Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3 beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signaling.
Transmembrane proteins of the tetraspanin superfamily are assembled in multimeric complexes on the cell surface. Spatial orientation of tetraspanins within these complexes may affect signaling functions of the associated transmembrane receptors (e.g. integrins, receptor-type tyrosine kinases). The structural determinants that control assembly of the tetraspanin complexes are unknown. We have found that various tetraspanins and the alpha(3) integrin subunit are palmitoylated. The stability and molecular composition of the palmitoylated alpha(3)beta(1)-tetraspanin complexes are not affected by adhesion. To assess the significance of palmitoylation in the function of the alpha(3)beta(1)-tetraspanin complexes we mapped the sites of palmitoylation for CD151. Mutation of six cysteines, Cys(11), Cys(15), Cys(79), Cys(80), Cys(242), and Cys(243) was necessary to completely abolish palmitoylation of CD151. The association of the palmitoylation-deficient mutant of CD151 (CD151Cys8) with CD81 and CD63 was markedly decreased, but the interaction of the alpha(3)beta(1)-CD151Cys8 complex with phosphatidylinositol 4-kinase was not affected. Ectopic expression of CD151Cys8 in Rat-1 cells impaired the interactions of the endogenous CD63 and CD81 with the alpha(3)beta(1) integrin. Although the expression of the palmitoylation-deficient CD151 does not change cell spreading on the extracellular matrix, the number of focal adhesions increased. Adhesion-induced phosphorylation of PKB/c-Akt is markedly increased in cells expressing a palmitoylation-deficient mutant, thereby providing direct evidence for the role of the tetraspanin microdomains in regulation of the integrin-dependent phosphatidylinositol 3-kinase signaling pathway. In contrast, activation of FAK and ERK1/2 were not affected by the expression of CD151Cys8. Our results demonstrate that palmitoylation of tetraspanins is critical not only for the organization of the integrin-tetraspanin microdomains but also has a specific role in modulation of adhesion-dependent signaling.
Cancer Research UK Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham B15 2TA, United Kingdom. f.berditchevski@bham.ac.uk
Fedor
F
Berditchevski
Elena
E
Odintsova
Shigeaki
S
Sawada
Elizabeth
E
Gilbert
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D001665:Binding Sites; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D003545:Cysteine; D005109:Extracellular Matrix / Q000502:physiology*; D039201:Integrin alpha3beta1 / Q000378:metabolism*; D021962:Membrane Microdomains / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology*; D060185:Tetraspanin 24; D014162:Transfection
D015703:Antigens, CD; C554717:Cd151 protein, rat; D039201:Integrin alpha3beta1; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D060185:Tetraspanin 24; D019308:Palmitic Acid; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D003545:Cysteine