Abstract
The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.
Highlights
Riched microdomains (TERM)2) which incorporate a number of tetraspanin-interacting receptors [1]
Flow cytometry and Western blotting have shown that down-regulation of CD151 did not affect surface and total levels of ␣31 and ␣6 integrins (␣71 is not expressed in epithelial cells)
These results indicated that the light chain of the ␣3 integrin subunit, which has three putative glycosylation sites, is modified by both complex and hybrid/high mannose oligosaccharides
Summary
The MDA-MB-231 and HeLa cell lines were purchased from the Cancer Research UK. The anti-CD63 (6H1) and anti-CD151 (5C11 and 11B1G4) mouse mAbs and rabbit anti-CD151 polyclonal Ab were described previously (50 –53). The anti-CD82 (TS82) mAbs were generously provided by Dr E. To generate stable MDA-MB-231/CD151(Ϫ) and HeLa/CD151(Ϫ) cell lines, transfections were carried out using Fugene (Roche Applied Science) for HeLa or GeneJammer (Stratagene) for MDA-MB-231 cells. Transfected cells were selected and maintained in DMEM containing 0.5–1.0 g/ml puromycin. CD151-positive and -negative populations were selected by cell sorting (BDFACSVantage SE) and confirmed by flow cytometry and Western blotting. To generate MDA-MB-231/rec series (MDA-MB-231/CD151(Ϫ) cells with the reconstituted expression of the CD151 wild-type or CD151 mutants) pZeoSV-based constructs were introduced into the cells using GeneJammer and selected in growth medium containing 100 –300 g/ml Zeocin. Various reconstituted CD151 cell lines were sorted to obtain a pool of cells expressing CD151 at levels similar to that of the control MDA-MB-231
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