RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity.

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.

Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.

Identification and characterization of GFRalpha-3, a novel Co-receptor belonging to the glial cell line-derived neurotrophic receptor family.

A new family of neuronal survival factors comprised of glial cell line-derived neurotrophic factor (GDNF) and neurturin has recently been described (Kotzbauer, P. T., Lampe, P. A., Heuckeroth, R. O., Golden, J. P., Creedon, D. J., Johnson, E. M., Jr., and Milbrandt, J. (1997) Nature 384, 467-470). These molecules, which are related to transforming growth factor-beta, are important in embryogenesis and in the survival of distinct neuronal populations. These molecules signal through a novel receptor system that includes the Ret receptor tyrosine kinase, a ligand (i.e. GDNF or neurturin), and an accessory glycosyl-phosphatidylinositol-linked molecule that is responsible for high affinity binding of the ligand. Two accessory molecules denoted GDNF family receptor 1 and 2 (GFRalpha-1 and GFRalpha-2) have been described that function in GDNF and neurturin signaling complexes. We have identified a novel co-receptor belonging to this family based on similarity to GFRalpha-1, which we have named GFRalpha-3. GFRalpha-3 displays 33% amino acid identity with GFRalpha-1 and 36% identity with GFRalpha-2. Despite the similarity of GFRalpha-3 to GFRalpha-1 and GFRalpha-2, it is unable to activate Ret in conjunction with GDNF, suggesting that there are likely additional undiscovered ligands and/or Ret-like receptors to be identified. GFRalpha-3 is anchored to the cell membrane by a phosphatidylinositol-specific phospholipase C-resistant glycosyl-phosphatidylinositol linkage. GFRalpha-3 is highly expressed by embryonic day 11 but is not appreciably expressed in the adult mouse. In situ hybridization analyses demonstrate that GFRalpha-3 is located in dorsal root ganglia and the superior cervical sympathetic ganglion. Comparison of the expression patterns of GFRalpha-3 and Ret suggests that these molecules could form a receptor pair and interact with GDNF family members to play unique roles in development.

Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.

Glial cell line-derived neurotrophic factor signals through the RET receptor and activates mitogen-activated protein kinase.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta family of growth factors, was first identified by its ability to promote the survival of midbrain dopaminergic neurons in culture. We demonstrate that GDNF treatment of several neuroblastoma cell lines leads to dose-dependent tyrosine phosphorylation of the RET receptor and that other transforming growth factor-beta family members are not able to activate the RET receptor. GDNF treatment of neuroblastoma cells also results in increased transcription of an Elk luciferase reporter gene, suggesting that GDNF activates the mitogen-activated protein kinase signal transduction pathway.

Oncogenic RET receptors display different autophosphorylation sites and substrate binding specificities.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild-type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities.

Transcriptional vs. posttranscriptional control of neuropeptide Y gene expression.

Human neuropeptide Y (NPY) gene expression occurs exclusively in the central and peripheral nervous systems requiring complex cell-specific regulation. In this study we have examined the effect of modulating the second messenger systems involving protein kinase A and protein kinase C on the expression of the NPY gene in different neuronal cell types. We report that the effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin on a neuroblastoma cell line (LA-N-5) and a pheochromocytoma cell line (PC12) are mediated through both increased transcription of the NPY gene and through stabilization of NPY messenger RNA (mRNA). After 8 h of treatment TPA and forskolin increase the steady-state level of NPY mRNA 10- and 12-fold in LA-N-5 and PC12 cells, respectively. This response in neuroblastoma cells is due to an increase in the half-life of NPY mRNA. The response in PC12 cells is mediated by both increased mRNA stability and increased transcription. Transient transfection analyses using PC12 cells indicate that only 51 base pairs 5' to the transcription start site are necessary for the TPA and forskolin induced transcriptional response. Thus, these experiments demonstrate that TPA and forskolin effect the regulation of the NPY gene via transcriptional and posttranscriptional mechanisms in a cell-specific manner.

Transcriptional regulation of the human neuropeptide Y gene by nerve growth factor.

Neuropeptide Y (NPY) is the most ubiquitously expressed peptide in the mammalian nervous system. Transcription of the NPY gene in PC12 cells is regulated by a number of agents, including the neurotrophic peptide nerve growth factor (NGF). In this paper, we define the cis-acting promoter elements which respond to NGF and characterize the trans-acting factors which interact with these sequences. The NGF-responsive elements of the NPY gene lie between nucleotides -87 and -36. At least four proteins interact with this promoter region. One of these proteins interacts with a CT-rich sequence centered at position -51, which closely abuts a binding site for transcription factor AP-2 centered at position -63. Two newly characterized factors bind between positions -87 and -58. These proteins are expressed in a tissue-specific manner and, together with the other binding activities, modulate the transcriptional activity of the NPY gene. These results suggest that the concerted interplay of these proteins, in response to NGF, increases the transcriptional activity of the NPY gene.