A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin.
Tetraspanins associate on the cell membrane with several transmembrane proteins, including members of the integrin superfamily. The tetraspanin CD9 has been implicated in cell motility, metastasis, and sperm-egg fusion. In this study we characterize the first CD9 conformation-dependent epitope (detected by monoclonal antibody (mAb) PAINS-13) whose expression depends on changes in the activation state of associated beta(1) integrins. MAb PAINS-13 precipitates CD9 under conditions that preserve the association of this tetraspanin with integrins, but not under conditions that disrupt these interactions. Induction of activation of beta(1) integrins by temperature, divalent cation Mn(2+), or mAb TS2/16 correlated with enhanced expression of the PAINS-13 epitope on a variety of cells. Through the use of different K562 myeloid leukemia transfectant cells expressing specific members of the beta(1) integrin subfamily we show that the expression of the PAINS-13 epitope depends on CD9 association with alpha(6)beta(1) integrin. The mAb PAINS-13 reactivity has been mapped to the CD9 region comprising residues 112-154 in the NH(2) half of the large extracellular loop. Also, we show that the CD9 conformation recognized by mAb PAINS-13 is functionally relevant in beta(1) integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation.
Gutierrez-Lopez
Maria Dolores
MD
Instituto de Farmacologia y Toxicologia CSIC-UCM, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain.
Ovalle
Susana
S
Yanez-Mo
Maria
M
Sanchez-Sanchez
Noelia
N
Rubinstein
Eric
E
Olmo
Nieves
N
Lizarbe
Maria Antonia
MA
Sanchez-Madrid
Francisco
F
Cabanas
Carlos
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D004789:Enzyme Activation; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D039121:Integrin alpha6beta1 / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011487:Protein Conformation; D011509:Proteoglycans / Q000378:metabolism; D060245:Tetraspanin 29
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554869:CD9 protein, human; C554870:Cd9 protein, mouse; D004338:Drug Combinations; D000939:Epitopes; D039121:Integrin alpha6beta1; D019012:Integrin beta1; D007797:Laminin; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D060245:Tetraspanin 29; C059485:matrigel; D003094:Collagen; D019869:Phosphatidylinositol 3-Kinases