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Vol. 15, Issue 2, 625-636, February 2004
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2
1 Integrin from Membrane Rafts to Caveolae and Subsequent Protein Kinase C-dependent Internalization
* Cell Biology, University of Jyväskylä, FIN-40351 Jyväskylä, Finland;
VTT Medical Biotechnology, FIN-20520 Turku, Finland;
Department of Microbiology, University of Oulu, FIN-90220 Oulu, Finland; and
Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland
Submitted August 13, 2003;
Revised October 29, 2003;
Accepted November 13, 2003
Monitoring Editor: Carl-Henrik Heldin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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2
1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface 21 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of 21 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally 21 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase C activity. Caveolae are known to be highly immobile structures and 21 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to 21 integrin, because the antibody-mediated formation of V integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to 21 and taking advantage of 21 internalization via caveolae. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Pelkmans and Helenius, 2002; Parton 2003). They have been described as highly immobile and not involved in constitutive endocytic trafficking (Thomsen et al., 2002). Their internalization can be activated, for example, by the phosphatase inhibitor okadaic acid (Parton et al., 1994). In endothelial cells the interaction of albumin docking protein gp60 and caveolin-1 initiates the endocytosis of caveolae (Minshall et al., 2000). Caveolins are membrane proteins that are molecular markers of caveolae and shown to be crucial for the formation of these structures (Fra et al., 1995). Caveolins can be associated with cell surface growth factor receptors (Couet et al., 1997) and cell adhesion receptors (Wary et al., 1996, 1998; Wei et al., 1999) and caveolae might therefore play a role in cellular signaling. In addition, caveolae have been suggested to participate in the uptake of folate by pinocytosis, but this is still under discussion (Parton, 2003). Caveolin-deficient mice have given novel insight into the function of caveolae. Caveolin-1 gene knockout leads to pulmonary defects, vasoconstriction, or dilatation abnormalities and resistance to diet-induced obesity (Drab et al., 2001; Razani et al., 2001; Schubert et al., 2001; Sotgia et al., 2002; Razani et al., 2002). Simian virus 40 (SV40) has been shown to be internalized through caveolae and detailed studies focused on the virus entry mechanism have produced a lot of new information about the biology of caveolae (Pelkmans et al., 2001, 2002). We have recently shown that human echovirus 1 (EV1) is another virus using caveolae in its entry (Marjomäki et al., 2002). Subsequent to internalization via caveolae, EV1 was shown to be localized in perinuclear structures, which contained caveolin-1 (Marjomäki et al., 2002). Such structures include caveosomes, recently identified endocytic organelles, which participate in the entry of SV40. However, the entry routes of SV40 and EV1 seem not to be identical (Marjomäki et al., 2002) and EV1 was found to be a unique tool to study the cell biology of caveolae. Importantly, EV1 uses 21 integrin, a collagen receptor, to bind to cell surface (Bergelson et al., 1992) and therefore, the entry of EV1 can be used to study the role of integrins in caveolae function. Previous studies have shown that integrins can be coprecipitated with caveolin-1 (Wary et al., 1996, 1998), suggesting that caveolae form a platform for integrin-mediated signaling.
Here, we show that 21 integrin is located in raft like membrane domains rather than in caveolae. However, the formation of 21 integrin clusters triggers their lateral redistribution along cell surface to caveolae and consequently activates the internalization of caveolae in a protein kinase C (PKC)-dependent manner. Integrin 21 represents a novel mechanism to activate caveolae-mediated endocytosis. The molecular mechanisms of endocytosis and recycling of 1, 2, and V integrins have been studied in detail (Bretscher 1992; Fabbri et al., 1999; Ng et al., 1999a; Laukaitis et al., 2001), but integrin trafficking has been described to take place in endosomes and in an endocytic recycling pathway. Thus 21 seems to have unique activities compared with other integrins. In agreement with this our results show that the clusters of V integrin are internalized by a different mechanism, namely in clathrin-coated entry vesicles and early endosomes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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2 integrin (SAOS-21 cells; Ivaska et al., 1999). Cells transfected with an empty expression vector (SAOS-pAW) were used as a control. EV1 (Farouk strain, ATCC) was propagated in GMK cells and purified in sucrose gradients as described previously (Marjomäki et al., 2002). The following antibodies were used: rabbit antisera against purified EV1 (Marjomäki et al., 2002), EEA1 (Mu et al., 1995), transferrin (Zymed, South San Francisco, CA), ERK (Santa Cruz Biotechnology, Santa Cruz, CA), caveolin-1 (Transduction Laboratories, Lexington, KY; N20 antibody, Santa Cruz), and phosphorylated protein kinase C (Upstate Biotechnology, Lake Placid, NY) as well as monoclonal antibodies (mAb) against total protein kinase C (Upstate), caveolin-1 (Transduction Laboratories and Zymed), tubulin (Sigma, St. Louis, MO), myc (9E10, ATCC), integrin 2 subunit (MCA2025, Serotec, Raleigh, NC), integrin V subunit (L230, ATCC), and phosphorylated ERK (Transduction Laboratories). Conjugation of anti-2 and anti-V antibodies to Alexa 488 was done using a mAb labeling kit (Molecular Probes, Inc., Eugene, OR).
