Das fokale Adhäsionsprotein Trip6 und seine nukleäre Isoform nTrip6 in der Maus - Charakterisierung und Manipulation der trip6-Genexpression zur selektiven Analyse von Trip6- und nTrip6 vermittelten Funktionen in vivo

Transduction of an extracellular signal from the plasma membrane to specific compartments in the cell requires the formation of multi protein complexes of distinct composition and localization. Adaptor proteins, such as LIM domain proteins, play an important role in the recruitment of proteins to their interaction partners within the protein network. The intracellular adhesion protein Trip6 and its smaller, nuclear isoform nTrip6 are both LIM domain proteins encoded by the gene trip6 but are localized in different cellular compartments. Trip6 regulates the formation of focal adhesion plaques at the plasma membrane while nTrip6 acts in the nucleus, as co-activator for the transcription factors AP-1 and NF-KB. Due to their specific intracellular distribution the two trip6 protein isoforms regulate different functional properties of the cell. Trip6 might modulate cell migration and adhesion and nTrip6 might regulate cell proliferation and differentiation. Any imbalance in Trip6 and nTrip6 functions might have significant consequences in pathological situations such as inflammation, cancer or metastasis. Neither the mechanism by which the two isoforms are generated, nor the in vivo relevance of Trip6 and nTrip6 functions is known. Thus, one aim of this work was to generate a conditional trip6 deficient mouse strain to study the functions of trip6 in vivo. I modified the trip6 gene by conditional gene targeting in murine embryonic stem cells. In these cells the trip6 gene was flanked by Cre-DNA-recombinase recognition sites (loxP-sites). Cre-expression leads to excision of loxP-sites flanked DNA-sequences. It is now possible to use these embryonic stem cells to generate a conditional trip6 deficient mouse line. In this mouse, both Trip6 isoforms will be knocked out. To discriminate the functions of the two isoforms in vivo, one strategy would be to reconstitute the knockout mouse with only one isoform. A prerequisite was thus to identify the mechanism of nTrip6 generation. In this work, I showed that nTrip6 is not generated by translation of an alternatively spliced trip6 mRNA but through alternative translation initiation at a secondary start site. In mutagenesis studies as well as by the use of specific morpholinos to sterically block translation initiation, I showed that the AUG codon at position 480 in the murine trip6 mRNA is utilized for the translation of nTrip6. The alternative translation initiation at this site was mRNA cap-dependent and did not require internal ribosome entry sites (IRES). Furthermore a morpholino targeting the secondary AUG and knocking down only nTrip6 repressed the TPA-induced activation of an AP-1 target promoter. Thus the selective manipulation of conventional or alternative translation initiation at the trip6 mRNA will now serve as a strategy to specifically block the generation and function of Trip6 or nTrip6, in order to study their respective roles in cell proliferation, differentiation and migration. Moreover, the knowledge about the generation of the two isoforms will be valuable to study their respective functions in vivo, by the specific rescue of the knockout mouse with either Trip6 or nTrip6.