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Address correspondence to Joanne E. Murphy-Ullrich, Dept. of Pathology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, VH 668 1530, 3rd Ave. South, Birmingham, AL 35294-0019. Tel.: (205) 934-0415. Fax: (205) 975-934. E-mail: Murphy{at}path.uab.edu
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Abstract |
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Key Words: thrombospondin; G proteins; ERK; focal adhesions; cell adhesion
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Introduction |
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Thrombospondins (TSPs)* 1 and 2, tenascin-C, and SPARC are matricellular proteins, which when presented to cells as soluble proteins, stimulate a transition in cell adhesion from a state of strong adhesion to one of intermediate adhesion. This is characterized by a restructuring of focal adhesion plaques with dispersal of vinculin and -actinin and reorganization of actin stress fibers while maintaining integrin clusters and a spread cell shape (Murphy-Ullrich, 2001). The intermediate adhesive state is regarded as the phenotype of an adaptive response that facilitates motility and potentially induces cell survival signals (Murphy-Ullrich, 2001).
TSP signals focal adhesion disassembly through interactions of a 19 amino acid sequence from its NH2-terminal heparin-binding domain with a cell surface form of calreticulin (CRT; Goicoechea et al., 2000, 2002). TSPCRT signaling of focal adhesion disassembly is pertussis toxin (PTX)sensitive and involves both Gi2 and Gß
subunits (Orr et al., 2002). TSP/hep I binding to CRT stimulates a PTX-dependent activation of phosphoinositide 3-kinase (PI3K) and extracellular signalrelated kinase (ERK), which are required for focal adhesion disassembly. Because CRT is not a transmembrane protein, it is likely that CRT acts in association with a coreceptor.
The low density lipoprotein receptorrelated protein (LRP), also known as CD91 or the 2-macroglobulin (
2M) receptor, is a member of a family of multi-ligand binding proteins involved in numerous biological functions (Herz and Strickland, 2001; Strickland et al., 2002). Among its ligands is the NH2-terminal heparin-binding domain of TSP; LRP mediates endocytic degradation of TSP (Godyna et al., 1995; Mikhailenko et al., 1997). There is also indirect evidence for the association of LRP with CRT. CRT competitively inhibited the association of LRP with heat shock protein gp96 (Basu et al., 2001). CRT and LRP are colocalized on the macrophage cell surface by immunohistochemistry, where they function as coreceptors for macropinocytosis of necrotic cells (Ogden et al., 2001). Although these data suggest that CRT and LRP can form bimolecular signaling complexes, there are actually no direct data to show binding of these two proteins.
Once considered primarily for its role in endocytosis, it is becoming clear that LRP and other family members associate with a number of cytoplasmic adaptor molecules and participate in signaling of multiple cellular functions (Hussain, 2001; Strickland et al., 2002). LRP is a 600-kD transmembrane protein composed of one 515-kD heavy () subunit and one 85-kD light (ß) subunit noncovalently attached to each other (Herz et al., 1990; Willnow et al., 1996). The light subunit has a transmembrane domain and a cytoplasmic tail that contains two NPxY sequences (Gliemann et al., 1994). These motifs are available for interactions with adaptor or scaffolding molecules, such as Shc, FE65, and Disabled-1 (Ermekova et al., 1997; Trommsdorff et al., 1998, 1999; Barnes et al., 2001). LRP is localized in caveolae, and is rapidly and transiently tyrosine phosphorylated by the PDGF receptor in a src- and PI3K-dependent manner (Boucher et al., 2002; Loukinova et al., 2002). PDGF-mediated phosphorylation of LRP's cytoplasmic domain results in increased association of the adaptor protein Shc with LRP. Expression of a chimeric protein with the LRP cytoplasmic domain increases JNK association at the plasma membrane, inhibiting JNK translocation to the nucleus and gene regulation (Lutz et al., 2002). Other LRP family members (LRP5 and LRP6) act as coreceptors with the G proteincoupled receptor Frizzled in the Wnt signaling pathway, suggesting a role in transcriptional regulation (Tamai et al., 2000; Wehrli et al., 2000). Finally,
2M binding to LRP on macrophages activates PI3K (Misra and Pizzo, 1998).
