CD9, but not other tetraspans, associates with the β1 integrin precursor

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Abstract
The molecules of the tetraspan superfamily have two unequal extracellular domains separated by four transmembrane (TM) domains. These molecules are associated on the cell surface with each other and with other partner molecules, in particular β1 integrins. We now show that CD9 associates with the precursor of the β1 integrin (preβ1). This association is detected as early as 15 min after metabolic labeling, and the use of Brefeldin A demonstrates that it does not require Golgi modifications of either CD9 or integrin β1. The specificity of this interaction is demonstrated by the fact that other tetraspans, CD63, CD81, and CD82, do not associate with preβ1, and CD9 does not associate with immature human histocompatibility leukocyte antigen class I. In order to localize the region of CD9 responsible for the association with the β1 integrin, we have generated two reciprocal chimeric CD9/CD82 molecules with the junction localized just after the third TM region. The large extracellular loop of CD9 or the fourth TM domain, or both, appear to be sufficient to mediate an association with the mature integrin with a high efficiency, compared to CD82. By contrast, association with preβ1 requires at least two regions of the molecule. Mutation of CD9 at the consensus site of the tetraspan superfamily, localized between the second and the third TM domain, did not impair the co-precipitation of preβ1. Finally, because preβ1 associates with calnexin, we have investigated a possible association of CD9 with this chaperone molecule. CD9 associates with calnexin independently of its association with the β1 integrin, suggesting that calnexin could be involved in the processing of CD9.