Analysis of Heregulin-Induced ErbB2 Phosphorylation with a High-Throughput Kinase Receptor Activation Enzyme-Linked Immunosorbant Assay
✍ Scribed by Michael D. Sadick; Mark X. Sliwkowski; Andrew Nuijens; Laura Bald; Nancy Chiang; Julie A. Lofgren; Wai Lee T. Wong
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 178 KB
- Volume
- 235
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
factor, heregulin (HRG) 2 (6). Heregulin and its rodent A rapid, sensitive, and high-throughput assay has homolog, neu differentiation factor (NDF; 7, 8), were been developed to quantify ligand-induced receptor originally purified based on their ability to stimulate tyrosine kinase activation in terms of receptor phosthe phosphorylation of a 185-kDa protein in the breast phorylation. The assay, termed a kinase receptor acticarcinoma cell lines MCF-7 and MDA-453, respecvation enzyme-linked immunosorbant assay (KIRAtively. Alternatively spliced forms of the HRG/NDF ELISA), consists of two separate microtiter plates, one gene are now known to account for the potent Schwann for cell culture, ligand stimulation, and cell lysis/recell mitogen, glial growth factor (GGF; 9, 10), and aceceptor solubilization and the other plate for receptor tylcholine receptor-inducing activity (ARIA; 11, 12). In capture and phosphotyrosine ELISA. The assay was response to heregulin, ErbB2 functions in conjunction developed for analysis of heregulin-induced ErbB2 acwith at least two other closely related receptors, ErbB3 tivation and utilizes the stimulation of intact receptor and ErbB4. The ErbB2 molecule likely associates as a on the adherent breast carcinoma cell line, MCF-7. hetero-oligomer with ErbB3 and/or ErbB4 (13, 14). Membrane proteins are solubilized via Triton X-100 ly-While heregulin does not bind directly to ErbB2, it does sis and the receptor is captured in ELISA wells coated interact with ErbB3 (15, 16) and/or ErbB4 (17-19).