Here, we report that LRP acts as the coreceptor for cell surface CRT to mediate focal adhesion disassembly in response to the hep I sequence of TSP. Our results demonstrate that LRP is necessary for focal adhesion disassembly by TSP because the LRP antagonist, receptor-associated protein (RAP), or antibodies against LRP block TSP/hep Iinduced focal adhesion disassembly, and LRP-deficient cells do not signal in response to TSP/hep I treatment. Furthermore, we provide the first direct evidence that LRP and CRT interact and that hep I, CRT, and LRP are complexed in cells. These results establish LRP as a signaling coreceptor for CRT and provide evidence for a novel function for LRP in regulation of cell adhesion.
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Results |
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To test this hypothesis, we used a pAb (anti-CD91) raised against a 7580-kD fragment of the LRP heavy chain (extracellular domain). This antibody blocked focal adhesion disassembly by hep I (Fig. 1). Similarly, a second LRP pAb (R2629) that recognizes multiple epitopes in the ligand-binding regions of the heavy chain of LRP also blocked hep I stimulation of focal adhesion disassembly (unpublished data). These antibodies also blocked focal adhesion disassembly by TSP (unpublished data). Incubation of the cells with the antibodies alone did not affect the number of cells positive for focal adhesions, demonstrating that blockade of LRP in the absence of a TSP stimulus is not sufficient to alter focal adhesion stability.
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LRP-deficient cells do not respond to stimulation by hep I
To further confirm whether LRP plays a role in focal adhesion disassembly, mouse embryonic fibroblasts (MEFs) genetically deficient in LRP were treated with TSP or hep I. Fibroblasts (MEF-1) wild type for LRP and fibroblasts heterozygous (PEA 10) or homozygous null (PEA 13) for LRP were used (Willnow and Herz, 1994). Cells were incubated with hep I peptide and analyzed for focal adhesions by interference reflection microscopy. Hep I was unable to stimulate focal adhesion disassembly in either the heterozygous or the homozygous LRP-null cells, although the wild-type parental line responds to TSP and hep I as previously observed for bovine aortic endothelial (BAE) cells and other MEF strains (Fig. 3). The PEA 10 cells, which are heterozygous for the LRP gene and express 50% of wild-type levels of LRP, do not respond to hep I, suggesting that there is a critical level of LRP expression on the cells necessary to mediate focal adhesion disassembly (Fig. 3). In contrast, both PEA 10 and PEA 13 MEFs were able to respond to the active fragment of tenascin-C, suggesting that the failure to respond to TSP/hep I is not due to a generalized defect in these cells (Murphy-Ullrich et al., 1991; Fig. 3).
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Treatment of cells with 1 µM hep I peptide stimulated a significant increase in ERK phosphorylation in wild-type cells (Fig. 7, A and B). In contrast, there was no stimulation of ERK phosphorylation in the homozygous LRP-deficient cell line PEA 13 (Fig. 7 A). The PEA 10 line heterozygous for LRP showed a reduced ability to phosphorylate ERK in response to hep I treatment. Further evidence for the role of LRP in hep I signaling is seen by the ability of RAP to block ERK phosphorylation in response to hep I in wild-type MEF cells (Fig. 7 B).
