The RET51/FKBP52 complex and its involvement in Parkinson disease.

The tyrosine kinase receptor RET51 is expressed in distinct families of neurons where it promotes different functions. FKBP52 is an immunophilin with neuroprotective effects on different kinds of neurons. In this paper, we demonstrate that RET51 activation by both glial cell line-derived neurotrophic factor (GDNF) and NGF triggers the formation of RET51/FKBP52 complex. The substitution of the tyrosine 905 of RET51, a key residue phosphorylated by both GDNF and NGF, disrupts the RET51/FKBP52 complex. NGF and GDNF have a functional role in dopaminergic (DA) neurons where RET51 and FKBP52 are expressed with a yet undefined function. To clarify if RET51/FKBP52 complex should exert its function in DA neurons, we used an indirect approach by screening the genes encoding for RET51 and FKBP52 in a group of 30 Parkinson's disease patients. The degeneration of DA neurons is the main feature of PD, which is associated to a complex multifactorial aetiology combining environmental, age-related and genetic factors. We found a compound heterozygous carrying two mutations in RET and FKBP52 that are sufficient to disrupt the RET51/FKBP52 complex, indicating its potential role in PD.

The tyrosine kinase receptor RET interacts in vivo with aryl hydrocarbon receptor-interacting protein to alter survivin availability.

CONTEXT: RET is a tyrosine kinase transmembrane receptor expressed in two main alternative isoforms: RET9 and RET51. RET transduces a positive signal leading to survival, differentiation, or migration in the presence of its ligand glial cell line-derived neurotrophic factor, whereas in its absence a proapoptotic fragment that initiates a negative signaling for apoptosis is generated. The signal transduction mechanisms leading to apoptosis are still unclear. OBJECTIVE: To shed light on the mechanisms of RET-induced apoptosis, we searched for novel interactors of RET51. DESIGN: The "split ubiquitin yeast two-hybrid system" was used with RET51 as bait against a human brain expression library. RESULTS: We identified aryl hydrocarbon receptor-interacting protein (AIP), a cochaperone recently found mutated in pituitary adenoma patients, as a novel interactor of RET. We showed that RET interacts specifically with AIP both in mammalian cell lines and in vivo in the pituitary gland, regardless of the presence of pituitary adenoma-specific mutations. AIP and RET genes were sequenced in 28 pituitary adenoma, but no relevant mutations were found. In addition, we identified the proapoptotic domain of RET as responsible for the interaction with AIP. Finally, we demonstrated that the AIP-RET interaction does not require RET kinase activity or kinase-dependent signal transduction and that it prevents the formation of the AIP-survivin complex. CONCLUSIONS: The identification of the AIP-RET complex represents a starting point to study key cellular processes involved in RET-induced apoptosis.

An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor.

The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol.

Signaling of human frizzled receptors to the mating pathway in yeast.

Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Galpha protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors.

A small-molecule-protein interaction system with split-ubiquitin as sensor.

The identification of receptors for small molecules is of great pharmaceutical importance for drug-discovery research. Several systems for the identification of protein-small-molecule interactions have been developed in the past. These were modifications of the classical yeast two-hybrid system, relying on a transcriptional read-out following nuclear translocation of the complex. Here we present a novel three-hybrid technology based on the split-ubiquitin system for the analysis of protein-small-molecule interactions independently of a nuclear translocation of the complex. The performance of the system is compared to a method based on the classical yeast two-hybrid system by using a chemical inducer of dimerization (CID) comprised of methotrexate linked to dexamethasone. Steric issues are addressed by varying the linker length of the compounds, as well as by comparing the orientation of fusion proteins. The system is further extended to the analysis of a small-molecule inhibitor of human PCTAIRE protein kinase 3, which is related to cyclin-dependent kinases (CDKs), an important class of pharmaceutical targets.

The Golgi localization of Arabidopsis thaliana beta1,2-xylosyltransferase in plant cells is dependent on its cytoplasmic and transmembrane sequences.

To investigate the targeting of proteins to the plant Golgi we studied Arabidopsis thaliana beta1,2-xylosyltransferase (XylT), a glycosyltransferase which is unique to plants and some invertebrates. Different deletion constructs of the putative cytoplasmic (C)-transmembrane (T)-stem (S) region of the enzyme were transiently expressed in the tobacco-related model plant species Nicotiana benthamiana. Subcellular localization of fusion proteins between CTS, CT, T, or C domains and the reporter molecule green fluorescent protein by fluorescence microcopy and density-gradient centrifugation revealed that the CT region alone is sufficient to sustain Golgi retention of XylT without the contribution of any luminal sequences. The finding of an incomplete retention by the T region alone suggests an important auxiliary role of the C domain in Golgi retention of the protein. However, the C segment did not confer any Golgi retention by itself, as the respective fusion protein was found exclusively in the cytoplasm. These results provide evidence that plant and mammalian cells rely on similar mechanisms to deliver glycosyltransferases to the Golgi apparatus.