A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover.
The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF-binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.
Defize
L H
LH
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht.
Arndt-Jovin
D J
DJ
Jovin
T M
TM
Boonstra
J
J
Meisenhelder
J
J
Hunter
T
T
de Hey
H T
HT
de Laat
S W
SW
D000017:ABO Blood-Group System / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001345:Autoradiography; D002294:Carcinoma, Squamous Cell; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002999:Clone Cells; D000939:Epitopes / Q000276:immunology; D066246:ErbB Receptors / Q000032:analysis* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D037102:Lectins; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D000939:Epitopes; D037102:Lectins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases