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α-arrestin-dependent endocytosis after cycloheximide treatment

ABSTRACT: To identify new plasma membrane (PM) proteins that require α-arrestins for endocytosis, we performed an iTRAQ-based quantitative proteomic screening. We analysed and compared the PM proteome for each arrestin knockout (art∆) strain to a WT strain after a 90 min cycloheximide (CHX) treatment. CHX is a translation inhibitor and can trigger the endocytosis of several transporters in a substrate-independent manner. We searched for proteins that underwent CHX-induced endocytosis in a WT strain but that remained stabilized at the PM in an art∆ strain.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Jérôme Savocco

LAB HEAD: Pierre Morsomme

PROVIDER: PXD014695 | Pride | 2019-09-25

REPOSITORIES: Pride

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			Action	DRS
	20180822_first-screening_S1R1_new-db.group	Other
	20180822_first-screening_S1R1_new-db_MGFPeaklist.mgf	Mgf
	20180822_first-screening_S1R2_new-db.group	Other
	20180822_first-screening_S1R2_new-db_MGFPeaklist.mgf	Mgf
	20180822_first-screening_S1R3_new-db.group	Other

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Project description:Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from estrogens and androgens into signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in cell adhesion, morphological tissue development, and sterol biosynthesis but suppress genes involved in growth factor signaling and cell adhesion. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were identified, and their enrichment in the glycolytic pathway was demonstrated. At the highest dose (100 nM), E2 induced genes enriched not only for cell adhesion but also steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF-β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas soluble growth factors might play significant roles when estrogen level is high. Primary culture urogenital sinus mesenchymal cells were isolated from prostate glands of gestation day 17 CD1 male mouse fetuses. Cells were then exposed to 10 pM or 100 pM 17beta-estradiol or vehicle (0.05% ethanol) for four days in the presence of 690 pM 5alpha-dihydrotestosterone. Total cellular RNA was then isolated for determination of transcriptomal profiles by Affymetrix mouse gene 1.0 ST array.

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α-arrestin-dependent endocytosis after cycloheximide treatment

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