Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors.
A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr approximately 180,000 EGF-receptor complex and larger Mr greater than or equal to 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd approximately 0.16 nM) and low- (Kd approximately 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the Mr approximately 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr approximately 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr approximately 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4 degrees C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.
D003907:Dexamethasone / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D010446:Peptide Fragments; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D066246:ErbB Receptors; D014357:Trypsin