Transfections
SAOS-21 cells were transfected with FuGENE reagent (Boehringer Mannheim, Indianapolis, IN) and the cells were used for experiments after an expression time of 25-40 h. The GFP constructs of actin (CLONTECH, Palo Alto, CA), caveolin-1 (caveolin-GFP; from Dr. Ari Helenius, Institute of Biochemistry, ETH-Hoenggerberg, Switzerland; Pelkmans et al., 2001) wild-type Eps15 (DIIId2; from Dr. Alice Dautry-Varsat, Pasteur Institute, Paris; Benmerah et al., 1998), dominant negative (DN) Eps15 (d95/925; from Dr. Alice Dautry-Varsat, Pasteur Institute, Paris; Benmerah et al., 1999), wild-type PKC (Dr. Peter J. Parker, Cancer Research, UK; Ng et al., 1999b), DN PKC (T497A kinase-dead, substrate-binding mutant; Dr. Peter J. Parker, Cancer Research, UK; Mostafavi-Pour et al., 2003), and DN PKC
(kinase dead form; Ivaska et al., 2002b) were used. DN MEK (1E8; from Dr. Natalie Ahn, University of Colorado; Holmström et al., 1999), constitutively active MEK (1R4F; from Dr. Natalie Ahn, University of Colorado; Holmström et al., 1999), and DN Ras (asn-17-ras; from Dr. Larry Feig, Tufts University; Feig and Cooper, 1988) were cotransfected to the cells with pEGFP-C2 (CLONTECH). A myc tagged AP180C (from Dr. Dieter Blaas, University of Vienna; Ford et al., 2001) was revealed from transfected cells by myc labeling.
Flotation Gradient Centrifugation
SAOS-21 cells were lysed with 1% Triton X-100 in PBS supplemented with protease inhibitors for 30 min on ice. The homogenate was adjusted to a sucrose density of 40.6%, overlaid with 35% sucrose and then filled with 5% sucrose. The gradient was centrifuged for 35,000 rpm at 4°C in a Beckman Sw 41Ti rotor for 18 h.
Integrin Clusters
Antibody against 2 integrin was added in DMEM (supplemented with 0.1% serum, low-DMEM), incubated for 1 h on ice, and washed. Cells were subsequently incubated with Alexa-conjugated goat anti-mouse IgG antibody on ice and washed. Formation of integrin clusters was allowed to occur at 37°C for 30-90 min. Clusters of V integrins were allowed to form using an anti-V subunit mAb and Alexa-conjugated anti-mouse IgG antibody. As a control, anti-2 and anti-V mAbs, directly labeled with Alexa 488, were used alone without the clustering secondary antibodies.
Measurement of Internalization
The internalization of EV1 or integrin clusters was estimated by confocal microscopy using the three-dimensional (3D) LSM program, version 1.4.2 (Carl Zeiss, Jena, Germany). Cells were incubated with EV1 for 1 h and fixed with 3% paraformaldehyde for 20 min. After fixation, EV1 was labeled without permeabilization using a primary antibody and then the anti-rabbit Alexa 546 conjugate. After a 5-min permeabilization with 0.2% Triton X-100 EV1 was again labeled but now using anti-rabbit Alexa 488 conjugate. Thus, plasma membrane associated EV1 was stained with both Alexa 546 and 488 conjugates and was seen as yellow when the red and green channels were merged. Green signal representing internalized EV1 was measured. First the double-labeled cells were imaged as z-stacks with the confocal microscope. Then, in the 3D for LSM program the volume of internalized vesicles and the total amount of fluorescence was measured. Alltogether 20 cells were measured in the experiment. In parallel experiments the clusters of 2 or V integrins were allowed to form in the presence of Alexa-conjugated secondary antibody and to be internalized. Samples were fixed as above. The internalized integrins had green color, and the plasma membrane associated integrins were labeled with Alexa 546 conjugate. Again yellow color in merge images represented integrin clusters on the plasma membrane and green signal represented internalized clusters.
