The Eph receptors represent the largest family of receptor tyrosine kinases identified to date, with fourteen structurally similar Eph receptors now identified in the human genome. Eph receptors and ephrins are both anchored to the cell surface and play a key role in cell-cell communication, regulating such processes as tissue patterning, axonal guidance, and synaptic plasticity, angiogenesis and tumorigenesis. Both the Eph receptors and the ephrins can be divided into two classes based on sequence conservation. There are nine human EphA receptors and five ephrin-A ligands in the A class and there are five human Eph receptors and three ephrin-B ligands in the B class. The ephrins of the A class are GPI-anchored while the ephrins of the B class function as transmembrane proteins that contain conserved carboxy-terminal cytoplasmic domains. Interestingly, there is high binding promiscuity in the interactions between Eph receptors and ephrins of the same class, and several Eph receptors/ephrins can also mediate interactions between classes. The purpose of Core A is to provide protein expression and purification services to support the crystallographic, NMR, biochemical, and cell culture studies of the program project. The Eph Protein Production Core will generate purified Eph receptor and ephrin protein domains for Project 1-3. The Core will use both baculovirus and bacterial expression systems to produce protein domains that will be purified for crystallization trials, NMR experiments, biochemical, cell culture and in vivo studies. Filtration and column-based puriflcation methodologies will be used to generate purified proteins and Eph receptor/ephrin complexes. Size-exclusion chromatography, dynamic light scattering and mass spectrometry techniques will be used to assess complex formation and homogeneity and monodispersity of the purified proteins. Furthermore, the Core will design, generate and store wild-type and mutant Eph receptor and ephrin protein constructs. Relevant data from protein production runs will be stored on a SharePoint Server to enable data sharing amongst project participants

Public Health Relevance

Significant expertise and effort are required to express and purify Eph receptors and ephrin protein domains for structural and biochemical studies to define the structural features of Eph receptor-ephrin binding specificity and their importance for tumor growth. Facilitating these studies through a centralized protein production core facility will also help the development of chemical compounds and peptides that will serve as potential cancer therapeutic starting points.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
1P01CA138390-01A1
Application #
7800001
Study Section
Special Emphasis Panel (ZCA1-GRB-P (O1))
Project Start
2009-12-01
Project End
2014-11-30
Budget Start
2009-12-01
Budget End
2011-01-31
Support Year
1
Fiscal Year
2010
Total Cost
$189,140
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
020520466
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Stammes, Marieke A; Prevoo, Hendrica A J M; Ter Horst, Meyke C et al. (2017) Evaluation of EphA2 and EphB4 as Targets for Image-Guided Colorectal Cancer Surgery. Int J Mol Sci 18:
Singec, Ilyas; Crain, Andrew M; Hou, Junjie et al. (2016) Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling. Stem Cell Reports 7:527-542
Bhaskar, Archana; Tiwary, Bhupendra Nath (2016) Hypoxia inducible factor-1 alpha and multiple myeloma. Int J Adv Res (Indore) 4:706-715
Pasquale, Elena B (2016) Exosomes expand the sphere of influence of Eph receptors and ephrins. J Cell Biol 214:5-7
Barquilla, Antonio; Lamberto, Ilaria; Noberini, Roberta et al. (2016) Protein kinase A can block EphA2 receptor-mediated cell repulsion by increasing EphA2 S897 phosphorylation. Mol Biol Cell 27:2757-70
Singh, Deo R; Pasquale, Elena B; Hristova, Kalina (2016) A small peptide promotes EphA2 kinase-dependent signaling by stabilizing EphA2 dimers. Biochim Biophys Acta 1860:1922-8
Singh, Deo R; Cao, QingQing; King, Christopher et al. (2015) Unliganded EphA3 dimerization promoted by the SAM domain. Biochem J 471:101-9
Wu, Bainan; Wang, Si; De, Surya K et al. (2015) Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells. Chem Biol 22:876-887
Barquilla, Antonio; Pasquale, Elena B (2015) Eph receptors and ephrins: therapeutic opportunities. Annu Rev Pharmacol Toxicol 55:465-87

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