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Discussion |
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It is becoming clear that beyond its role as a molecular chaperone, CRT also regulates multiple cellular functions as a cell surface protein (Eggleton et al., 1994; Arosa et al., 1999; Johnson et al., 2001; Seddiki et al., 2001). In addition to mediating focal adhesion disassembly in response to TSP, CRT modulates melanoma cell spreading on laminin substrates (White et al., 1995) and fibroblast proliferation in response to fibrinogen (Gray et al., 1995). The mechanisms whereby cell surface CRT mediates cellular signals have not been previously described. Evidence that CRT signaling is PTX-sensitive in at least three different cell types suggests that heterotrimeric G protein signaling might be a common mechanism (Cho et al., 1999, 2001). Signaling by an antibacterial peptide in U937 monocytes to generate O2- occurs in a G proteindependent manner (Cho et al., 2001). Furthermore, cell surface CRT similarly mediates the effects of this anti-microbial peptide on neutrophils through PTX-sensitive G proteins (Cho et al., 1999). These findings are consistent with our data showing that TSP stimulates focal adhesion disassembly through activation of PTX-sensitive Gi2 proteins and ERK phosphorylation (Orr et al., 2002), and with the current data showing that hep I stimulates association of LRP with the G
i2 subunit.
Typically, heterotrimeric G proteins are associated with seven transmembrane-spanning receptors (Gilman, 1987). Although it might be atypical for the Gi2 subunit to associate with LRP and mediate signaling, there is precedent for such signaling mechanisms. G proteins are known to associate with single transmembrane types of receptors such as the receptors for insulin, erythropoietin, EGF, and IGF-I and IGF-II (Yang et al., 1991; Krieger-Brauer et al., 1997; Fedorov et al., 1998; Hallak et al., 2000; Guillard et al., 2001). LRP has been shown to associate with heterotrimeric G proteins, primarily G
s, in melanoma cells (Goretzki, and Mueller, 1998). There is also evidence that LRP is involved in G protein signaling; apoE4 binding to LRP stimulates PTX-sensitive neuronal cell apoptosis in association with G
i subunits (Hashimoto et al., 2000). ApoE peptides or activating antibodies to LRP stimulate an increase in cytosolic calcium in an LRP- and PTX-sensitive manner (Wang and Gruenstein, 1997; Misra et al., 1999). Our present work provides further evidence for the involvement of LRP in G protein signaling and shows that LRPG protein interactions are involved in regulation of a defined biological response, focal adhesion disassembly.
Evidence suggests that CRT and LRP interactions are physiologically important. CRT plays a role in C1q signaling of apoptotic cell ingestion through association with LRP, and the two proteins colocalize on the surface of macrophages (Ogden et al., 2001). Furthermore, CRT and another LRP family member, megalin, both bind C1q (Sim et al., 1998). Other data show that interactions of heat shock proteins gp96, hsp90, and hsp70 with LRP are competitively blocked by CRT, and antibody to LRP blocks antigen presentation by CRT (Basu et al., 2001). Our data are consistent with these observations that CRTLRP interactions are important in cellular processes and demonstrate a new role for LRP in focal adhesion disassembly.
LRP has signaling functions distinct from its role as a cargo transport protein (Hussain, 2001; for review see Strickland et al., 2002). LRP has two NPxY signaling motifs in its cytoplasmic domain; however, endocytosis depends primarily on a YXXL sequence immediately after the second NPxY motif (NPVYxxL). Both NPxY motifs appear to interact with signaling adaptor molecules (Li et al., 2000). LRP associates with numerous adaptor molecules including Fe65 (Ermekova et al., 1997), Disabled-1 (Dab-1; Trommsdorff et al., 1998, 1999), JIP 1 and 2 (Gotthardt et al., 2000), and Shc (Barnes et al., 2001). A tyrosine-phosphorylated form of LRP copurified with Shc in v-src transformed cells (Barnes et al., 2001). This finding is intriguing, as Shc activates the Ras signaling pathway and downstream ERK activation through recruitment of Grb2-Sos (Nakamura et al., 2002). Recently, we showed that TSP/hep I signaling through CRT stimulates ERK activity, and that ERK activity is required for focal adhesion disassembly (Orr et al., 2002). LRP also appears to be involved in PI3K signaling as 2M binding to LRP on peritoneal macrophages activates PI3K (Misra and Pizzo, 1998). This is consistent with our observations that cells deficient in LRP do not activate PI3K in response to hep I stimulation. Together, these observations support a direct role for LRP in transmitting signals from TSP bound to CRT.
