WITHDRAWN: RhoB-dependent modulation of early endocytic traffic in Madin-Darby canine kidney cells.
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Journal Article↗
Biomedical subjects
Rebecca P Hughey
Publications and source records attributed to Rebecca P Hughey.
Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the gamma-subunit.
Epithelial sodium channels (ENaC) are expressed in the apical membrane of high resistance Na(+) transporting epithelia and have a key role in regulating extracellular fluid volume and the volume of airway surface liquids. Maturation and activation of ENaC subunits involves furin-dependent cleavage of the ectodomain at two sites in the alpha subunit and at a single site within the gamma subunit. We now report that the serine protease prostasin further activates ENaC by inducing cleavage of the gamma subunit at a site distal to the furin cleavage site. Dual cleavage of the gamma subunit is predicted to release a 43-amino acid peptide. Channels with a gamma subunit lacking this 43-residue tract have increased activity due to a high open probability. A synthetic peptide corresponding to the fragment cleaved from the gamma subunit is a reversible inhibitor of endogenous ENaCs in mouse cortical-collecting duct cells and in primary cultures of human airway epithelial cells. Our results suggest that multiple proteases cleave ENaC gamma subunits to fully activate the channel.
Animals↗
The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit.
Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.
Animals↗
Mechanism underlying flow stimulation of sodium absorption in the mammalian collecting duct.
Vectorial Na(+) absorption across the aldosterone-sensitive distal nephron plays a key role in the regulation of extracellular fluid volume and blood pressure. Within this nephron segment, Na(+) diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial Na(+) channel (ENaC), which is considered to be the rate-limiting step for Na(+) absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net Na(+) absorption and that increases in laminar shear stress activate ENaC expressed in oocytes by increasing channel open probability. We therefore examined whether flow stimulates net Na(+) absorption (J(Na)) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated J(Na) in CCDs were mediated by ENaC, as J(Na) was inhibited by benzamil. Flow-dependent increases in J(Na) were observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal Ca(2+) concentration ([Ca(2+)]) or chelating intracellular [Ca(2+)] with BAPTA did not prevent the flow-dependent increase in J(Na). Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced J(Na) when tubules were perfused at a slow flow rate. However, trypsin did not further enhance J(Na) in CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive J(Na) in oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in J(Na) accompanying increases in luminal flow rates reflects an increase in channel open probability.
Amiloride↗
Recycling of MUC1 is dependent on its palmitoylation.
MUC1 is a mucin-like transmembrane protein expressed on the apical surface of epithelia, where it protects the cell surface. The cytoplasmic domain has numerous sites for phosphorylation and docking of proteins involved in signal transduction. In a previous study, we showed that the cytoplasmic YXXphi motif Y20HPM and the tyrosine-phosphorylated Y60TNP motif are required for MUC1 clathrin-mediated endocytosis through binding AP-2 and Grb2, respectively (Kinlough, C. L., Poland, P. A., Bruns, J. B., Harkleroad, K. L., and Hughey, R. P. (2004) J. Biol. Chem. 279, 53071-53077). Palmitoylation of transmembrane proteins can affect their membrane trafficking, and the MUC1 sequence CQC3RRK at the boundary of the transmembrane and cytoplasmic domains mimics reported site(s) of S-palmitoylation. [3H]Palmitate labeling of Chinese hamster ovary cells expressing MUC1 with mutations in CQC3RRK revealed that MUC1 is dually palmitoylated at the CQC motif independent of RRK. Lack of palmitoylation did not affect the cold detergent solubility profile of a chimera (Tac ectodomain and MUC1 transmembrane and cytoplasmic domains), the rate of chimera delivery to the cell surface, or its half-life. Calculation of rate constants for membrane trafficking of wild-type and mutant Tac-MUC1 indicated that the lack of palmitoylation blocked recycling, but not endocytosis, and caused the chimera to accumulate in a EGFP-Rab11-positive endosomal compartment. Mutations CQC/AQA and Y20N inhibited Tac-MUC1 co-immunoprecipitation with AP-1, although mutant Y20N had reduced rates of both endocytosis and recycling, but a normal subcellular distribution. The double mutant chimera AQA+Y20N had reduced endocytosis and recycling rates and accumulated in EGFP-Rab11-positive endosomes, indicating that palmitoylation is the dominant feature modulating MUC1 recycling from endosomes back to the plasma membrane.
Animals↗
Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition.
Epithelial Na+ channels (ENaC) are inhibited by extracellular Na+, a process referred to as Na+ self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in alpha, or one site in gamma, blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant alpha-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+ self-inhibition response, while channels with a mutant gamma-subunit showed a modestly enhanced Na+ self-inhibition response. Analysis of Na+ self-inhibition at varying [Na+] indicates that channels containing mutant alpha-subunits exhibit an increased Na+ affinity. At the single-channel level, channels with a mutant alpha-subunit had a low open probability (P(o)) in the presence of a high external [Na+] in the patch pipette. P(o) dramatically increased when trypsin was also present, or when a low external [Na+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na+ self-inhibition and that activation requires that the alpha-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC P(o) and extracellular [Na+], supporting the notion that Na+ self-inhibition reflects a P(o) reduction due to high extracellular [Na+].
Allosteric Regulation↗
Role of N- and O-glycans in polarized biosynthetic sorting.
