Integrin Signaling and Lipid Rafts

[Cell Cycle 3:6, 725-728; June 2004]; ©2004 Landes Bioscience

Integrin Signaling and Lipid Rafts Extra Views

KEY WORDS integrins, membrane domains, Rho GTPases, signaling, migration, anchorage-dependent growth

ACKNOWLEDGEMENTS

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INTEGRIN SIGNALING AND ANCHORAGE INDEPENDENCE OF CELL GROWTH Integrins are transmembrane receptors that mediate cell-cell adhesion, cell attachment to the extracellular matrix (ECM), cell spreading and cytoskeletal organization.1 Integrin-mediated adhesion initiates transduction of signals towards the cell interior and also regulates transmission of signals from growth factor receptors.2,3 Thus, activation of most signaling pathways, including the Erk, PI 3-kinase, FAK, Src and Rho GTPases pathways depend on integrins.4 Integrin-mediated signal transduction regulates cell migration, cell cycle progression, gene expression and survival.5 Alteration of these processes is key to tumorigenesis. In normal cells, integrin signals leading to survival and proliferation mediate anchorage-dependence of growth.2 In cancer cells, by expression of oncogenes and/or loss of tumor suppressors, integrin signaling pathways are altered or bypassed, leading to anchorage-independent cell growth and enhanced migration and invasion.6 Anchorageindependent cell growth is indeed the in vitro characteristic that best correlates with tumorigenesis in vivo.7

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I am thankful to Juan M. Zapata for critical reading of the manuscript. This work was supported by the MCYT (Ministerio de Ciencia y Tecnología, Spain) through grant SAF2002-02425 and the Ramón y Cajal Program, and by the Leukemia and Lymphoma Society of America.

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=952

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Received 04/26/04; Accepted 04/28/04

Integrin-mediated adhesion regulates the recruitment of the small GTPase Rac to the plasma membrane and subsequent activation of downstream signaling. We recently reported that Rac binds preferentially to cholesterol-rich membranes (“lipid rafts”), and integrins regulate Rac function by preventing the internalization of its binding sites within these domains. Regulation of lipid rafts by integrins may be important for the spatial control of cell migration and signaling pathways involved in anchorage-dependent cell growth.

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Correspondence to: Miguel A. Del Pozo; Centro Nacional de Investigaciones Cardiovasculares; Madrid, Spain/ Departments of Immunology and Cell Biology; The Scripps Research Institute; La Jolla, California USA; Tel.: +34.91.806.1880x1132; Fax: +34.91.803.5258; Email: [email protected]

ABSTRACT

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RHO GTPASES, TRANSFORMATION AND INTEGRIN SIGNALING

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The Rho family of small GTPases plays a central role in integrating signals from integrins and growth factor receptors.8 Rho GTPases regulate cell polarization and migration, membrane trafficking, cell cycle progression, gene expression and oncogenic transformation.9 These events require interactions with effector molecules, which occurs only when the GTPase is bound to GTP. Loading with GTP is tightly regulated by guanine nucleotide exchange factors (GEFs) that induce activation and GTPase activating proteins (GAPs) that catalyze inactivation.8 Additionally, guanine nucleotide dissociation inhibitors (Rho-GDI’s) keep Rho GTPases soluble in the cytoplasm by shielding the geranyl-geranyl moiety10 and blocking effector binding.11 The small GTPase Rac1 is a member of this family with a key role in survival, gene expression and cell cycle progression.12 Rac controls the cell cycle through effects on cyclin D1 transcription and translation13,14 and can stimulate retinoblastoma protein (pRb) phosphorylation,15 leading to G1-phase progression. Recent studies suggest that Rac mediates integrin-stimulated translation of cyclin D1.16 Rac effects on survival are mediated through activation of NF-κB and PI 3-kinase.17-19 Rac is required for transformation induced by Ras, v-Abl, Brc-Abl and Src oncogenes.9 Rac and its GEFs, such as Tiam-1, Vav and GEFT, are involved in tumorigenic growth and metastasis in several systems.9,20 Therefore, accumulated evidence shows that Rac is deregulated in some tumors. Thus, the control of Rac function by integrins and its deregulation in cancer are key events in anchorage dependent and independent cell growth, respectively. In addition to their effect on GTP loading,16,21-23 we recently reported that integrins independently control the translocation of GTP-bound Rac and Cdc42 to the plasma membrane and its coupling to effectors.11,22 GTP-Rac in nonadherent cells remains in the Cell Cycle

