Palmitoylation supports assembly and function of integrin–tetraspanin complexes

As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (α3, α6, and β4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of β4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient β4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core α6β4–CD151 complex formation was unaltered. There is also a functional connection between CD9 and β4 integrins, as evidenced by anti-CD9 antibody effects on β4-dependent cell spreading. Notably, β4 palmitoylation neither increased localization into “light membrane” fractions of sucrose gradients nor decreased solubility in nonionic detergents—hence it does not promote lipid raft association. Instead, palmitoylation of β4 (and of the closely associated tetraspanin CD151) promotes CD151–α6β4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

[1]  Richard T. Lee,et al.  Direct Extracellular Contact between Integrin α3β1 and TM4SF Protein CD151* , 2000, The Journal of Biological Chemistry.

[2]  J. Kreidberg,et al.  (Alpha)3(beta)1 integrin regulates epithelial cytoskeletal organization. , 1999, Journal of cell science.

[3]  P. Steinlein,et al.  T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their definition by antibody affinity and antigen density. , 1999, International immunology.

[4]  A. Sonnenberg,et al.  The Tetraspan Molecule Cd151, a Novel Constituent of Hemidesmosomes, Associates with the Integrin α6β4 and May Regulate the Spatial Organization of Hemidesmosomes , 2000, The Journal of cell biology.

[5]  M. Resh,et al.  Compartmentalization of integrin α6β4 signaling in lipid rafts , 2003, The Journal of cell biology.

[6]  A. Pepe,et al.  The Intracellular Functions of Ot 6 ~ 4 Integrin Are Regulated by EGF , 2002 .

[7]  J. Kreidberg,et al.  Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology. , 2002, Molecular biology of the cell.

[8]  Y. Poumay,et al.  The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility. , 2000, European journal of cell biology.

[9]  T. Südhof,et al.  Fatty acylation of synaptotagmin in PC12 cells and synaptosomes. , 1996, Biochemical and biophysical research communications.

[10]  T. V. Kolesnikova,et al.  Functional domains in tetraspanin proteins. , 2003, Trends in biochemical sciences.

[11]  Leslie M Shaw,et al.  Activation of Phosphoinositide 3-OH Kinase by the α6β4 Integrin Promotes Carcinoma Invasion , 1997, Cell.

[12]  A. Sacchi,et al.  Cooperative Signaling between α6β4Integrin and ErbB-2 Receptor Is Required to Promote Phosphatidylinositol 3-Kinase-dependent Invasion* , 2000, The Journal of Biological Chemistry.

[13]  A. Toker,et al.  Protein Kinase C–Dependent Mobilization of the α6β4 Integrin from Hemidesmosomes and Its Association with Actin-Rich Cell Protrusions Drive the Chemotactic Migration of Carcinoma Cells , 1999, The Journal of cell biology.

[14]  M. Hemler,et al.  Function of the Tetraspanin CD151–α6β1 Integrin Complex during Cellular Morphogenesis , 2002 .

[15]  R. Yauch,et al.  Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. , 1998, Molecular biology of the cell.

[16]  L. Shaw,et al.  The α6β4 integrin and epithelial cell migration , 2001 .

[17]  L. Ashman,et al.  Regulation of Endothelial Cell Motility by Complexes of Tetraspan Molecules CD81/TAPA-1 and CD151/PETA-3 with α3β1 Integrin Localized at Endothelial Lateral Junctions , 1998, The Journal of cell biology.

[18]  T. V. Kolesnikova,et al.  EWI-2 regulates α3β1 integrin–dependent cell functions on laminin-5 , 2003, The Journal of cell biology.

[19]  M. Klagsbrun,et al.  The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor , 1995, The Journal of cell biology.

[20]  R. Derynck,et al.  The Tetraspanin Cd9 Associates with Transmembrane TGF-α and Regulates TGF-α–Induced Egf Receptor Activation and Cell Proliferation , 2000, The Journal of cell biology.

[21]  L. Trusolino,et al.  A Signaling Adapter Function for α6β4 Integrin in the Control of HGF-Dependent Invasive Growth , 2001, Cell.

[22]  R. Yauch,et al.  Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase. , 2000, The Biochemical journal.

[23]  Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions. , 1999, The Biochemical journal.

[24]  M. Akashi,et al.  C-kit associated with the transmembrane 4 superfamily proteins constitutes a functionally distinct subunit in human hematopoietic progenitors. , 2002, Blood.

[25]  M. Hemler,et al.  Transmembrane-4 Superfamily Proteins Associate with Activated Protein Kinase C (PKC) and Link PKC to Specific β1 Integrins* , 2001, The Journal of Biological Chemistry.

[26]  T. V. Kolesnikova,et al.  Evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking. , 2004, The Biochemical journal.

[27]  T. V. Kolesnikova,et al.  EWI-2 modulates lymphocyte integrin α4β1 functions , 2004 .

[28]  E. Odintsova,et al.  Expression of the Palmitoylation-deficient CD151 Weakens the Association of α3β1 Integrin with the Tetraspanin-enriched Microdomains and Affects Integrin-dependent Signaling* , 2002, The Journal of Biological Chemistry.

[29]  P. Marchisio,et al.  The MSP Receptor Regulates α6β4 and α3β1 Integrins via 14-3-3 Proteins in Keratinocyte Migration , 2003 .

