Kristy J. Wilson

Functional selectivity of EGF family peptide growth factors: Implications for cancer

Breast, prostate, pancreatic, colorectal, lung, and head and neck cancers exploit deregulated signaling by ErbB family receptors and their ligands, EGF family peptide growth factors. EGF family members that bind the same receptor are able to stimulate divergent biological responses both in cell culture and in vivo. This is analogous to the functional selectivity exhibited by ligands for G-protein coupled receptors. Here we review this literature and propose that this functional selectivity of EGF family members is due to distinctions in the conformation of the liganded receptor and subsequent differences in the sites of receptor tyrosine phosphorylation and receptor coupling to signaling effectors. We also discuss the roles of divergent ligand activity in establishing and maintaining malignant phenotypes. Finally, we discuss the potential of mutant EGF family ligands as cancer chemotherapeutics targeted to ErbB receptors.

EGFR ligands exhibit functional differences in models of paracrine and autocrine signaling.

Epidermal growth factor (EGF) family peptides are ligands for the EGF receptor (EGFR). Here, we elucidate functional differences among EGFR ligands and mechanisms underlying these distinctions. In 32D/EGFR myeloid and MCF10A breast cells, soluble amphiregulin (AR), transforming growth factor alpha (TGFα), neuregulin 2 beta, and epigen stimulate greater EGFR coupling to cell proliferation and DNA synthesis than do EGF, betacellulin, heparin-binding EGF-like growth factor, and epiregulin. EGF competitively antagonizes AR, indicating that its functional differences reflect dissimilar intrinsic activity at EGFR. EGF stimulates much greater phosphorylation of EGFR Tyr1045 than does AR. Moreover, the EGFR Y1045F mutation and z-cbl dominant-negative mutant of the c-cbl ubiquitin ligase potentiate the effect of EGF but not of AR. Both EGF and AR stimulate phosphorylation of EGFR Tyr992. However, the EGFR Y992F mutation and phospholipase C gamma inhibitor U73122 reduce the effect of AR much more than that of EGF. Expression of TGFα in 32D/EGFR cells causes greater EGFR coupling to cell proliferation than does expression of EGF. Moreover, expression of EGF in 32D/EGFR cells causes these cells to be largely refractory to stimulation with soluble EGF. Thus, EGFR ligands are functionally distinct in models of paracrine and autocrine signaling and EGFR coupling to biological responses may be specified by competition among functionally distinct EGFR ligands.

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop.

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse-chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.

UNC-89 (obscurin) binds to MEL-26, a BTB-domain protein, and affects the function of MEI-1 (katanin) in striated muscle of Caenorhabditis elegans

UNC-89 (obscurin) interacts with MEL-26, a BTB-domain protein/adaptor for cullin-3. MEL-26 colocalizes with UNC-89 at M-lines. Mutations in MEL-26, CUL-3 (cullin-3), and MEI-1 (katanin) result in a muscle phenotype similar to that of unc-89 mutants. The level of MEI-1 is reduced in unc-89 mutants, suggesting that normally UNC-89 inhibits CUL-3/MEL-26 in muscle.

Immunofluorescent Localization of Proteins in Caenorhabditis elegans Muscle

Caenorhabditis elegans is a premier model genetic system for discovering new information about the assembly and maintenance of striated muscle. The localization of a protein within a nematode muscle cell can reveal important clues to its function. In C. elegans, proteins can be localized by two different methods at the light microscopy level: GFP tagged proteins and indirect immunofluorescence. Although there are advantages and disadvantages of each method, antibodies can be used to localize proteins expressed at endogenous levels and without tags that might interfere with function. Immunolocalization requires efficient and effective methods of fixation. Here, we describe in detail two different methods for fixation of adult worms, the Nonet method and the Constant Spring method. We also discuss the advantages and the disadvantages of each, and how to choose between them. These methods are also useful for localizing proteins expressed in other cell types.

The Q43L mutant of neuregulin 2β is a pan-ErbB receptor antagonist

The ErbB4 receptor tyrosine kinase possesses both tumour suppressor and oncogenic activities. Thus pharmacological agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2β (neuregulin 2β) stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus in the present paper we hypothesize that NRG2β/Q43L may be an ErbB4 antagonist. NRG2β/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2β/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2β. In addition, NRG2β stimulation of cell proliferation requires PI3K (phosphoinositide 3-kinase) activity and NRG2β stimulates greater Akt phosphorylation than does NRG2β/Q43L. Moreover, EGFR [EGF (epidermal growth factor) receptor] kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2β and NRG2β/Q43L may differentially stimulate ErbB4 coupling to the transcriptional co-regulator YAP (Yes-associated protein). Finally, NRG2β/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2β/Q43L is a pan-ErbB antagonist. Thus we postulate that NRG2β/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2β/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.

Helping Practitioners and Researchers Identify and Use Education Research Literature.

This article introduces the Evidence-Based Teaching Guides feature. Each guide links to and summarizes research on a specific pedagogy and associated practical recommendations. Each guide consists of a website with a visual map of instructional choices, linked pages with relevant research, and an instructor checklist of recommendations.