ABSTRACT
The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Spermatozoa/metabolism , Swine/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Animals , Antibodies , Cell Line , Gene Expression Profiling , Gene Expression Regulation, Developmental , Humans , Male , Mice , Multidrug Resistance-Associated Proteins/genetics , Protein Processing, Post-Translational , Protein Transport , Rats , Spermatozoa/cytology , Testis/cytology , Tissue Distribution , TransfectionABSTRACT
Alkylphospholipids, such as the antitumor ether lipid 1-O-octadecyl-2-O-methylglycero-3-phosphocholine, modulate cancer cell invasion through changes in the adherens junction E-cadherin complex, a major organizer of epithelia. We wanted to know whether alkylphospholipids would also change tight junctions, molecular complexes that seal cell-to-cell contacts in polarised epithelia. Therefore, human colorectal cancer cell layers T84 were established in two-compartment culture chambers and the functional integrity of tight junctions was evaluated through their transepithelial electrical resistance. Incorporation of alkylphospholipids causes a rapid and reversible decrease of transepithelial electrical resistance. This decrease is due to an increased paracellular permeability and is temperature-independent. Unlike methyl-beta-cyclodextrin, alkyl-phospholipids do not specifically displace lipids from raft-like membrane domains. Nevertheless, alkylphospholipids change the detergent-solubility of zonula occludens-protein and occludin. Our data, together with the literature, indicate that non-toxic doses of alkylphospholipids affect more than one cell-cell adhesion complex, probably through their incorporation into the plasma membrane lipid bilayer.
Subject(s)
Antineoplastic Agents/pharmacology , Phospholipid Ethers/pharmacology , Phosphorylcholine/analogs & derivatives , Tight Junctions/drug effects , beta-Cyclodextrins , Blotting, Western , Cell Membrane/drug effects , Cell Membrane/metabolism , Cholesterol/metabolism , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Cyclodextrins/pharmacology , Detergents/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Mannitol/pharmacokinetics , Microscopy, Confocal , Phosphorylcholine/pharmacology , Precipitin Tests , Tritium , Tumor Cells, CulturedABSTRACT
BACKGROUND: Transforming growth factor-beta (TGF-beta) initiates intracellular signalling by inducing the formation of a heteromeric complex between TGF-beta type I (TbetaR-I) and TGF-beta type II serine/threonine kinase receptors (TbetaR-II). After the activation of TbetaR-I kinase by TbetaR-II kinase, specific receptor-regulated Smads (R-Smads) are phosphorylated by TbetaR-I kinase. Smad anchor for receptor activation (SARA), which contains a FYVE finger domain, regulates the subcellular localization of R-Smads and presents them to TbetaR-I. However, it is unclear where SARA is localized in the cell and which phospholipid(s) interacts with its FYVE domain. RESULTS: Wild-type SARA and the FYVE domain of SARA (FYVE(SARA)) reveal a punctate staining pattern and co-localize with the early endosomal markers, early endosomal antigen-1 (EEA1) and hepatic growth factor-regulated tyrosine kinase substrate (Hrs). The ectopic expression of dominant negative rab5, a critical regulatory molecule in endosome function, redistributes SARA from punctate to a diffuse cytosolic staining pattern. A lipid binding assay demonstrated that the recombinant FYVE domain from SARA predominantly interacts with phosphatidylinositol 3-phosphate (PtdIns(3)P). Consistent with this result, wortmannin, a PI3 kinase inhibitor, resulted in both a redistribution of SARA from the endosomal compartment to the cytosol and the attenuation of both TGF-beta-induced R-Smad phosphorylation and transcriptional activation. Ectopic expression of the FYVE domain of SARA also induced the redistribution of wild-type SARA and inhibited TGF-beta as well as BMP/Smad-induced transcriptional responses. CONCLUSION: The FYVE domain is sufficient and necessary for the early endosomal localization of SARA, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-beta/Smad signalling.
