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Abstract

 
Abstract No.:B-A2003
Country:Canada
  
Title:THE NEURONAL ADAPTOR PROTEIN FRS3 IS A NOVEL MICROTUBULE ASSOCIATED PROTEIN
  
Authors/Affiliations:1 Todd Hryciw; 1 Todd Hryciw*; 1 Sara LeMay; 1 Sara Lemay; 1 Susan Meakin; 1 Susan Meakin;
1 Robarts Research Institute, London, ON, Canada
  
Content:Frs3 is a member of the Fibroblast Growth Factor Receptor Substrate family of adaptor proteins. Frs proteins are characterized by an N-terminal myristylation consensus sequence followed by a phosphotyrosine-binding domain, 3 (Frs3) or 4 (Frs2) tyrosines that form Grb2 docking sites and 2 tyrosines that form Shp2 docking sites when phosphorylated by an active receptor tyrosine kinase such as FGFR or the Trk family of neurotrophin receptors. Recruitment of Grb2/Shp2 leads to activation of the Ras/MAPK and PI-3K pathways. While Frs2 and Frs3 have high sequence similarity, they have differential expression patterns: Frs2 is expressed early in development and is ubiquitous, whereas Frs3 is expressed later and is restricted to the developing and mature nervous system.
Objectives: In order to help understand the function of Frs3 in the nervous system we sought to determine its subcellular locale both in vivo and in cultured neurons.
Materials and methods: Immunohistochemistry was performed on paraffin-embedded sections of various aged mouse tissues to determine the expression pattern of Frs3 protein in the developing mouse nervous system. Immunocytochemistry was performed on cultured postnatal hippocampal neurons to determine where Frs3 localizes in neurons. Subcellular fractionation techniques (differential detergent solubility, lipid raft purification, microtubule purification) were employed to further define the compartmentalization of Frs3 in transfected 293T and PC12 cells, as well as in cultured neurons and neural tissues.
Results: In the developing mouse central nervous system, Frs3 protein expression coincides with neurogenesis, peaks in the first week postnatal, and declines to a low level by adulthood. While in transfected 293T cells EGFP-Frs3 was found specifically at the plasma membrane, in neurons and neural tissue, immunocytochemistry indicated that Frs3 was predominantly intracellular and particularly concentrated in axonal tracts. Differential detergent solubility showed that transfected Frs3 in 293T cells is in an SDS-soluble compartment, whereas neuronal Frs3 exists in freely soluble and Triton X-100 soluble compartments. As is expected for a myristylated protein, transfected Frs3 in 293T cells is found almost exclusively in lipid rafts; however, in neurons, the endogenous Frs3 protein is excluded from lipid rafts. Mutating the N-terminal glycine residue to an alanine caused transfected Frs3 in 293T cells to localize to freely soluble and Triton X-100 soluble compartments and to be excluded from lipid rafts, mimicking the results observed for neuronal Frs3. Finally, since Frs3 seems to be concentrated in axonal tracts, we examined whether Frs3 is associated with microtubules. Neuronal Frs3 precipitates out with reconstituted microtubules from postnatal cortical homogenates, indicating that in neurons Frs3 is a novel microtubule-associated protein.
Conclusions: Although Frs3 has a myristylation signal sequence and localizes to lipid rafts in forced expression systems, we believe that Frs3 evades myristylation in neurons. Neuronal Frs3 is instead a soluble protein with a strong affinity for microtubules. These results suggest that the Frs3 adaptor is a novel microtubule-associated protein.
  
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