Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains of epithelial FRT cells.

Dpl is a paralog of the PrPC, whose misfolded conformer (PrPSc) is responsible for the onset of transmissible spongiform encephalopathies (TSEs), or prion diseases. It has been shown that the ectopic expression of Dpl in the brain of some lines of PrP knock out mice provokes cerebellar ataxia that can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where and under which conditions, this event may occur. In the present study we addressed this issue by analyzing the intracellular localization and the interaction between Dpl and PrPC in FRT cells stably expressing the two proteins separately, or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both singly and doubly transfected clones. Interestingly we found that they associate with DRMs or lipid rafts, from where they can be co-immunoprecipitated in a cholesterol-dependent fashion. Although the interaction between Dpl and PrPC has been suggested before, our results provide the first clear evidence that this interaction occurs in rafts and is dependent on the integrity of these membrane microdomains. Furthermore, both Dpl and PrPC could be immunoprecipitated with flotillin-2, a raft protein involved in endocytosis and cell signaling events suggesting that they share the same lipid environment.