The goal of this K02 proposal is to allow the Principal Investigator to understand cellular signaling mechanisms for vascular pattern formation in heart development, providing him with greater than 75% of time that he will devote to research over the next five years. Parallels between plexin signaling in the nervous system and in the developing cardiovascular system have only recently become clear. Plexins and associated proteins are involved in congenital heart defects (CHD), which remains a significant challenge to pre- and neonatal health care. Specifically the Principal Investigator will focus on elucidating similarities and differences in functional interactions of plexins-A1, -B1 and -D1 with small GTPases. He will try to identify novel, vasculature specific binding partners. The long term aim is to understand how similar mechanisms are used in different cells and cellular functions, and how signaling specificity arises from the details of the molecular interactions. This basic knowledge will eventually allow the design polypeptides or other agents that can be used to monitor and manipulate cell signaling events, diagnose and affect CHDs early via gene therapy. The complex formation between the small GTPase Rac1 (and homologous Rnd1 protein) and the cytoplasmic region of plexin-B1 is one of the first documented cases of a direct interaction between a small GTPase and a transmembrane receptor. A range of biophysical studies on the Rac1 binding cytoplasmic domain of plexin-B1 are funded by NIGMS R01 grant (GM073071). Stimulated by the new reports of plexins role in cardiovascular development, the Principal Investigator would like to expand the project to the characterization of the structure and function of plexin-A1 and -D1 receptors.
Specific aims of the K02 project are: 1) Characterization of the GTPase binding and regulatory domains in the cytoplasmic regions of plexin-A1 and -D1, including modeling or structure determination by NMR/X-ray. 2) Identification of other plexin-B1 and -D1 cytoplasmic domain binding proteins by yeast two hybrid screening and computational modeling. 3) Manipulation of GTPase binding affinity and specificity by use of site directed mutagenesis and computational modeling. As part of the K02 project new techniques will be introduced into an already interdisciplinary laboratory. The Principal Investigator will undergo additional training and interactions with cellular and cardiovascular biologists will be expanded.
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