PDGFR – HCMV GB · Structural Studies of the Human Cytomegalovirus Glycoprotein B Activation Mechanism of PDGFR alpha

Structural Studies of the Human Cytomegalovirus Glycoprotein B Activation Mechanism of PDGFR alpha

During the funded period, my laboratory brought to conclusion two projects. The first project, structural investigations of KIT, was a continuation of the PI postdoctoral fellowship. The second project focuses on structure-function investigations of the Slit-Robo signaling system. KIT: A broad range of fundamental cellular processes including cell proliferation, differentiation, cell survival and metabolism are mediated by signaling pathways that are activated by the 58 members of the receptor tyrosine kinases (RTK) family of membrane receptors. The receptor tyrosine kinase KIT was initially discovered as the viral oncogenic protein of a feline sarcoma virus. Subsequent studies have demonstrated that KIT and its ligand stem cell factor (SCF) play an important role in the control of cell proliferation, differentiation and survival of a variety of cell types including germ cells, hematopoietic cells, melanocytes, intestinal pacemaker cells and sensory neurons in the CNS. Using electron microscopy and fitting of crystal structures, we determined the three dimensional reconstruction of ligand induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions between membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions that occur during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT. Slit-Robo: Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain Socio-economic impact of the project KIT: The results from the funded research revealed the exact molecular surfaces in the extracellular juxtamembrane D4-D5 region that are most critical for KIT activation. These identified sites will be used as targets for anti-cancer neutralizing antibodies.Also, reliable structural information of the dimeric intracellular domain of KIT will allow the development of a new generation of synthetic kinase inhibitors that will be designed based on the kinase environment, rather than merely the structural properties of the isolated kinase domain. Slit-Robo: Slit-Robo deregulation is associated with many types of cancers and several neuropathologies, including schizophrenia and autism. However, some of the normal and defective functional aspects of this signaling system remain enigmatic. This gap in knowledge is at least partially due to the limited mechanistic understanding of the Robo-receptor activation process. Our overall goal is to elucidate all aspects of Slit-Robo activation and signaling and by now we have marked two notable achievements, including the crystal structure of the Robo1 juxtamembrane domains.

Data: CORDIS, © European Union