FUNCTIONAL-ROLE OF N-GLYCOSYLATION IN ALPHA-5-BETA-1 INTEGRIN RECEPTOR - DE-N-GLYCOSYLATION INDUCES DISSOCIATION OR ALTERED ASSOCIATION OF ALPHA-5 AND BETA-1 SUBUNITS AND CONCOMITANT LOSS OF FIBRONECTIN-BINDING ACTIVITY

Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizin g the CS-1 peptide sequence of FN) integrin receptors. Integrin-depend ent cell adhesion to FN is greatly promoted by optimal G(M3) concentra tion at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji , T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-222 2), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 be ta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yam ada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studi ed the specific role of N-glycosylation in alpha 5 beta 1 function. Ke y findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetyl glucosaminidase F and peptide-N-4-(N-acetylglucosaminyl) asparagine am idase F (endo F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K56 2 cell surface tended to dissociate into alpha 5 and beta 1 subunits w hen an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, a nd the beta 1 subunit was preferentially precipitated by anti-beta 1 m onoclonal antibody ZH1. When intact cells were extracted and treated w ith either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, in dicating that the two subunits are normally tightly associated with ea ch other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosp hatidylcholine:cholesterol liposomes incorporating purified alpha 5 be ta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated wi th endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, t he alpha 5 or beta 1 subunit was precipitated separately, respectively . In contrast, both subunits were always coprecipitated when intact pu rified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. T hese findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for ass ociation of these subunits and for optimal binding to FN.