Assaying the biological role of the PKD1L1-PKD2 interaction

Polycystins are a family of transmembrane proteins found in metazoan species that play a plethora of roles, at multiple stages, throughout the lifespan of the animal organism. In human, two loci encoding polycystins, PKD1 and PKD2, were found to be causative of autosomal dominant polycystic kidney disease. Later studies identified these two genes in other organisms, such as mouse and zebrafish, which served as models for investigating the function and regulation of the two proteins. PKD1 and PKD2 behave as interacting partners and form cation-permeable channels, which are thought to control cellular proliferation. The interaction of the two proteins was shown to involve the respective coiled-coil (CC) domains of each protein, a typical protein-protein interaction domain. Later studies then determined that PKD2 is also able to interact with other PKD2 molecules through the CC domain, as well as other domains distributed throughout the protein. PKD2 has also been shown to play a role in left-right (L-R) asymmetry determination, in partnership with a PKD1 paralog, PKD1L1 (PKD1-like-1). The two have also been suggested to interact, presumably through their respective CC domains. The aim of the work described in this thesis is to test the differences and similarities between the interaction mechanisms of PKD1-PKD2 and PKD1L1-PKD2, in order to better understand how these molecules perform their function. I first investigated the molecular mechanisms involved in the PKD1L1-PKD2 interaction, through the use of biochemical techniques, by comparing the interaction of PKD2 WT or mutant proteins fragments with destabilized or truncated CCs. The results obtained suggest that the molecular mechanisms of interaction are likely different between the PKD1-PKD2 and PKD1L1-PKD2 complexes, in that the PKD1L1-PKD2 interaction does not appear to require the CC domain for interaction. I performed phenotypic analysis on embryos carrying truncation or insertion mutations in the region of Pkd2 encoding the CC region. The phenotypic data indicates, for the first time, a separation between the role played by PKD2 in L-R and kidney development. Furthermore, the results described in this thesis also indicate that the PKD2 CC performs distinct functions, at the molecular level, in different contexts.