title: Protein 4.1-mediated membrane targeting of human discs large in epithelial cells.
abstract: Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R.
authors.affiliation: Department of Medicine, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Hanada
authors.forename: Atsuko
authors.initials: A
authors.lastname: Takeuchi
authors.forename: Gautam
authors.initials: G
authors.lastname: Sondarva
authors.forename: Athar H
authors.initials: AH
authors.lastname: Chishti
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D000074288:Discs Large Homolog 1 Protein; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005091:Exons; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D051528:Guanylate Kinases; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009479:Neuropeptides; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D009703:Nucleoside-Phosphate Kinase; C496468:DLG2 protein, human; D051528:Guanylate Kinases