Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cell-cell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.
A major obstacle for studying membrane proteins by biophysical techniques is the difficulty to produce sufficient amount of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially a eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacteria and insect cells, solubilization screening, large-scale production as well as commonly used affinity chromatography purification methods. We discuss general optimisation conditions such as promoters, tags and describe current techniques that can be used in any laboratory without specialised expensive equipment. Especially for insect cells, GFP-fusions are particularly useful for localisation and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.