The research in our laboratory is aimed at structural characterization of the molecular mechanisms of cell-cell interactions and signal transduction in the nervous system. We are particularly interested in understanding the processes of generating correct neuronal connectivity in the developing mammalian CNS. We use x-ray crystallography combined with other biophysical and biochemical techniques to study axon guidance molecules and their interactions with neuronal cell-surface receptors.
Axon guidance molecules are molecules outside the growth cone that assist in selection of the correct pathway and target by changing growth direction. Axon guidance cue readout and interpretation present an attractive system for studying both molecular recognition and signal transduction. Our efforts have been focused on the genetically well-characterized ligand/receptor systems, including ephrins/Eph receptor tyrosine kinases, netrins/DCC (UNC-5), and semaphorins/neuropilins.
Structure of the Ligand-binding Domain of Receptor Tyrosine Kinase EphB2
Eph receptors represent the largest subfamily of receptor tyrosine kinases (RTKs). The Eph RTKs and their ligands, the ephrins, play important roles during the formation of many embryonic boundaries. They are predominantly expressed in the developing and adult nervous system, where they are involved in contact-mediated axon guidance, axon fasciculation, and cell migration.
Understanding Eph signaling is, therefore, essential for development of methods for treatment of brain and spinal cord injuries. In addition to their roles in nervous system development, the Eph receptors and ephrins were recently shown to be molecular determinants that distinguish arteries and veins. Their actions are very similar to those of the angiopoietins and their endothelial-specific RTKs, the Ties, thus putting the Eph RTKs center-stage in the search for anticancer strategies.
Eph receptors are unique among other RTKs in that they fall into 2 subclasses with distinct ligand specificities and can also function as ligands activating bi-directional signaling. We recently determined the crystal structure of the ligand-binding domain of EphB2. We further employed structure-based mutagenesis to locate the ligand-binding site on the surface of the receptor.
The netrins are secreted diffusible proteins that form gradients in the developing embryo to guide migrating axons to their targets. They are bifunctional molecules, which can act both as attractants and repellents, and are structurally and functionally conserved in worms, flies, and vertebrates. The netrin signal is read by 2 classes of transmembrane receptors: repulsion-specific (UNC-5) and attraction-specific (UNC-40 or Deleted in Colorectal Cancer) receptors.
The semaphorins constitute a large family of axon guidance molecules found in both insects and vertebrates. Their distinctive feature is a 500 amino acid semaphorin domain, strictly required for activity, and characterized by 16 conserved cysteins. The semaphorins are grouped in 7 subclasses that include both transmembrane and secreted members. The neuropilins, a family of membrane-associated proteins, were recently identified as semaphorin receptors.