Identification of Ret motifs required for development

Columbia University Health Sciences, New York NY

Investigators

Abstract

Ret receptor tyrosine kinase (RTK) is necessary for kidney and enteric nervous system (ENS) development. Ret gain-of function mutations cause cancer, whereas loss-of-function mutations are the basis of congenital megacolon disease. Two alternatively spliced isoforms, Ret9 and Ret5 1, differ in their C-terminal tails. Knock-in mice expressing Ret9 alone are normal, whereas mice expressing Ret51 alone are characterized by defects in the ENS and kidney - specifically reduced growth and branching of collecting ducts. The specific goal of this proposal is to identify the structural motif in Ret9 that confers the ability to support normal development. One aim focuses on proteins binding to pY1062, a multipurpose docking site for signaling proteins. Residues N-terminal to pYlO62 are identical in both isoforms, but splicing leads to unique C-terminal binding specificities. We propose to mutate the C-terminal binding site of Ret51 to that of Ret9 in a knock-in mouse to determine its effect on development. A second approach focuses on a PDZ domain consensus binding site in the Ret9 C-terminal tail. We propose to add a PDZ domain binding site to Ret5 1 in a knock-in mouse to determine whether the presence of a PDZ binding domain is sufficient to rescue normal development. In addition, experiments will be performed to demonstrate the importance of this PDZ binding domain to Ret isoform localization, and to clone PDZ domain proteins that interact with Ret9 during kidney development. These experiments will enhance the understanding of the role of Ret isoforms during the development of the ENS and kidney.