Protein Kinase C Activation
SAOS-21 cells were starved overnight in low-DMEM. Cells were then treated in different ways: 1) stimulated with 1 µM PMA 30 min at 37°C; 2) incubated with EV1 for 1 h on ice, washed, and incubated for 30 min at 37°C; 3) pretreated with 10 µM safingol at 37°C for 30 min, followed by EV1 treatment together with 10 µM safingol as described above; 4) incubated with anti-2 mAb Fab fragment (2 µg) for 1 h on ice, washed, and incubated for 30 min at 37°C; 5) 21 integrin clustering as explained above and incubated for 30 min at 37°C; 6) control SAOS-21 cells were treated with low-DMEM for 1 h on ice and then incubated for 30 min at 37°C. The treated cells were lysed with 30 mM octylglucopyranoside (Sigma) with protease inhibitors for 30 min on ice.
SDS-PAGE and Immunoblotting
Samples were separated in 12% SDS-polyacrylamide gels and electroblotted onto PVDF membrane (Millipore, Bedford, MA). Aerolysin overlay assay was performed as described (Fivaz et al., 2002). Primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Richmond, CA) were used. Bands were detected by chemiluminescence (Pierce, Rockford, IL).
Immunofluorescence and Confocal Microscopy
Subconfluent SAOS-21 cultures were incubated with EV1 for various time periods and then fixed with methanol at -20°C for 6 min or with 4% paraformaldehyde for 20 min at room temperature. Cross-absorbed goat secondary antibodies against rabbit (Alexa 488, 546, or 633 nm; Molecular Probes, Inc.) and mouse (Alexa 488, 546, or 633 nm; Molecular Probes, Inc.) antibodies were used for labeling. The cells were examined with an Axiovert 100 M SP epifluorescence microscope (Carl Zeiss) equipped with a confocal setup (Zeiss LSM510). Images were acquired using a Plan Neofluar objective (63x, 1.25 oil) and a digital resolution of 512 x 512. False colocalization signals were avoided by scanning fluorescence from different excitation wavelengths separately.
Live Cell Microscopy
SAOS-21 cells were stained with Alexa 546-conjugated mutant aerolysin (ASSP; Fivaz et al., 2002) together with an anti-2 mAb on ice for 1 h. Alternatively, cells transfected with cDNA encoding caveolin-1-GFP (green fluorescent protein) or actin-GFP were treated with anti-2 mAb. After washes the cells were transferred to a preheated sample stage (27/37°C) at a confocal microscope (Carl Zeiss Axiovert 100M with LSM510). Anti-mouse Alexa 488 (aerolysin-containing cells) or anti-mouse Alexa 546 (GFP-containing cells) was added and z-stacks through the cells were imaged at 5- or 10-min intervals. CO2 independent medium (Sigma) was used in all steps. 3D projections of the selected images were created for each time point using LSM 510 3.0 and AxioVision Inside4D 3.0 (Carl Zeiss). These image series were processed and edited using Corel Photo-Paint 8, Ulead Media Studio ProVideo Editor 5.02a VE, and Quick Time Pro.0.2.
Electron Microscopy
SAOS-21 cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h, and then postfixed in 1% osmium tetroxide, dehydrated, stained with uranyl acetate, and embedded in LX-112. For visualizing clusters of 2 or V integrin, cells were treated with anti-2 or V mAb for 1 h on ice, then with rabbit antibodies against mouse IgG (Sigma), and finally with protein A gold (10-nm particles, G. Posthuma and J. Slot, Utrecht, The Netherlands), both for 1 h on ice. Cells were then either fixed immediately or incubated for 1 min to 2 h at 37°C in complete culture medium. Further preparation for electron microscopy was performed as described above.
