BIOSYNTHESIS, SURFACE EXPRESSION AND FUNCTION OF THE FIBRONECTIN RECEPTOR AFTER RAT-LIVER CELL-TRANSFORMATION TO TUMORIGENICITY

Zajdela hepatoma cells are poorly-adherent cells derived from an undif ferentiated tumour and transplanted into rat. We compared the biosynth esis, structure and function of the fibronectin receptor in normal rat hepatocytes with that in Zajdela hepatoma cells. The rat hepatocyte f ibronectin receptor has been isolated. It is composed of two subunits: alpha5 (molecular mass 155 kDa) and beta1 (molecular mass 115 kDa). H owever, its biosynthesis has not yet been described. Using polyclonal antibodies raised against each of the subunits of the receptor, we obs erved that the alpha5-subunit was sythesized as a 155-kDa polypeptide in normal rat hepatocytes and Zajdela hepatoma cells. In contrast, the molecular mass of the beta1-subunit was 130 kDa in Zajdela hepatoma c ells versus 115 kDa in normal rat hepatocytes. Pulse-chase experiments showed that the apparent transition time from the 100-kDa beta1-precu rsor to the 130-kDa mature form was abnormally prolonged in Zajdela he patoma cells since the latter was not detected until 24 h, while the t ransition from the 100-kDa precursor to the 115-kDa mature form began within 3 h in normal rat hepatocytes. Digestion of both the normal rat hepatocytes and Zajdela hepatoma cells 100-kDa beta1-precursors with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase yielded products from 100 kDa to 84 kDa and 82 kDa, respectively, as judged by SDS/PAGE, suggesting that the same polypeptide chain is synthesized i n normal rat hepatocytes and in Zajdela hepatoma cells. Incubation of the mature normal rat hepatocyte beta1-subunit with peptide N-glycosid ase reduced its molecular mass from 115 kDa to 82 kDa, as judged by SD S/PAGE, while the molecular mass of the abnormal mature Zajdela hepato ma cell beta1-subunit decreased from 130 to 110 kDa. Thus, in addition to alterations in the Asn-linked oligosaccharide processing, 'ascitic growth' induced other post-translational modifications in the Zajdela hepatoma cell beta1-subunit. Furthermore, both the abnormal mature 13 0-kDa and precursor 100-kDa beta1-subunits were detected on the surfac e of Zajdela hepatoma cells, associated with the alpha5-subunit. The r elationship between these structural alterations in the fibronectin re ceptor and the impaired Zajdela hepatoma cell binding to soluble fibro nectin or to a coated fibronectin matrix that was observed in this stu dy is discussed.