Contrasting effects of EWI proteins, integrins, and protein palmitoylation on cell surface CD9 organization.
CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of alpha3beta1 and alpha6beta4 integrins and by palmitoylation of the CD9 and beta4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression.
Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Xiuwei H
XH
Yang
Oleg V
OV
Kovalenko
Tatiana V
TV
Kolesnikova
Milena M
MM
Andzelm
Eric
E
Rubinstein
Jack L
JL
Strominger
Martin E
ME
Hemler
D015703:Antigens, CD / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D018450:Disease Progression; D006801:Humans; D039201:Integrin alpha3beta1 / Q000378:metabolism*; D039161:Integrin alpha6beta4 / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D019308:Palmitic Acid / Q000737:chemistry*; D011485:Protein Binding; D060245:Tetraspanin 29
D015703:Antigens, CD; C554869:CD9 protein, human; C440028:IGSF8 protein, human; D039201:Integrin alpha3beta1; D039161:Integrin alpha6beta4; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C486331:PTGFRN protein, human; D060245:Tetraspanin 29; D019308:Palmitic Acid