The basolateral sorting signal of the polymeric immunoglobulin receptor contains two functional domains

1996 ◽  
Vol 109 (8) ◽  
pp. 2133-2139
Author(s):  
V. Reich ◽  
K. Mostov ◽  
B. Aroeti
Keyword(s):  
Mdck Cells ◽  
Minor Effect ◽  
Polymeric Immunoglobulin Receptor ◽  
Type I ◽  
Apical Surface ◽  
Alanine Scanning Mutagenesis ◽  
Beta Turn ◽  
Immunoglobulin Receptor ◽  
Sorting Signal ◽  
Basolateral Targeting

Basolateral sorting of the polymeric immunoglobulin receptor (pIgR) expressed in Madin-Darby canine kidney (MDCK) cells is mediated by a 17-residue sorting signal that resides in the cytoplasmic domain. We have recently analyzed the sequence requirements of the signal by alanine scanning mutagenesis. We found that basolateral sorting is mediated primarily by three amino acids: H656, R657 and V660. Individual mutations of each of these residues to Ala caused a substantial decrease in basolateral sorting and a corresponding increase in targeting to the apical surface. Structural analysis of 17-residue peptides corresponding to the signal revealed that V660 is in a beta-turn (probably type I) secondary structure, and its mutation to Ala destabilized the turn. H656 and R657 were not part of the turn and substitution of Arg657 to Ala had no effect on the turn stability. These results suggested that the signal is comprised of two structurally distinct domains: a critical V660 in the context of the beta-turn and an additional two residues (H656 and R657) that are not in the turn and probably are unimportant for its stability. Here we provide evidence suggesting that the two domains are distinguishable not only by their structure but also by their function. Basolateral targeting of pIgR mutants bearing Ala mutations at either 656 or 657 was not affected by treatment with brefeldin A (BFA), while basolateral targeting of pIgR containing an Ala substitution at position 660 was markedly and uniquely stimulated by BFA. Compared to single Ala substitutions, simultaneous mutations of H656 and R657 to Ala caused an additional minor effect on basolateral and apical sorting, whereas double mutations of V660 and either H656 or R657 resulted in a maximal decrease in basolateral targeting and corresponding increase in apical targeting. These results suggest the existence of two domains in the signal. When both domains are destroyed, basolateral targeting is maximally inhibited. The results also imply that V660 mediates basolateral sorting by a different mechanism from H656 and R657. We suggest that V660 and perhaps more generally the beta-turn may interact with BFA-sensitive adaptor complexes.

scholarly journals Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

1993 ◽  
Vol 123 (5) ◽  
pp. 1149-1160 ◽  
Author(s):  
B Aroeti ◽  
P A Kosen ◽  
I D Kuntz ◽  
F E Cohen ◽  
K E Mostov
Keyword(s):  
Polymeric Immunoglobulin Receptor ◽  
Terminal Portion ◽  
Alanine Scanning Mutagenesis ◽  
Beta Turn ◽  
Immunoglobulin Receptor ◽  
Helix Structure ◽  
Targeting Signal ◽  
The Stability ◽  
Basolateral Targeting ◽  
Structure In Solution

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide.

Opposite sorting and transcytosis of the polymeric immunoglobulin receptor in transfected endothelial and epithelial cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1197-1206
Author(s):  
T. Su ◽  
K.K. Stanley
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Epithelial Cells ◽  
Cell Line ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Secretory Component ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Immunoglobulin Receptor

We have transfected a polarised endothelial cell line, ECV 304, and an epithelial cell line, MDCK, with a well characterised epithelial protein, the rat polymeric immunoglobulin receptor (pIgR), in order to study the protein sorting and transcytosis in endothelial cells. The expressed protein was normally processed and the steady state distribution between apical and basolateral surfaces was similar in both cell types. MDCK cells, however, showed a marked polarity in the delivery of newly synthesised pIgR to the cell surface, and in the release of secretory component. 88% of newly synthesised pIgR in MDCK cells was first delivered to the basolateral surface and 99% of secretory component was released from the apical surface. In contrast the basolateral targeting signal of pIgR was only partially recognised in endothelial cells, with 63% of the newly synthesised pIgR being first delivered to the basolateral surface. At steady state only 43% of the pIgR was found on the basolateral membrane. The direction of dimeric IgA transcytosis in endothelial cells was from apical to basolateral surfaces, opposite to that in MDCK cells. These data suggest that endothelial cells poorly recognise the targeting signals of proteins from epithelial cells, and that the direction of transcytosis is linked to the biological role of the cells.

scholarly journals Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells.