[30]  M. Linder,et al.  Signalling functions of protein palmitoylation. , 1998, Biochimica et biophysica acta.

[31]  R. Jaenisch,et al.  Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. , 1996, Development.

[32]  M. Hemler,et al.  Evaluation of Prototype Transmembrane 4 Superfamily Protein Complexes and Their Relation to Lipid Rafts* , 2001, The Journal of Biological Chemistry.

[33]  K. Sekiguchi,et al.  Laminin-10/11 and Fibronectin Differentially Regulate Integrin- dependent Rho and Rac Activation via p130Cas-CrkII-DOCK180 Pathway* , 2001, The Journal of Biological Chemistry.

[34]  M. Resh Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. , 1999, Biochimica et biophysica acta.

[35]  Eric Rubinstein,et al.  Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation , 2002, FEBS letters.

[36]  M. Mann,et al.  Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Hemler,et al.  Specific tetraspanin functions , 2001, The Journal of cell biology.

[38]  M. Hemler,et al.  Transmembrane-4-superfamily proteins CD151 and CD81 associate with α3β1 integrin, and selectively contribute to α3β1-dependent neurite outgrowth , 2000 .

[39]  G. O'Neill,et al.  Integrin signalling: a new Cas(t) of characters enters the stage. , 2000, Trends in cell biology.

[40]  M. Stepp,et al.  Integrins as receptors for laminins , 2000, Microscopy research and technique.

[41]  A. Sonnenberg,et al.  Association of the tetraspanin CD151 with the laminin-binding integrinsα 3β1, α6β1, α6β4 and α7β1 in cells in culture and in vivo , 2002 .

[42]  T. S. Panetti,et al.  Tyrosine phosphorylation of paxillin, FAK, and p130CAS: effects on cell spreading and migration. , 2002, Frontiers in bioscience : a journal and virtual library.

[43]  D. Azorsa,et al.  Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions. , 1999, The Biochemical journal.

[44]  M. Hemler,et al.  Transmembrane-4-superfamily proteins CD151 and CD81 associate with alpha 3 beta 1 integrin, and selectively contribute to alpha 3 beta 1-dependent neurite outgrowth. , 2000, Journal of cell science.

[45]  R. Hynes,et al.  α3Aβ1 integrin localizes to focal contacts in response to diverse extracellular matrix proteins , 1995 .

[46]  Y. Shimizu,et al.  Stimulation of beta1-integrin function by epidermal growth factor and heregulin-beta has distinct requirements for erbB2 but a similar dependence on phosphoinositide 3-OH kinase. , 1999, Molecular biology of the cell.

[47]  M. Hemler Integrin associated proteins. , 1998, Current opinion in cell biology.

[48]  F. Giancotti,et al.  The intracellular functions of alpha6beta4 integrin are regulated by EGF , 1996, The Journal of cell biology.

[49]  L. Shaw,et al.  Activation of phosphoinositide 3-OH kinase by the alpha6beta4 integrin promotes carcinoma invasion. , 1997, Cell.

[50]  M. Hemler Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. , 2003, Annual review of cell and developmental biology.

[51]  M. Hemler,et al.  An extracellular site on tetraspanin CD151 determines α3 and α6 integrin–dependent cellular morphology , 2002, The Journal of cell biology.

[52]  F. Watt,et al.  Functional significance of CD9 association with beta 1 integrins in human epidermal keratinocytes. , 1996, Cell adhesion and communication.

[53]  V. Quaranta,et al.  Inhibitory Role of α6β4-Associated Erbb-2 and Phosphoinositide 3-Kinase in Keratinocyte Haptotactic Migration Dependent on α3β1 Integrin , 2001, Journal of Cell Biology.

[54]  S. Fitter,et al.  Analysis of the CD151·α3β1 Integrin and CD151·Tetraspanin Interactions by Mutagenesis* , 2001, The Journal of Biological Chemistry.

[55]  R. Hynes,et al.  alpha 3A beta 1 integrin localizes to focal contacts in response to diverse extracellular matrix proteins. , 1995, Journal of cell science.

[56]  J. Kreidberg,et al.  SUMMARY α 3 β 1 integrin regulates epithelial cytoskeletal organization , 2022 .

[57]  Y. Yazaki,et al.  Tyrosine phosphorylation of p130Cas in cell adhesion and transformation. , 1996, Human Cell.

[58]  F. Giancotti,et al.  EGF-R signaling through Fyn kinase disrupts the function of integrin α6β4 at hemidesmosomes , 2001, The Journal of Cell Biology.

[59]  S. Funderud,et al.  The nature of the subset of MHC class II molecules carrying the CDw78 epitopes. , 1999, International immunology.

[60]  J. Hanke,et al.  Discovery of a Novel, Potent, and Src Family-selective Tyrosine Kinase Inhibitor , 1996, The Journal of Biological Chemistry.

[61]  S. Levy,et al.  CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. , 1998, Annual review of immunology.

[62]  C. Gundersen,et al.  Extensive lipidation of a Torpedo cysteine string protein. , 1994, The Journal of biological chemistry.

[63]  F. Berditchevski Complexes of tetraspanins with integrins: more than meets the eye. , 2001, Journal of cell science.