Also, we report here that LRP binds CRT and forms complexes with CRT in cells. TSP/hep I binds aa 1936 of the N-domain of CRT, and this interaction mediates focal adhesion disassembly (Goicoechea et al., 2002). Our in vitro binding analyses suggest that hep I binding to CRT does not interfere with the ability of LRP to bind CRT (unpublished data). On the contrary, hep I increases CRTLRP association at the cell membrane. PAI-1 binding to uPAR exposes a cryptic binding site on uPAR for LRP (Stefansson et al., 1998). TSP/hep I binding to CRT might also alter CRT structure, exposing a new or additional LRP binding sites. Hep I potentially induces CRT clustering at the membrane, thereby increasing CRT affinity for LRP. This model is consistent with the fact that CRT binding to cells in the absence of hep I is insufficient to signal focal adhesion disassembly (Goicoechea et al., 2000, 2002), and the present observation that LRP co-complexes with a higher mol wt form of CRT.
The extracellular binding domains of LRP involved in binding CRT are not known. Interestingly, the ligand-binding clusters of LRP are comprised of cysteine-rich complement-like repeats (Herz and Strickland, 2001). C1q is a known ligand of CRT, and CRT has been referred to as the cC1q receptor (Eggleton et al., 1998). C1q binds to a site spanning the N and P domains (aa 160283) of CRT (Stuart et al., 1997; Kovacs et al., 1998). It is interesting to speculate that CRT recognizes these complement-like domains of LRP.
Although the NH2-terminal heparin-binding domain of TSP binds LRP, and LRP mediates the endocytic degradation of TSP1 and 2 (Godyna et al., 1995; Mikhailenko et al., 1997), our analyses failed to demonstrate binding between LRP and the hep I sequence from the NH2-terminal heparin-binding domain of TSP. It remains to be seen as to whether hep I signaling through LRP modulates endocytic degradation of TSP, or whether LRP down-regulates TSPCRT signaling of focal adhesion disassembly through endocytosis of this complex.
In summary, these works identify a novel mechanism of CRT signaling from the cell surface through TSP-stimulated association with LRP. Furthermore, these data link LRP to the Gi signaling pathway activated by TSP/hep I and its downstream effectors, PI3K and ERK. Finally, these data implicate LRP in a novel biologic response, modulation of the intermediate cell adhesion.
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Materials and methods |
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Antibodies
Rabbit anti-CRT antiserum was purchased from Affinity BioReagents, Inc. Goat anti-CRT IgG (calregulin, sc-6468) was purchased from Santa Cruz Biotechnology, Inc. Anti-CD91 rabbit pAb against LRP (used at 1:500-1000) was a gift from Dr. R. Binder (University of Connecticut School of Medicine, Farmington, CT; Binder et al., 2000). R2629 rabbit pAb against LRP, developed in the Strickland lab, and mouse anti-LRP 8G1 and 5A6 antibodies (purchased from Maine Biotechnology) have been described previously (Strickland et al., 1990; Kounnas et al., 1992b). Rabbit anti-phospho-ERK, rabbit anti-ERK and mouse anti-EGF receptor antibodies were purchased from Cell Signaling Technology. Rabbit anti-ß3 integrin was purchased from CHEMICON International. Mouse antibody to PI3K (p85 subunit) and mouse anti-Gi antibody were purchased from Upstate Biotechnology. Mouse anti-G
i2 antibody was purchased from BIOMOL Research Laboratories, Inc. Mouse antibody to the Ang II type 1 receptor was purchased from Abcam, Ltd.