1989 ◽  
Vol 109 (2) ◽  
pp. 475-486 ◽  
Author(s):  
P P Breitfeld ◽  
J M Harris ◽  
K E Mostov
Keyword(s):  
Mdck Cells ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Immunoglobulin Receptor ◽  
Intracellular Sorting ◽  
Polymeric Immunoglobulins ◽  
Darby Canine Kidney ◽  
Canine Kidney

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.

scholarly journals Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells.

1990 ◽  
Vol 111 (6) ◽  
pp. 2365-2373 ◽  
Author(s):  
P P Breitfeld ◽  
W C McKinnon ◽  
K E Mostov
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Membrane Domains ◽  
Immunoglobulin Receptor ◽  
Vesicular Traffic ◽  
Protein Traffic ◽  
Microtubule Disruption ◽  
Recycling Pathway

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.

scholarly journals MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells

2002 ◽  
Vol 159 (1) ◽  
pp. 37-44 ◽  
Author(s):  
María C. de Marco ◽  
Fernando Martín-Belmonte ◽  
Leonor Kremer ◽  
Juan P. Albar ◽  
Isabel Correas ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Mdck Cells ◽  
Functional Characterization ◽  
Essential Element ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Immunoglobulin Receptor ◽  
Indirect Route ◽  
Hepatoma Hepg2

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.

scholarly journals An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells.

1987 ◽  
Vol 105 (5) ◽  
pp. 2031-2036 ◽  
Author(s):  
K E Mostov ◽  
P Breitfeld ◽  
J M Harris
Keyword(s):  
Mdck Cells ◽  
Secretory Protein ◽  
Site Directed Mutagenesis ◽  
Polymeric Immunoglobulin Receptor ◽  
Apical Surface ◽  
Basal Secretion ◽  
Madin Darby Canine Kidney ◽  
Immunoglobulin Receptor ◽  
Darby Canine Kidney ◽  
Canine Kidney

The polymeric immunoglobulin receptor is expressed in a variety of polarized epithelial cells. Newly made receptor travels first to the basolateral surface. The receptor is then endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. We have now deleted the membrane spanning and cytoplasmic portions of the receptor by site-directed mutagenesis, thus converting the receptor to a secretory protein. When expressed in polarized Madin-Darby canine kidney (MDCK) cells the truncated protein is secreted at both surfaces, with a ratio of apical-to-basal secretion of 3.4. In contrast, when the exogenous secretory protein chicken lysozyme is expressed in these cells, it is released at both sides with a ratio of apical-to-basal secretion of 0.43. (Koder-Koch, C., R. Bravo, S. Fuller, D. Cutler, and H. Garoff, 1985, J. Cell Biol., 43:297-306). Lysozyme is thought to lack a signal that targets it to one surface or the other, and so its secretion may represent a default, bulk flow pathway to both surfaces. When compared with lysozyme, the truncated polymeric immunoglobulin receptor is preferentially secreted apically by a factor of 3.4:0.43 or 7.8. We suggest that the lumenal portion of the polymeric immunoglobulin receptor contains a signal that targets it to the apical surface.

scholarly journals A novel juxtamembrane basolateral targeting motif regulates TGF-β receptor signaling in Drosophila

2020 ◽  
Author(s):  
Aidan J. Peterson ◽  
Stephen J. Murphy ◽  
Melinda G. Mundt ◽  
Maryjane Shimell ◽  
Edward B. Leof ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Wing Disc ◽  
Female Sterility ◽  
Epithelial Tissue ◽  
Type I ◽  
Apical Surface ◽  
Type Ii ◽  
Apical Side ◽  
Basolateral Targeting

AbstractIn polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the two interacting components giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue that requires the TGF-β family member Dpp for growth and patterning. Dpp protein has been observed in a gradient within the columnar cells of the disc, but also uniformly in the disc lumen leading to the question of how graded signaling is achieved in the face of two distinctly localized pools. We find the Dpp type II receptor Punt, but not the type I receptor Tkv, is enriched at the basolateral membrane, and depleted at the junctions and apical surface. Wit, a second type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies enabled us to identify the BLT, a novel, short juxtamenbrane sequence that confers basolateral targeting of Punt in both wing discs and mammalian MDCK cells. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA

Cell ◽  
1986 ◽  
Vol 46 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Keith E. Mostov ◽  
David L. Deitcher
Keyword(s):  
Mdck Cells ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor

Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

1995 ◽  
Vol 108 (1) ◽  
pp. 369-377 ◽  
Author(s):  
K.L. Soole ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
B.H. Hirst
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Gpi Anchor ◽  
Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

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