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Blood, 15 December 2002, Vol. 100, No. 13, pp. 4502-4511
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Identification of CD9 extracellular domains important in
regulation of CHO cell adhesion to fibronectin and fibronectin
pericellular matrix assembly
George A. Cook,
Celia M. Longhurst,
Svetozar Grgurevich,
Shila Cholera,
Joseph T. Crossno Jr, and
Lisa K. Jennings
From the Vascular Biology Center of Excellence,
Department of Medicine; and the Departments of Molecular Sciences and
Surgery, The University of Tennessee Health Science Center, Memphis,
TN.
CD9, a 24-kDa member of the tetraspanin family, influences cellular
growth and development, activation, adhesion, and motility. Our
investigation focuses on the hypothesis that the CD9 second extracellular loop (EC2) is important in modulating cell adhesive events. Using a Chinese hamster ovary (CHO) cell expression
system, we previously reported that CD9 expression inhibited
cell adhesion to fibronectin and fibronectin matrix assembly. For the
first time, a functional epitope on CD9 EC2 that regulates these
processes is described. Binding of mAb7, an EC2-specific anti-CD9
monoclonal antibody, reversed the CD9 inhibitory activity on CHO
cell adhesion and fibronectin matrix assembly. This reversal of
cell phenotype also was observed in CHO cells expressing CD9 EC2
truncations. Furthermore, our data showed that the EC2 sequence
173LETFTVKSCPDAIKEVFDNK192 was
largely responsible for the CD9-mediated CHO cell phenotype. Two
peptides, 135K-V172 (peptide 5b) and
168P-I185 (peptide 6a), selectively blocked
mAb7 binding to soluble CD9 and to CD9 on intact cells. These active
peptides reversed the influence of CD9 expression on CHO cell adhesion
to fibronectin. In addition, confocal microscopy revealed that CD9
colocalized with the integrin  5 1 and
cytoskeletal F-actin in punctate clusters on the cell surface,
particularly at the cell margins. Immunoprecipitation studies confirmed
CD9 association with 1 integrin. The cellular distribution and colocalization of focal adhesion kinase and
-actinin with cytoskeletal actin was also influenced by CD9
expression. Thus, CD9 may exhibit its effect by modulating the
composition of adhesive complexes important in facilitating
cell adhesion and matrix assembly.
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