Statistical Analysis
For analyzing the transfected samples in Figures 5C and 6B the binomial t test (comparison of two experimental percentual figures) was used. For analyzing the differences in binding of EV1 to cells (Figure 7B) multivariate analysis of variance (MANOVA) was used with the least significant difference (LSD) as a post hoc test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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21 Integrin Is in Membrane Rafts21 integrin in SAOS-21 cells (human osteosarcoma cells transfected with 2 integrin cDNA) is found in fractions containing detergent resistant membranes (DRM; Figure 1A). The same fractions contained glycosyl phosphatidyl inositol anchored proteins (GPI-APs), well-known components of raft type cholesterol-sphingolipid-rich microdomains and caveolin-1, a hallmark protein for caveolae and caveosomes (Figure 1A). GPI-APs were recognized using Alexa 546-conjugated mutant aerolysin (ASSP; Fivaz et al., 2002). Because flotation gradient centrifugation cannot be used to separate different DRM domains, confocal microscopy was used. When cells were plated on plastic in the presence of serum, patches of ASSP were found at the cell peripheries, suggesting the presence of raft domains. In addition, some intracellular staining was seen (Figure 1B). In SAOS-21 cells 21 integrin colocalized with ASSP positive membrane domains (Figure 1B), indicating that ligand free 21 is present in raft domains. Caveolin-1 was mainly seen inside the cell, most clearly around the nucleus. Some caveolin-1 was also seen on the cell surface in accordance with the fact that the SAOS cell surface is rich in caveolae (Marjomäki et al., 2002). No clear colocalization of caveolin-1 and 21 integrin was detected (Figure 1B).
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Clustering of 21 Integrin Leads to Lateral Redistribution following Actin Filaments and Internalization in Caveolae-like Structures
Cluster formation was first induced using primary anti-2 integrin antibodies together with secondary antibodies. The fate of the clustered integrins was followed by live cell confocal microscopy, and the data were processed to obtain projections of the 3D images of the cells at different time points. Concomitant staining of cells with Alexa 546-conjugated ASSP was used to mark the GPI-APs containing domains. During the follow-up period integrins formed clusters (Figure 2A) that started to move along the cell surface. Concomitantly, small clusters fused together (Figure 2B, Video 1). Moving integrin clusters followed cortical actin microfilaments, i.e., filaments close to the cell surface (Figure 2, C-E, Video 2). Destruction of microfilaments with cytochalasin D (5 µg/ml) inhibited the movement of 21 integrins out of lipid rafts (our unpublished results). The presence of 21 integrins on the cell surface was also confirmed by measuring the relative distribution of internalized vs. cell surface integrins using differential staining before and after permeabilization (our unpublished results).
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Integrin clusters were subsequently internalized (Figure 3A, Movie 3). Importantly, under the same conditions no integrin clustering or internalization was detected if cells were treated with fluorophore-labeled primary antibody without the secondary antibody (Figure 3B). Furthermore, the conditions used in live cell microscopy had no effects on cell viability or morphology (our unpublished results). Integrin clusters were internalized in caveolin-1-positive vesicles (Figure 3C, Video 4). Integrin 21 was immunostained before and after permeabilization of the cells to distinguish between internalized and cell surface integrin. Two hours after antibody-mediated clustering 71 ± 3% of 21 integrin was intracellular. Destruction of microfilaments with cytochalasin D (5 µg/ml), which inhibited the movement of 21 integrins out of lipid rafts, did not completely prevent the 21-mediated EV1 entry and infection (our unpublished results). GPI-APs were also internalized to some extent. However, this is not evident in the images because of the "shadow" rendering method used. In accordance with our previous observations (Marjomäki et al., 2002), EV1 was rapidly internalized into similar vesicles that contained caveolin-1 and 21 integrin, and the vesicles had the tendency to accumulate close to the nucleus (Figure 3, C and D). The internalization of clustered integrins was further studied by EM using secondary antibodies and protein A gold. The cell surface invaginations containing clustered integrins appeared to be similar to caveolae and, accordingly, the integrin containing intracellular vesicles resembled caveosomes (Pelkmans et al., 2001; Figure 3E). EV1 labeled with gold particles was shown to accumulate into identical vesicles (Figure 3F). Our data indicate that the formation of 21 clusters alone may trigger accumulation of the receptors into caveosome like organelles and that EV1 may behave as a multivalent ligand that activates the same internalization process as the antibodies.