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INTEGRIN SIGNALING AND LIPID RAFTS

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The plasma membrane contains domains that are enriched in cholesterol, sphingolipids and a variety of proteins including caveolin, flotillins, src-family kinases and GPI-linked proteins.25-28 These domains are more ordered than bulk plasma membranes and have been isolated by a variety of techniques based on their resistance to solubilization in nonionic detergents or low buoyant density.29 In spite of intense research on the subject, there is still considerable uncertainty about the size and composition of these structures in vivo,27,30-36 and there is evidence for the existence of different types.28,37,38 Collectively referred to as “lipid rafts”, they have been proposed to function in signal transduction by compartmentalizing components at the plasma membrane.25 The best defined subtype of lipid raft, caveolae, contains the protein caveolin. They have been shown to localize and organize signal transduction at the plasma membrane.39 Much evidence has now accumulated to show that RhoA and Rac are concentrated in lipid rafts and caveolae.40-43 Thus, we investigated the involvement of these domains in integrin regulation of Rac membrane targeting.24 Our first approach was to disrupt lipid rafts from NIH-3T3 cells with methyl-β-cyclodextrin, a carbohydrate containing a hydrophobic pocket that binds and extracts cholesterol from membranes.25 Depletion of membrane cholesterol mimicked the effects of loss of cell adhesion, impairing Rac membrane translocation and activation of the Rac effector Pak (p21-activated kinase), but not Rac activation itself. Consistently, similar results were reported in both human neutrophils and A431 epidermoid carcinoma cells.44,45 To further investigate the role of cholesterol-rich domains in Rac1 targeting, we incubated a recombinant, isoprenylated Rac1/ RhoGDI complex46 with plasma membrane fractions purified from human fibroblasts by using a detergent-free density gradient centrifugation method.47 Rac bound preferentially to the cholesterol/ caveolin-enriched fraction. Moreover, Rac bound specifically to liposomes prepared with an equimolar mixture of phosphatidylcholine (PC), cholesterol and sphingomyelin (Sph), that is in a liquid ordered state similar to that of lipid rafts.48,49 Membrane domain binding specificity is therefore determined somehow by the physical state of the lipids. We next used cholera toxin subunit B (CTxB) to label ganglioside GM1, a standard marker for lipid rafts.50 Active Rac colocalized strongly with GM1 in NIH-3T3 cells, primarily at cell edges. Other studies have shown that both cholesterol and Rac are highly concentrated in the leading edge of migrating cells.51,52 Moreover, we found that clustering of GM1 with CTxB-coated beads induced coclustering of Rac. Thus, Rac preferentially associates in vivo with membrane domains enriched in GM1. Consistent with our findings, it was recently reported that a peptide encoding Rac-1 C-terminus localizes to lipid rafts and inhibits Rac localization and function, as measured by membrane ruffling.53