Proteins
TSP was purified from human platelets purchased from the American Red Cross as described previously (Murphy-Ullrich and Höök, 1989). LRP was isolated from human placenta as described previously (Ashcom et al., 1990). Recombinant human RAP expressed as a GST fusion protein was purified as described previously (Williams et al., 1992). Recombinant tenascin-C fnIIIA-D was a gift from Dr. Harold Erickson (Duke University, Durham, NC; Aukhil et al., 1993). cDNA for GST-CRT was a gift from Dr. M. Michalak. Fusion proteins were expressed in Escherichia coli and purified as described previously (Baksh and Michalak, 1991; Goicoechea et al., 2000). Hep I peptide, biotin-tagged CRT binding peptide (GQPMYGQPMY), and the membrane-permeable G protein inhibitory peptides were synthesized, purified, and analyzed by AnaSpec (Murphy-Ullrich et al., 1993; Jorgensen et al., 2000; Orr et al., 2002). Hep I peptide with an NH2-terminal biotin tag was synthesized at the University at Alabama, Birmingham Peptide Synthesis Core.
LRP-CRT binding assays
GST-CRT was expressed as described previously (Goicoechea et al., 2000). The GST tag was cleaved using the Restriction Protease Xa cleavage and removal kit (Roche). 20 nM recombinant CRT was incubated with 10 nM LRP with or without 10 nM RAP for 1 h at 4°C in DTO buffer (DMEM, 0.5% Tween 20, and 0.1% ovalbumin). Complexes were immunoprecipitated (1 h at 4°C) with anti-CRT anti-serum (1:30) or 15 µg/ml nonimmune rabbit IgG conjugated to GammaBind G Sepharose (Amersham Biosciences). As a control for nonspecific binding, LRP was incubated with 20 nM BSA in the presence or absence of RAP, and then subjected to immunoprecipitation with rabbit anti-BSA serum (1:30; Sigma-Aldrich) conjugated to GammaBind G Sepharose. Complexes were washed 7x in DTO buffer and resuspended in Laemmli buffer. Samples were separated by SDS-PAGE (6%), transferred to nitrocellulose, and then probed with anti-LRP antibody (8G1) at 1 µg/ml and goat antimouse IgG-HRP (1:10,000). Blots were developed using Western Lightning Chemluminescence Reagent Plus (PerkinElmer). Band intensity was quantified by densitometry (One-DScan software v1.31; Scanalytics).
Coprecipitation of LRP and CRT from cell extracts
BAE cells (4 x 100 mM) were grown to near confluence, washed three times in DMEM, and cells were then treated with 1 µM hep I or the modified hep I peptide for 10 min before harvesting by scraping. Cells were pelleted, and pellets were washed twice with DMEM plus protease inhibitors. Cells were disrupted by homogenization with a tissue grinder (20 strokes) on ice, and insoluble proteins were pelleted before detergent extraction. The pellet was resuspended in 0.5 ml 100 mM N-octylglucopyranoside on ice for 4060 min. Insoluble proteins were removed by centrifugation, and equal amounts of protein from the detergent-soluble supernatant (extract) were used for the coimmunoprecipitation assays.
GammaBind G Sepharose beads were blocked overnight in blocking buffer (0.1% ovalbumin in DMEM), and were then incubated for 12 h with 15 µl rabbit anti-CRT antiserum or nonimmune rabbit serum. Beads were washed four times and were then incubated for 1 h at 4°C with 360 µg of BAE cell extract in an equal volume of 2x binding buffer (0.1% Triton X-100 in DMEM). Beads were washed four times with binding buffer and immune complexes were extracted in 30 µl 2x nonreducing Laemmli buffer. LRP was detected in anti-CRT immunoprecipitated complexes by immunoblotting with mouse anti-LRP antibody (8G1) at 1 µg/ml. Alternately, CRT was detected in samples immunoprecipitated with 5 µg mouse anti-LRP (5A6) antibody or nonimmune mouse serum by immunoblotting with goat anti-CRT IgG (1:1,000).