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Importantly, in EM we could not see EV1 or 21 clusters inside coated vesicles, suggesting that clathrin-dependent processes are not involved in their entry. This was further studied by transient transfection with cDNA coding for dominant negative (DN) Eps15, known to inhibit the internalization of transferrin (Benmerah et al., 1999). As shown in Table 1, DN Eps15 had no effect on 21 internalization, although it could inhibit the entry of transferrin. In SAOS-21 cells, transfected AP180C (Ford et al., 2001) proved to be an even more effective inhibitor of transferrin internalization, but without an effect on 21 entry (Table 1).
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The Clusters of V Integrins Are Internalized in Coated Vesicles and Endosomes
We repeated the same experiment with antibodies against V integrin subunit and secondary antibodies. In 2 h 93 ± 1% of V clusters had been internalized. If primary anti-V antibodies were used alone, no clustering or internalization of V integrins was detected (our unpublished results). The clusters of V integrins were internalized in caveolin-1-negative vesicles (Figure 4A). However, some vesicles containing V clusters were positive for early endosome associated protein (EEA1; Figure 4B) and for endocytosed transferrin (Figure 4C). Importantly, internalized 21 clusters were in EEA1 and transferrin negative vesicles (Figure 4, B and C). Finally, electron microscope images revealed that V clusters labeled with gold particles were found in structures that were morphologically identical to clathrin-coated vesicles (Figure 4D). Picornaviruses that bind to V integrins, such as parechovirus-1, are known to enter their host cells via the clathrin-dependant pathway (Joki-Korpela et al., 2001).
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Protein Kinase C Activity Is Essential for the Internalization of 21 Integrin via Caveolae
The fact that integrins can mediate cellular signals (Schwartz and Ginsberg, 2002) may influence their internalization. We have previously shown that mitogen activated protein kinases (MAPKs) are regulated by the collagen receptor integrins (Ravanti et al., 1999). Here, extracellular signal regulated kinase (ERK) was activated at very early time points after 21 binding to EV1 (Figure 5A). Because a selective inhibitor of ERK activation (20 µM PD98059, Calbiochem, La Jolla, CA; Figure 5B) or transient transfections with cDNAs coding for dominant negative or constitutively active MEK (DN MEK and CA MEK, respectively) had no effect on EV1 infection (Figure 5C), and DN MEK inhibited the internalization of 21 integrin only 10% (Figure 5C), we tried to specify other components of the same pathway, upstream of ERK and MEK, using selective inhibitors of Ras and protein kinase C (PKC). A Ras inhibitor (500 µM perillyl alcohol, POH, Aldrich, Milwaukee, WI) had little effect on ERK activation and its consequent transport into nucleus (Figure 5D). In addition, transfections with DN Ras had no effect on 21 internalization or EV1 infection (Figure 5C). In contrast, a PKC inhibitor (5 µM bisindolylmaleimide, Calbiochem) almost completely prevented accumulation of ERK in nucleus (Figure 5D). The experiment was repeated with another PKC inhibitor (10-25 µM safingol, Calbiochem) with the same results (Figure 5D).
On the basis of these findings, we deduced that PKC activity is needed for the activation of signaling pathways by 21 integrin. Furthermore, as shown in Figure 6A, the presence of PKC inhibitors reduced the number of EV1-infected cells to background levels. Safingol has been considered to be a selective inhibitor of the isoform of PKC (Masur et al., 2001). Here, we also performed transfections with DN PKC cDNA to confirm the role of this isoform in the process (Figure 6B). DN PKC inhibited EV1 entry and infection, whereas overexpression of wild-type PKC promoted it (Figure 6B). DN PKC was used as a control and it had no effect (Figure 6B). The critical role of PKC in EV1 entry and infection was also confirmed in a third set of experiments (Figure 6C), in which chronic treatment of cells with a phorbol ester (PMA, 6 h) was used to downregulate PKC expression. Chronic PMA treatment is known to induce the degradation of both classical and novel PKCs (Srivastava et al., 2002), and it is not specific for PKC. The effectiveness of the treatment was confirmed by Western blotting (Figure 6C). Concomitantly with a decrease in PKC level, EV1 infection was prevented (Figure 6C). The amount of active (phosphorylated) PKC in SAOS-21 cells was analyzed by Western blotting with phosphospecific antibodies. Control SAOS-21 cells contained phosphorylated PKC and the fact that short PMA treatment (30 min) could only slightly increase the phosphorylation (in five experiments on average 1.5-fold) suggested that most of it might be in the active form (Figure 6D). Some increase (maximally 1.5 fold) in the phosphorylation of PKC was seen after the cells were exposed to EV1 (Figure 6D). A similar small increase in phosphorylation was seen in the presence of primary anti-2 and secondary antibodies when compared with the effect of Fab fragments of the same anti-2 antibody (Figure 6D). We suggest that the increase in PKC phosphorylation may be a part of the signaling events after integrin clustering. In any case, its activity seems to be critical for integrin signaling.
We next tested the mechanism used by PKC inhibitors to affect the 21 integrin-mediated entry of EV1. If safingol was added to cells 5 min after EV1 almost complete inhibition was still obtained, whereas if added 30 min after EV1 it could no longer prevent infection (Figure 7A). This result limited the action of PKC to the first minutes of the process. Experiments measuring the number of radioactive EV1 bound to the cell surface indicated that safingol does not inhibit ligand binding to 21 integrin (Figure 7B). Actually, in repeated experiments safingol seemed to slightly increase the number of cell bound EV1 viruses (Figure 7B), making it possible to speculate that safingol may actually affect receptor recycling. However, the increase was not statistically significant (multivariate analysis of variance), and therefore it was not studied further. In the presence of safingol EV1 binding to 21 integrin could still initiate the lateral redistribution of the integrin out of lipid rafts (our unpublished results). Furthermore, in the presence of safingol clustering antibodies could still initiate the process leading to the co-localization of 21 integrin and caveolin-1 (Figure 7C). Thus, safingol had no effect on the lateral movement of 21 integrin after cluster formation. Finally, we used confocal microscopy to quantify the number of EV1 particles inside cells and safingol could remarkably reduce the entry of EV1 (Figure 7D). Furthermore, safingol had no effect on the entry of clustered V integrins (Figure 7E). Thus, the results indicate that the crucial role of PKC in the process is limited to the activation of caveola entry by 21 integrins that have moved from lipid rafts to caveolae.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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M2 integrin in monocytoid cells (Simon et al., 1993) and by IIb3 integrin in platelets (Wencel-Drake et al., 1996). Integrins V3 and V5 can mediate the internalization of vitronectin (Pijuan-Thompson and Gladson, 1997). Some integrins can also recycle (Bretscher 1992; Fabbri et al., 1999). This process seems to be important for migrating epithelial cells (Ng et al., 1999a; Laukaitis et al., 2001). During cell migration 1 integrin trafficking has been described to take place in endosomes and the endocytic recycling pathway (Ng et al., 1999a; Laukaitis et al., 2001) in a PKC-dependent manner (Ng et al., 1999a). We have recently reported that 21 integrin is internalized via caveolae in the process of EV1 entry (Marjomäki et al., 2002). This represents a novel internalization mechanism for integrins. Integrin 21 is a collagen receptor. It is expressed on platelets, epithelial cells, and also on many mesenchymal cell types, including fibroblasts, chondrocytes, osteoblasts, and endothelial cells. Integrin 21 mediates internalization of collagen and therefore it may participate in the regulation of matrix turnover (Segal et al., 2001). In endothelial cells 21-mediated collagen endocytosis may play a special role in the formation of capillary tube and lumen (Davis and Camarillo, 1996). It is not known whether integrins that are internalized in caveolae can recycle.
Many members of the integrin family are used by different viruses for attachment to the cell surface and for subsequent internalization into the host cell (Wang, 2002; Bomsel and Alfsen, 2003). It has been suggested that viruses have evolved to recognize cell adhesion receptors because of their relatively low affinity to natural ligands (Wang, 2002). The integrins may, however, play an active role in the entry process. For example, Kaposi's sarcoma associated herpes virus (human herpes virus 8) has been shown to use 31 integrin as its receptor and to activate 31-dependent signaling pathways, including focal adhesion kinase (Akula et al., 2002). Thus, virus binding may activate integrin associated cellular signaling, which could promote or modulate virus entry. Integrin 21 differs from other integrin type virus receptors by being the only integrin known to mediate virus entry through caveolae instead of clathrin-coated vesicles (Marjomäki et al., 2002). Caveolae are also involved in the entry of SV40 (Pelkmans et al., 2001; Pelkmans et al., 2002), mouse polyoma virus (Richterova et al., 2001), filoviruses (Empig and Goldsmith, 2002), and some bacteria (Shin et al., 2000).
Despite the fact that 21 integrin is internalized in caveolae our results indicate that after synthesis it is targeted to lipid rafts together with GPI-APs. The binding of both natural ligands and EV1 to 21 integrin may cause the formation of a receptor cluster. Here, using antibodies to induce the cluster formation we were able to show that 21 clustering alone can initiate the lateral redistribution of 21 out of raft domain. The integrins seem to follow actin microfilaments and during the process smaller clusters fuse together forming larger ones. Lateral redistribution of 51 integrin on the cell surface has been detected during electric field directed fibroblast locomotion (Brown and Loew, 1994). When integrins moved toward the cathode they also seemed to form large clusters (Brown and Loew, 1994). However, typically 51 is redistributed in endocytic vesicles in which the integrin colocalizes with transferrin receptor (Laukaitis et al., 2001). Thus, the lateral movement of 21 on cell surface from rafts to caveolae indicates that 21 acts in a different way than fibronectin receptors or other integrins studied so far. In migrating cells it is not known, whether 21 is among the 1 integrins that are guided to the endocytic recycling pathway (Ng et al., 1999a). However, integrin clustering after the binding of multivalent ligands seems to initiate a very different process.
When 21 integrin clusters were found in caveolae like invaginations on the cell surface their colocalization with caveolin-1 was observed. Previous studies have shown that integrins can be coprecipitated with caveolin-1 (Wary et al., 1996, 1998; Wei et al., 1999). Our results indicate, that in the case of 21 this association is obvious only after integrin clustering and redistribution. Caveolae are immobile and their internalization requires an activating stimulus (Thomsen et al., 2002). For example, the interaction of albumin-docking protein gp60 and caveolin-1 in endothelial cells (Minshall et al., 2000) and the phosphatase inhibitor okadaic acid (Parton et al., 1994) can trigger the internalization of caveolae. Clustering of 21 integrin represents a novel mechanism to activate caveolae.
The formation of integrin clusters is known to be important for signaling events (Miyamoto et al., 1995). Furthermore, caveolin-1 may participate in integrin-mediated signaling (Wary et al., 1996, 1998). The interaction of 21 with collagen can activate several signaling pathways, including MAPKs (Ivaska et al., 1999; Ravanti et al., 1999), the 
isoform of PKC (PKC; Xu and Clark, 1997), and protein phosphatase 2A (PP2A; Ivaska et al., 2002a). Here, PKC activity was needed for the internalization of 21 integrin in caveolae. Inhibition of PKC could not prevent the cluster formation or the lateral movement of 21 on cell surface. Its inhibitory effect was seen only during a limited time period, suggesting that PKC activity is obligatory only for the entry of caveolae. Interestingly, PKC may directly bind to the cytoplasmic domain of integrin 1 subunit (Ng et al., 1999a). Furthermore, PKC seems to be a resident protein of caveolae, which are major cell surface locations for this enzyme (Smart et al., 1994; Mineo et al., 1998). In caveolae PKC might be constantly active (Smart et al., 1994; Mineo et al., 1998). Accordingly, control SAOS-21 cells contained phosphorylated PKC, and PMA could only slightly increase its phosphorylation. A similar small increase in the phosphorylation of PKC was seen after the cells were exposed to EV1 or antibodies forming 21 clusters. Previous studies support the idea that PKC is needed for caveola-related signaling functions (Smart et al., 1994; Mineo et al., 1998).
We have identified the formation of 21 integrin clusters as a key event leading to the relocation of 21 to caveolae. Furthermore, 21 clusters trigger the internalization of caveolae (the process has been summarized in Figure 8). Endocytosis via caveolae may be utilized by the natural ligands of 21 integrin. In addition, EV1 has evolved to take advantage of this phenomenon by binding to 21 on the cell surface. PKC appears to be an important regulator of the entry process. The data indicate that the function of 21 is unique when compared with any other integrin.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: CA, constitutively active; DN, dominant negative; DRM, detergent resistant membranes; EEA1, early endosome associated protein; ERK, extracellular signal regulated kinase; EV1, echovirus-1; GPI-APs, glycosyl phosphatidyl inositol anchored proteins; MAPK, mitogen activated protein kinase; MOI, multiplicity of infection; PKC, protein kinase C; SV40, simian virus 40.
¶ Corresponding author. E-mail address: jyrki.heino{at}utu.fi.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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31 (CD 49c/29) is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 108, 407-419.[CrossRef][Medline]
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1 h (Western blot). (B) Inhibitors of ERK activation (PD) and p38 kinase (SB) do not inhibit infection in contrast to methyl 