These results beg the question of whether cholesterol-rich domains are the targets for integrin control of Rac membrane targeting. To gain additional insight into this question, we studied the effects of integrins on the distribution of raft markers. Strikingly, we observed that loss of integrin-mediated adhesion caused a rapid internalization of several raft markers, including GM1 and GPI-anchored proteins.24 These effects were reversed specifically by replating cells on integrin ligands or antibodies. Therefore, internalization of raft markers is reversible and its regulation integrin-specific. Importantly, similar results were obtained when studying the subcellular distribution of cholesterol, the main component of these microdomains. Thus, loss of integrin-mediated adhesion induces internalization of cholesterol-rich membrane domains. Rac does not seem to bind to internal membranes after rafts have internalized in nonadherent cells, since it dissociates from the total membrane fraction after detachment22 and remains in the cytoplasm bound to RhoGDI.11 This is most likely explained by alterations in the internalized raft domains, as suggested by biochemical analysis of GM1 distribution in cold detergent extracts.24 Integrins affect membrane domains in a spatial fashion. Local clustering of integrins with beads specifically induced local accumulation of both GM1 and Rac,24 consistent with the fact that integrins locally regulate Rac membrane targeting and binding to effectors.11 This finding further supports the hypothesis that integrin effects on raft domains may mediate Rac membrane targeting. Effects on rafts may also account for the previous observation that integrin occupancy and clustering induce localization of many signaling proteins now known to associate with these domains.54 It may be significant, however, that in all these instances, accumulation of GM1, Rac or other signaling proteins 54 is not sharply confined to the bead surface but is often noticeable at a moderate distance. This result may indicate that integrins do not necessarily interact directly with components of these membrane domains, but more likely initiate a signaling cascade that affects their targeting or trafficking. It also correlates with the fact that Rac-induced lamellipodia typically form at cell edges close to but not within focal adhesions or focal complexes. In any case, these findings underscore the importance of understanding the precise spatiotemporal regulation of signaling events in cell migration. It is tempting to speculate that integrin-regulated local Rac effector coupling via effects on rafts could crucially control cell migration. In agreement with this hypothesis, recent studies highlight the importance of the polarization of lipid rafts and membrane microviscosity (i.e., the physical properties of the membrane itself ) in the regulation of cell migration.45,51,52,55 This is certainly an interesting area of future research. To directly test the hypothesis that integrin-regulated raft internalization mediates loss of Rac membrane targeting, we artificially prevented internalization of GM1 by treating cells with CTxB-beads before detachment. Under these circumstances, nonadherent cells maintained Rac association to the plasma membrane and coupling to its effector Pak.24 This experiment demonstrates that internalization of cholesterol-rich domains from the membrane mediates the loss of Rac membrane targeting and downstream signaling after detachment from the ECM.

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LIPID RAFTS AND RAC MEMBRANE TARGETING

INTEGRIN-MEDIATED ADHESION REGULATES THE LOCATION OF CHOLESTEROL-RICH MEMBRANE DOMAINS

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cytoplasm bound to RhoGDI rather than being recruited to the cell surface and thus is uncoupled from downstream signaling. GTP-Rac binds better to membranes from adherent cells than from suspended cells, indicating that integrins regulate Rac membrane binding sites at the cell surface. Rac membrane targeting requires both the geranyl modification and the polybasic sequence in the hyper variable C-terminus but is independent of effector binding.11 After these studies, the mechanism how integrins regulate Rac binding sites at the plasma membrane remained unsolved. However, we have recently obtained evidence involving certain plasma membrane microdomains termed “lipid rafts”.24

