Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor.
An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.
L
L
Beguinot
D
D
Werth
S
S
Ito
N
N
Richert
M C
MC
Willingham
I
I
Pastan
D001355:Alpharetrovirus; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D002642:Chick Embryo; D003593:Cytoplasm / Q000032:analysis; D004789:Enzyme Activation; D000939:Epitopes / Q000276:immunology; D066246:ErbB Receptors; D005347:Fibroblasts / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; D011956:Receptors, Cell Surface; D014764:Viral Proteins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases