Direct linkage of EGF to its receptor: Characterization and biological relevance

Abstract

A small portion of the 125I-EGF that binds specifically to intact cells or isolated membrane from a variety of sources becomes directly and irreversibly linked to EGF receptors. This provides a simple technique for affinity labeling the EGF receptor. Membranes isolated from the human epidermoid carcinoma cell line A431, which possesses extraordinarily high numbers of EGF receptors, gave rise to three major direct linkage complexes of MW = 160,000, 145,000, and 115,000. The time course for information of each is similar, showing that 125I-EGF can form direct linkage complexes with several preexisting forms of the EGF receptor. The direct linkage of EGF to receptor is slow in comparison to 125I-EGF binding, but both processes have similar susceptibilities to competition by unlabeled EGF. EGF was modified chemically with the amino site-specific reagent, N-hydroxysuccinimidyl biotin. The biotinyl-EGF had a reduced capacity to engage in direct linkage complex formation with no concomitant reduction in its ability to bind to EGF receptors. Since native and biotinyl EGF have identical abilities to stimulate the uptake of 3H-thymidine into DNA when incubated with cultured murine 3T3 cells, the direct linkage of EGF to its receptor does not appear to play an important role in EGF-stimulated mitogenesis.

Effect of oxidative iodination of epidermal growth factor on its binding and secretion by hepatocytes

“…However, a portion of intact EGF in bile was associated with a 66 kD band (unpublished observations), analogous to the band observed in Figure 5 (with native, unlabeled EGF). A similar 66 kD protein with which EGF formed a covalent-like association was observed by Linsley and Fox [5] in 3T3 cell membranes, and was assumed to represent a breakdown product of the higher molecular weight EGF receptor.…”

Preclinical modeling of EGFR inhibitor resistance in head and neck cancer

“…T24 is derived from a transitional bladder carcinoma 44 and A431 is an epidermoid carcinoma of the vulva. 45 UM-22A and UM-22B cells were a generous gift from Dr Tom Carey (University of Michigan) and PCI-52 and PCI-15B cells were a generous gift from Dr Theresa Whiteside (University of Pittsburgh). HeLa cells are a cervical cancer cell line maintained in DMEM + 10% FBS.…”

Antibody microarray and immunoblotting analyses of the EGF signaling phosphorylation network in human A431 epidermoid carcinoma cells

“…It should be noted that all of these phosphosite-specific antibodies had undergone negative purification with phosphotyrosine-agarose twice in order to reduce non-specific phosphotyrosine-binding. This crossreactivity with phosphosite-specific antibodies may reflect the very high level of over-expression of the EGF receptor in A431 cells and the extensive phosphorylation of this receptor in response to EGF [5,6]. Databases such as PhosphoNET (www.phosphonet.ca) and PhosphoSitePlus (www.phosphosite.org) [3] document at least 32 serine-, 23 tyrosine-, and 17 threonine-phosphorylation sites that have been identified in the human EGF receptor by mass spectrometry studies, and phosphorylation is often also induced at serine and threonine sites in addition to tyrosine sites with engagement of the EGF receptor with its ligands.…”

“…To preserve the phosphorylation of proteins in lysates from cells and tissues, the inclusion of cocktails of protein phosphatase and protease inhibitors in homogenization buffers has been routinely used for over three decades [4]. To explore how well this practice actually worked, we focused on tracking the phosphorylation of the EGF receptor and downstream signalling proteins in A431 cells, which over-express this receptor-tyrosine kinase and undergo apoptosis with longer term exposure to EGF [5][6][7][8][9][10]]. …”