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was the only raft-domain marker affected by the integrin-FAK pathway,56 whereas we found that also cholesterol and GPI-linked proteins internalize upon detachment.24 In this regard, evidence indicating that multiple types of membrane microdomains coexist within the plasma membrane 28,34,37,38 supports the idea that different domains maybe involved in the regulation of the Rho and Rac pathways. An intriguing hypothesis is that raft internalization could be a mechanism by which integrins influence signaling pathways other than Rac and Rho. Lipid rafts play a prominent role in many signal transduction pathways,25,26,29 and many of these pathways are shut down upon loss of integrin-mediated cell adhesion.3,4 Indeed, function of the Ras/Erk pathway, JNK, PI 3-kinase, FAK and Src family kinases are all highly dependent on integrins and have been linked to lipid rafts.57-60 These pathways are also involved in cell cycle progression and anchorage dependence of Figure 1. Model for Integrin regulation of Rac targeting via an effect on lipid rafts. We propose growth.2,4 Therefore, normal control of raft interthat integrins regulate Rac targeting and signaling via a pathway that controls the internalization nalization by integrins and its alteration could be of Rac binding sites within rafts. The cell has been artificially divided into two zones: (A) Regions a key event in anchorage dependent and indepenof the cell where integrins are occupied by ligand and/or activated are designated as “adherent”. In plasma-membrane edges of these areas, lipid rafts mediate binding of activated Rac, which is dent cell growth, respectively. These studies also raise questions about the able to couple to effectors and trigger downstream signaling. The exact nature of the Rac binding sites has not been precisely identified yet. (B) Areas of the cell where integrins are not occupied/ kinetics of Rac activation and membrane targeting. activated are designated as “non-adherent”. Cells detached from the ECM emulate these zones. Although our results show that Rac activation by In B areas, rafts undergo internalization, and then accumulate in a central compartment of the cell. GEFs and stable membrane targeting can be Internalization of rafts hampers Rac plasma membrane localization. Thus, Rac remains in the cytoseparated,11,22 under other conditions activation plasm bound to RhoGDI and uncoupled from downstream signaling mediated by effector moleand targeting are coupled. GEFs generally must cules. Anchorage-independent cells could potentially loose this mechanism of control by integrins localize to membranes to promote activation,8 and thus exhibit abnormal, ectopic Rac signaling in B zones. most likely because membranes facilitate dissociation of Rac from RhoGDI. But whether the exchange occurs in A MODEL FOR INTEGRIN REGULATION OF RAC SIGNALING proximity to membrane targeting sites or if once activated Rac All these data suggest a model in which we can distinguish two translocates between membrane sites and the cytoplasmic, elements (Fig. 1). First, Rac binding sites are components of choles- RhoGDI-bound state remains unclear. Elucidating the precise terol-rich membrane domains (lipid rafts). The selectivity of Rac for dynamics of nucleotide exchange, RhoGDI displacement and lipid rafts is determined, at least in part, by the lipids themselves. membrane targeting should be addressed in future studies. However, it is unlikely that lipids alone completely account for Rac References binding, suggesting that other components that remain to be identi1. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992; fied may contribute to Rac recruitment (see Fig. 1). Second, integrins 69:11-25. 2. Assoian RK, Schwartz MA. Coordinate signaling by integrins and receptor tyrosine kinasmaintain lipid raft domains at the plasma membrane. In the absence es in the regulation of G1 phase cell-cycle progression. Curr Opin Genet Dev 2001; of integrin-mediated adhesion, lipid rafts are cleared from the cell 11:48-53. surface through internalization. Preventing internalization maintains 3. Yamada KM, Even-Ram S. Integrin regulation of growth factor receptors. Nat Cell Biol 2002; 4:E75-E6. Rac plasma membrane association and coupling to effectors in nonMartin KH, Slack JK, Boerner SA, Martin CC, Parsons JT. Integrin connections map: To 4. adherent cells. Together, these results show that integrins regulate infinity and beyond. Science 2002; 296:1652-3. Rac targeting and signaling by inhibiting internalization of Rac 5. Giancotti FG, Ruoslahti E. Integrin signaling. Science 1999; 285:1028-32. binding sites in rafts. 6. Schwartz MA. Integrins, oncogenes, and anchorage independence. J Cell Biol 1997; 139:575-8. Palazzo et al. concomitantly found that integrins regulate 7. Freedman VH, Shin S. Cellular tumorigenicity in nude mice: Correlation with cell growth Rho-mediated microtubule stabilization (an essential event in cell in semisolid medium. Cell 1974; 3:355-9. migration) by localizing GM1 domains at the leading edge of the 8. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature 2002; 420:629-35. cell.56 Together, the two studies24,56 show that specific membrane 9. Sahai E, Marshall CJ. Rho-GTPases and cancer. Nat Rev Cancer 2002; 2:133-42. 10. Hoffman GR, Nassar N, Cerione RA. Structure of the Rho family GTP-binding protein microdomains mediate regulation of both Rac and Rho by integrins, Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 2000; 100:345-56. significantly reinforcing the proposed model (see Fig. 1). Palazzo 11. del Pozo MA, Kiosses WB, Alderson N, Meller N, Hahn KM, Schwartz MA. Integrins reget al. also found that focal adhesion kinase (FAK) is a crucial mediulate GTP-Rac localized effector interactions through dissociation of RhoGDI. Nature Cell Biol 2002; 4:232-9. ator of integrins in the pathway controlling GM1-localization. It 12. Ridley AJ. Cyclin’ round the cell with Rac. Dev Cell 2001; 1:160-1. remains to be tested whether FAK also mediates integrin-regulated 13. Joyce D, Bouzahzah B, Fu M, Albanese C, D’Amico M, Steer J, et al. Integration of raft-dependent effects on Rac targeting. Some differences between Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-κB-depenthe two studies suggest that this might not be the case. Thus, GM1 dent pathway. J Biol Chem 1999; 274:25245-9. www.landesbioscience.com

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