Biosynthesis and acquisition of biological activity of the fibronectin receptor

Abstract

The biosynthesis of the 140-kilodalton fibronectin receptor complex by cultured 3T3 mouse cells was characterized and compared with that of chick embryo fibroblasts. Three murine glycoprotein components of 140-150 kilodaltons (band 1), 125 kilodaltons (band 2), and 105 kilodaltons (band 3) could be immunoprecipitated from metabolically labeled 3T3 cells using polyclonal antibodies. In pulse-chase experiments, bands 1 and 3 of the mouse receptor were labeled to maximal levels immediately after completion of the labeling pulse. However, band 2 was not detected at short chase times, and it reached maximal labeling only after approximately 12 h of chase. The appearance of band 2 occurred at the same rate as the disappearance of band 3. Only bands 1 and 2 could be affinity purified by binding to immobilized fibronectin cell-binding fragment, indicating that they represent mature functional receptor components. When 3T3 cells were incubated with radioactive sugars, band 1 of the receptor labeled well with both [3H]mannose and [3H]glucosamine. However, band 2 labeled well with [3H]glucosamine but contained low amounts of mannose, and band 3 labeled well with [3H]mannose but contained low amounts of glucosamine. Digestion of both bands 2 and 3 with endoglycosidase F yielded similar-sized products of approximately 88,000 daltons, suggesting that post-translational asparagine-linked oligosaccharide processing can account for most of the size difference between these bands. These data suggest that in the mouse fibronectin receptor, band 3 is a biologically inactive precursor of band 2 that does not appear on the cell surface. In contrast, pulse-chase experiments using chicken embryo fibroblasts indicated that the three components of the chicken 140k complex were distinct moieties. Our results demonstrate distinct types of processing for fibronectin receptor complexes from different species. In mammalian cells, this receptor undergoes a surprisingly long (20 h) maturation process involving asparagine-linked oligosaccharides before reaching its final, biologically active form.