Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression.
Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.
Lax
I
I
Rorer Biotechnology, Inc., King of Prussia, Pennsylvania.
Bellot
F
F
Honegger
A M
AM
Schmidt
A
A
Ullrich
A
A
Givol
D
D
Schlessinger
J
J
D000494:Allosteric Regulation; D000818:Animals; D002478:Cells, Cultured; D002872:Chromosome Deletion; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D008565:Membrane Proteins; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases