Analysis of RET and co‐receptor Down‐Regulation

RET is a mammalian receptor tyrosine kinase that is activated by association with a protein co‐receptor GFR‐α (1–4) and its corresponding ligand (GDNF, Neurturin, Artemin or Persephin). GDNF/RET signal transduction is an indispensable component for kidney and enteric nervous system development and receptor activation has been studied in some detail. However, it is much less clear how this down‐regulation works for RET or its co‐receptor. This issue is complicated by the fact that RET is found as one of two main isoforms, RET‐9 and RET‐51, both of which appear to have distinct functions. In order to determine how RET activity is down‐regulated transcriptionally, primers were created to measure the levels of human RET, RET‐51, RET‐9 and GFRα‐2 mRNA. These primers are gene specific and have been shown to work in both RT‐PCR and real time PCR assays. Using 18S RNA primers for normalization, expression of RET and its co‐receptor have been measured in SK‐N‐SH cells. In addition to transcriptional regulation, the importance of ligand dependent receptor/co‐receptor degradation was also analyzed. A protein proposed to be involved in RET degradation, Sprouty‐1, has been cloned and both a full length Sprouty‐1 and a C’ truncation expression vector have been created. Preliminary studies have suggested that Sprouty 1 associates with RET. (Supported by NIH Grant R15‐NS048043)