The RET Receptor Is Linked to Stress Response Pathways

Abstract

RET is a transmembrane receptor required for the development of neuroendocrine and urogenital cell types. Activation of RET has roles in cell growth, migration, or differentiation, yet little is known about the gene expression patterns through which these processes are mediated. We have generated cell lines stably expressing either the RET9 or RET51 protein isoforms and have used these to investigate RET-mediated gene expression patterns by cDNA microarray analyses. As seen for many oncogenes, we identified altered expression of genes associated generally with cellcell or cell-substrate interactions and up-regulation of tumor-specific transcripts. We also saw increased expression of transcripts normally associated with neural crest or other RET-expressing cell types, suggesting these genes may lie downstream of RET activation in development. The most striking pattern of expression was up-regulation of stress response genes. We showed that RET expression significantly up-regulated the genes for heat shock protein (HSP) 70 family members, HSPA1A, HSPA1B, and HSPA1L. Other members of several HSP families and HSP70-interacting molecules that were associated with stress response protein complexes involved in protein maturation were also specifically up-regulated by RET, whereas those associated with the roles of HSP70 in protein degradation were down-regulated or unaffected. The major mechanism of stress response induction is activation of the heat shock transcription factor HSF1. We showed that RET expression leads to increased HSF1 activation, which correlates with increased expression of stress response genes. Together, our data suggest that RET may be directly responsible for expression of stress response proteins and the initiation of stress response.

Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

“…Constructs were validated for binding of known RET substrates (data not shown). Site-specific RET mutants were generated in WT-RET constructs by overlapping PCR, as described (15). Glutathione S-transferase (GST) fusion constructs encoding residues 664 to 1,072 of WT-RET and 2B-RET (with the M918T mutation) were generated in a modified pGEX-4T-3 vector.…”

“…Constructs were verified by direct sequencing (Cortec, Kingston, Ontario, Canada). Expression and GST fusion constructs for GFRa1 (15) Protein purification and biophysical analyses. Purification of GST-RET proteins and biophysical analyses are described in Supplementary Methods.…”

“…Constructs were transiently transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Full-length RET constructs were cotransfected with a GFR1 expression construct (15) and treated with 100 ng/mL GDNF (Promega, Madison, WI) for 15 minutes before harvesting. AP20187 dimerizer (1 Amol/L; ARIAD, Cambridge, MA) was added to induce icRET dimerization 30 minutes before harvesting.…”

“…Total protein lysates were harvested 48 hours after transfection and suspended in 20 mmol/L Tris-HCl (pH 7.8), 150 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 1% Igepal, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mL aprotonin, and 10 Ag/mL leupeptin (15). Protein assays were carried out using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL).…”

RET ligand-induced internalization and its consequences for downstream signaling

“…(a) SH-SY5Y neuroblastoma cells were transiently transfected with GFRa1 using DIMRIE C (Invitrogen, Burlington, ON, Canada), treated with retinoic acid for 24 h, serum-starved overnight, then stimulated with 50 ng/ml GDNF (PeproTech Canada Inc., Ottawa, ON, Canada) for the indicated times. Proteins were harvested in Igepal lysing buffer, as previously described (Myers and Mulligan, 2004), separated by SDS-PAGE, transferred to nitrocellulose membrane (Bio Rad, Mississaugua, ON, Canada) and probed for either RET (H300, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), phospho-RET (a-Phospho-Ret ( . FRET was measured using the FRET-sensitized emission wizard of the Leica Confocal Spectrum Express 03 software package in conjunction with a Leica TCS SP2 inverted confocal microscope.…”