Biochemical characterization of mutant EGF receptors expressed in the hemopoietic cell line BaF/3

Abstract

The Epidermal Growth Factor (EGF) receptor appears to require a fully active tyrosine kinase domain to transmit mitogenic signals. However, waved-2 mice carrying a mutation in the alpha-helix C of their EGF-R, which abolishes tyrosine kinase activity, only display a mild phenotype and are fully viable. This suggests that the mutant EGF-R signals through heterodimerization with endogenous, kinase active members of the EGF-R family such as ErbB-2 or ErbB-4. We have examined the biochemistry of EGF-Rs carrying mutations in the alpha-helix C of the human EGF-R (V741G and Y740F), in the ATP binding site (K721R) and at the C-terminus (CT957), by expression in BaF/3 cells which are devoid of EGF-R family members. The in vitro kinase activity of the alpha-helix C EGF-R mutants was severely impaired as a result of reduced phosphotransfer activity without appreciable changes in the affinity for either ATP or peptide substrate. Surprisingly, EGF stimulation of cells carrying the different mutant or wild type EGF-Rs resulted in tyrosine phosphorylation of EGF-R proteins; this phosphorylation was abolished in crude plasma membrane preparations, and appears to be due to activation of a membrane-associated or a cytosolic kinase. Receptor-mediated internalization of EGF was profoundly suppressed in the V741G, K721R and CT957 receptor mutant, and high affinity EGF binding was undetectable in the V741G and K721R receptors. We conclude that specific residues in the C-helix of the EGF-R kinase are essential for full kinase activity; mutations in this region do not affect ATP binding, but impair the receptors' phosphotransfer ability. High affinity binding of EGF is not dependent on tyrosine kinase activity or sequences in the C-terminus.

The tumor-specific de2–7 epidermal growth factor receptor (EGFR) promotes cells survival and heterodimerizes with the wild-type EGFR

“…Like BaF/3.D2-7 À2 cells, BaF/3 cells transfected with wt EGFR and stimulated with ligand can proliferate at IL-3 concentrations insufficient to induce proliferation of the parental cell line (Walker et al, 1998a). Interestingly, the EGF-dependent survival of BaF/3 cells expressing the wt EGFR was completely abrogated by PD 098059, implicating MAPK as a keysignalling molecule in survival (Walker et al, 1998b).…”

“…Parental and transfected BaF/3 cells were maintained in RPMI 1640 (GibcoBRL, Grand Island, NY, USA), 10% FCS (CSL, Melbourne, Australia), 2 mM glutamine (Sigma Chemical Co., St Louis, MO, USA), penicillin/streptomycin (GibcoBRL, Grand Island, NY, USA) and 10% WEHI 3BD À -conditioned medium as a source of IL-3. The generation of BaF/3 cells expressing the wt EGFR has been described previously (Walker et al, 1998a). The vector pRc/RSV, which contains wt EGFR cDNA, was cotransfected with a hygromycin-resistance vector (pTK/ Hyg) into BaF/3 cells expressing the de2-7 EGFR to generate BaF/3 cells expressing both receptors.…”

Molecular mechanisms underlying ErbB2/HER2 action in breast cancer

“…(3) Genetic studies implicate essential roles for ErbB1 in both homo-and heterodimeric complexes. Mice harboring a kinase defective mutant of ErbB1, thus unable to form active ErbB1 homodimers but able to form active heterodimers, exhibit a rather mild phenotype which is very similar to that for mice mutant for the ErbB ligand TGFalpha (Fowler et al, 1995;Luetteke et al, 1994;Mann et al, 1993;Walker et al, 1998). However ErbB1 knockout mice exhibit extremely severe embryonic abnormalities, implicating an additional requirement for ErbB1 heterodimeric complexes in some developmental processes (Sibilia and Wagner, 1995;Threadgill et al, 1995).…”

The Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor AG1478 Increases the Formation of Inactive Untethered EGFR Dimers

“…BaF/3.wt is an interleukin-3-dependent murine hemopoietic cell line that has been transfected to overexpress functional wt EGFR, which is capable of inducing cell proliferation in response to ligand (31). BaF/3.V741G and BaF/3.K721R overexpress two kinase-impaired mutants (31,32). 293T cells, which expresses low levels of endogenous wt EGFR (Ͻ1 ϫ 10 4 EGFR per cell), were transfected to overexpress wt EGFR (293T.wt) or EGFR.⌬CR1 (293T.⌬CR1) (23,33).…”