisms.


Figure 7
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  		FIGURE 7.
SR-A degradation but not synthesis is affected by HK3. A, HEK293 cells stably expressing V5-hSR-A were transfected with or without HK3 siRNA or negative control siRNA, and incubated with [35S]methionine for 3 h. V5-tagged proteins were precipitated from the cell lysates and subjected to SDS-PAGE, followed by autoradiography. B, cell surface proteins on HEK293 cells expressing hSR-A transfected with or without HK3 siRNA were labeled with Sulfo-NHS biotin at 4 °C. The cells were then incubated at 37 °C in the presence of fucoidan for the indicated periods, and biotinylated proteins in cell lysates were precipitated with streptavidin-agarose. The precipitated SR-A was detected by Western blot analysis using {alpha}-V5 Ab. The amount of biotinylated SR-A was quantified using Luminous Imager as shown in the lower panel. Values are expressed as percentages of the initial value. *, p < 0.05, compared between HK3 siRNA transfection and mock transfection (mean ± S.E. of three experiments).

 


Figure 8
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  		FIGURE 8.
Possible roles of Hook3 in the intracellular trafficking of SR-A. After the post-translational processing in the Golgi, SR-A localizes to the plasma membrane. The receptor is endocytosed with its ligands, and either returns to the cell surface via a recycling pathway or is directed to lysosomes where they are degraded. The current study suggests the possible roles of HK3 in SR-A trafficking (gray arrow). The most likely mechanism is that HK3 may positively participate in the process of SR-A degradation and may down-regulate the total and surface amount of SR-A.

 
In this report, we suggest that the negatively charged residues, Glu12, Asp13, and Asp15 in the hSR-A cytoplasmic domain may interact with the C-terminal Val614–Ala717 region of hHK3. Because the C terminus of hHK3 is positively charged, binding of this region to the acidic hSR-A region seems reasonable. The Hook family of proteins is composed of the acidic N-terminal globular domain, the central coiled-coil, and the basic C-terminal domain. The N-terminal and the central coiled-coil regions are highly conserved among human HK1, HK2, HK3, together with dHK. In contrast, the basic C terminus is the most divergent region. The conserved N-terminal domain of Hook proteins are involved in microtubule binding (28), whereas the divergent C-terminal domains mediate the binding to different specific organelles. The C-terminal domain of hHK3, but not hHK1 or hHK2 has been demonstrated to localize to the cis-Golgi (28). From these observations, it is therefore believed that hSR-A may specifically bind to hHK3, although the interactions of hSR-A with hHK1 and hHK2 have not been examined yet.

Several studies have focused on the cytoplasmic region of SR-A required for cell surface trafficking or receptor internalization. Kosswig et al. (18) demonstrated that murine SR-A{Delta}1–49 in which the cytoplasmic domain was deleted, except for the six amino acids (KLKSFK) proximal to the membrane, localized to the cell surface but failed to internalize AcLDL. The normal processing of the protein, increased surface expression, and impaired internalization of murine SR-A{Delta}1–49 seem to be consistent with the current study indicating that human SR-A{Delta}1–24 localizes to the cell surface to a greater extent than full-length hSR-A but fails to internalize AcLDL. The KLKSFK motif may also be sufficient for cell surface trafficking of human SR-A, and the first 24 amino acids must be necessary for receptor internalization. The substitution of alanine for Val21, Phe23, or Asp24 in hSR-A has been demonstrated to reduce both cell surface localization and internalization (20). However, those residues, named as a VXFD motif, may not be required for surface trafficking, because SR-A{Delta}1–24 was sufficiently expressed on the cell surface in our study. Furthermore, we also found that the EDXD motif, consisting of Glu12, Asp13, and Asp15, may not be necessary for endocytosis. The phosphorylation of serine residues proximal to the membrane has also been demonstrated to be necessary for SR-A internalization (19, 21), indicating the complicated nature of regulation of receptor internalization.

Tian et al. (24) recently reported that the surface expression and half-life of murine SR-A were significantly increased by N-butyldeoxynojirimycin, an inhibitor of the N-glycan processing enzymes {alpha}-glucosidases I and II. In the presence of N-butyldeoxynojirimycin, SR-A was unable to interact with calnexin and became Endo H-sensitive. It is interesting that the immature glycosylated form of SR-A enhances protein stability. Because we did not see an effect of HK3 knock-down on SR-A processing and maturation (Fig. 7A), our data suggest another mechanism by which HK3 down-regulates SR-A stability independently of altering SR-A glycosylation. It is also interesting that the expression and stability of SR-A were promoted by interferon-{gamma} in the study by Tian et al. (24). The interferon-inducible GTPase, has been demonstrated to interact with hHK3 (38), suggesting the possibility that hHK3 may be involved in interferon-{gamma}-inducible membrane trafficking.

In conclusion, we identified HK3 as a novel interacting partner of the SR-A cytoplasmic domain. Our data indicated that HK3 may positively regulate the degradation process but negatively modulate the expression of SR-A. Physiologically, HK3 may take part in clearance of endocytosed ligand and receptors, and the down-regulatory effects of HK3 on SR-A expression might contribute to a suppressive role in the atherogenetic process. Although further studies are required, we consider there to be evidence that the microtubule-binding protein HK3 participates in the mechanisms involved in the intracellular trafficking of SR-A.


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan, and the Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Supported by Welch Foundation Grant I-1300 and National Institutes of Health Grant NS43406. Back

1 To whom correspondence should be addressed: South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. Tel.: 81-11-611-2111; Fax: 81-11-611-2236; E-mail: sanohito{at}sapmed.ac.jp.

3 The abbreviations used are: SR-A, scavenger receptor A; AcLDL, acetylated low density lipoprotein; hSR-A, human SR-A; HK3, Hook3; hHK3, human Hook3; mAb, monoclonal antibody; HEK293 cells, human embryonic kidney 293 cells; CD, cytoplasmic domain; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Ab, antibody. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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