Der Ko-Rezeptor CD44 v6: Ein Beitrag zur Signaltransduktion und Etablierung einer CD44-negativen Maus

Abstract

The cell adhesion molecule CD44 is involved in many cellular processes. Beside its function in cell-matrix adhesion CD44 is involved in the formation of a signaling complex involving the isoform CD44 v6, the receptor tyrosin kinase (RTK) c-Met and the ERM-protein ezrin in many cell types. Indeed, the CD44 tail recruits ERM proteins as well as the cytoskeleton in order to promote signaling from the c-Met RTK. In order to test whether the CD44 tail is there to bring ERM proteins to the membrane, fusion proteins were generated in which ezrin was fused directly to the transmembrane domain of CD44 v6. These fusion proteins were able to promote signaling indicating that ERM proteins have to be maintained at the membrane to fulfill their function. The cytoplasmic domain of CD44 seems to be responsible to bring ezrin into a complex with c-Met. To determine the role of ezrin in the signaling complex, this protein was modified in different ways in the fusion protein. Deletion of the F-actin binding domain in ezrin blocked HGF/SF induced signaling pointing towards an essential role of actin in this process. The aim of the second part of my PhD work was to establish a conditional knockout of CD44 in mice. The classical CD44 knockout mouse showed no overt phenotype, whereas a transgenic antisense knockout resulted in a drastic phenotype. To determine the role of CD44 in embryogenesis and to test whether a substitution had occurred in the classical knockout mouse, a conditional knockout mouse should be generated. A construct containing exon 3 flanked by LoxP sites included in genomic DNA was transfected into ES cells. ES cell clones harboring homologous integration of the LoxP sites containing sequences were injected into blastocytes and transferred into surrogate mothers. Five chimeric mice were born, the strongest chimera, a female, died after three weeks. The remaining mice were crossed with BL/6 mice to establish a homozygote CD44 knockout mouse line. The next step is the inactivation of CD44 by crossing the homozygote knockout mouse with a Cre recombinase expressing mouse. The deletion of exon 3 results in the production of a nonsense-mRNA which is degraded by NMD (nonsense-mediated mRNA decay). The expression of the Cre recombinase should be under the control of a time specific inducible or tissue specific promoter. These crossings will help to explain the functions of CD44 in embryogenesis.