Association of LRP with biotin-tagged hep I peptide bound to cells
Wild-type and CRT-null MEFs were incubated with biotin-tagged hep I peptide, untagged peptide for 10 min, washed in DMEM, and cells were then harvested by scraping. Membrane proteins were solubilized with 50 mM N-octylglucopyranoside. Equal amounts of protein (1 mg) from each cell type were analyzed. Proteins associated with biotinylated hep I were coprecipitated by incubation overnight at 4°C with a 100-µl slurry of neutravidin beads (Pierce Chemical Co.). Samples were washed seven times in DTO buffer. Bound proteins were solubilized in Laemmli buffer and separated by SDS-PAGE on 6% gels. After transfer to nitrocellulose, LRP coprecipitating with the biotin peptide complexes was detected by immunoblotting with 1 µg/ml rabbit anti-LRP antibody (R2629). In replicate experiments, membranes were probed with rabbit antibody to ß3 integrin and then stripped and reprobed with chicken anti-CRT. Equal amounts (50 µg) of detergent extract from the wild-type and CRT-null MEFS were probed for LRP by immunoblotting with rabbit anti-LRP antibody (R2629). In some experiments, replicate blots were probed with anti-CRT antiserum to confirm peptide binding. A biotin-tagged peptide known to bind CRT (GQPMYGQPMY; Jorgensen et al., 2000), but which does not stimulate focal adhesion disassembly was also tested.
Focal adhesion assay
The focal adhesion assays were performed as described previously (Murphy-Ullrich and Höök, 1989). Cells were preincubated under serum-free conditions for 30 min before treatment for 30 min at 37°C with hep I or TSP. Endogenous TSP production during the course of these assays is not a factor because mouse fibroblasts derived from TSP1 and TSP2 double knockout animals respond to TSP1 and hep I as do wild-type cells (fibroblasts were a gift of Dr. Paul Bornstein, University of Washington, Seattle, WA.
ERK activation assay
ERK activity was determined by Western blotting using phosphorylation statespecific antibodies of whole-cell lysates as described previously (Orr et al., 2002).
Immunoprecipitation and PI3K assay
Phosphatidylinositol (4,5)-bisphosphate (PIP2) was obtained from American Radiolabeled Chemicals, and [32P]ATP was purchased from Amersham Biosciences. PI3K activity was assessed in an in vitro kinase assay in anti-p85 immunoprecipitates using a PIP2 substrate as described previously (Greenwood et al., 1998).
G protein pull-down assay
BAE cells were grown to near confluence, serum-deprived, and incubated with either DMEM or hep I, modified hep I, or TSP. Cells were lysed and immunoprecipitated with mouse anti-LRP (8G1) antibody for 2 h at 4°C as described previously (Orr et al., 2002). In some experiments, lysates were immunoprecipitated with antibodies to either the angiotensin II type 1 receptor or to the EGF receptor. As an additional control, cells were preincubated for 1 h with 50 nM RAP before addition of hep I. Immunoprecipitates were analyzed for Gi2 association by immunoblotting with mouse anti-G
i2 antibodies followed by HRP-conjugated goat antimouse IgG (1:5,000). Bands were visualized with chemiluminescence reagent (NEN Life Science Products).
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL44575 to J.E. Murphy-Ullrich, and HL50784 and HL54710 to D.K. Strickland; by the Cardiovascular Pathology training grant (T 32 HL07918) to A.W. Orr; by the Bone Biology and Disease training grant (T32 AR47512) to C.A. Elzie; and by the American Heart Association, Southeastern Affiliate, post-doctoral fellowships to C.E. Pedraza (0120251B) and to S. Goicoechea (0020534B).
Submitted: 12 February 2003
Revised: 7 May 2003
Accepted: 9 May 2003
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