The maintenance of proper epithelial function requires efficient sorting of newly synthesized and recycling proteins to the apical and basolateral surfaces of differentiated cells. Whereas basolateral protein sorting signals are generally confined to their cytoplasmic regions, apical targeting signals have been identified that localize to luminal, transmembrane, and cytoplasmic aspects of proteins. In the past few years, both N- and O-linked glycans have been identified as apical sorting determinants. Glycan structures are extraordinarily diverse and have tremendous information potential. Moreover, because the oligosaccharides added to a given protein can change depending on cell type and developmental stage, the potential exists for altering sorting pathways by modulation of the expression pattern of enzymes involved in glycan synthesis. In this review, we discuss the evidence for glycan-mediated apical sorting along the biosynthetic pathway and present possible mechanisms by which these common and heterogeneous posttranslational modifications might function as specific sorting signals.
Animals↗
Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles.
The human mucin MUC1 is expressed both as a transmembrane heterodimeric protein complex that recycles via the trans-Golgi network (TGN) and as a secreted isoform. To determine whether differences in cellular trafficking might influence the O-glycosylation profiles on these isoforms, we developed a model system consisting of membrane-bound and secretory-recombinant glycosylation probes. Secretory MUC1-S contains only a truncated repeat domain, whereas in MUC1-M constructs this domain is attached to the native transmembrane and cytoplasmic domains of MUC1 either directly (M0) or via an intermitting nonfunctional (M1) or functional sperm protein-enterokinase-agrin (SEA) module (M2); the SEA module contains a putative proteolytic cleavage site and is associated with proteins receiving extensive O-glycosylation. We showed that MUC1-M2 simulates endogenous MUC1 by recycling from the cell surface of Chinese hamster ovary (CHO) mutant ldlD14 cells through intracellular compartments where its glycosylation continues. The profiles of O-linked glycans on MUC1-S secreted by epithelial EBNA-293 and MCF-7 breast cancer cells revealed patterns dominated by core 2-based oligosaccharides. In contrast, the respective membrane-shed probes expressed in the same cells showed a complete shift to patterns dominated by sialyl core 1. In conclusion, glycan core profiles reflected the subcellular trafficking pathways of the secretory or membranous probes and the modifying activities of the resident glycosyltransferases.
Amino Acid Sequence↗
Gamma-glutamyltranspeptidase: disulfide bridges, propeptide cleavage, and activation in the endoplasmic reticulum.
gamma-Glutamyltranspeptidase (gammaGT) is found primarily on the apical surface of epithelial and endothelial cells, where it degrades reduced and oxidized glutathione (gamma-GluCysGly) by hydrolysis of the unique gamma-glutamyl bond. Glutathione plays a key role in disulfide rearrangement in the endoplasmic reticulum (ER) and acts as a redox buffer. Previous work has shown that overexpression of gammaGT or an inactive splice variant gammaGTDelta7 mediates a redox stress response in the endoplasmic reticulum (ER) characterized by increased levels of BiP and induction of CHOP-10. To determine whether a CX(3)C motif might be the common feature of gammaGT and gammaGTDelta7 that mediates this response, we characterized disulfide bridges in gammaGT that might form between the six highly conserved Cys residues. Using site-directed mutagenesis of gammaGT, expression in Chinese Hamster Ovary (CHO) cells, metabolic labeling, and immunoblotting, our data predict disulfide formation between Cys49 and Cys73 and between Cys191 and Cys195 (the CX(3)C motif). Potential functions for this CX(3)C motif are discussed. In the course of defining the disulfides, we also noted that propeptide cleavage correlated with enzymatic activity. Because recent reports indicate that the homologous Escherichia coli gammaGT is a member of the N-terminal nucleophile (Ntn) hydrolase family, where the amino acid at the new N-terminus functions as the nucleophile for both autocatalytic cleavage and enzymatic activity, the rat gammaGT was similarly characterized. As predicted, mutations at the propeptide cleavage site coincidentally inhibit both heterodimer formation and gammaGT enzymatic activity. Analysis of early cleavage events using cell extraction into SDS indicates that propeptide cleavage occurs while gammaGT is still within the ER. Because activation and cleavage are coincident events, this raises the new question of whether an active glutathionase is present within the ER and what role gammaGT plays in modulating ER glutathione levels that are so critical for proper redox balance and disulfide formation in this compartment.
Animals↗
MUC1 membrane trafficking is modulated by multiple interactions.
MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [(35)S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr(20) and Tyr(60) were both required for efficient endocytosis. Mutation of Tyr(20) significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y(20)HPM is recognized as a YXXphi motif by the mu2 subunit. The tyrosine-phosphorylated Y(60)TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr(60) blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1.
Adaptor Protein Complex 2↗
Distinct pools of epithelial sodium channels are expressed at the plasma membrane.
The epithelial Na+ channel (ENaC) is assembled in the endoplasmic reticulum from three structurally related subunits (alpha, beta, and gamma). Channel maturation within the biosynthetic pathway involves cleavage of the alpha and gamma subunits by furin and processing of N-linked glycans on alpha, beta, and gamma to complex type. Both mature and immature subunits have been observed at the surface of stably transfected Madin-Darby canine kidney cells. We have examined whether channel maturation is an all-or-none event or whether heterogeneous processing of channel subunits occurs within an individual channel complex. Using an immobilized lectin to isolate proteins with complex type N-glycans, we found that individual channel complexes with mature subunits lack immature subunits. Furthermore, terminal processing of N-glycans on ENaC subunits was not dependent on cleavage of ENaC subunits, and proteolysis of channel subunits was not dependent on prior processing of N-glycans. Our results suggest that processing of subunits within an individual channel complex is an all-or-none event such that channels present on the cell surface contain either all mature or all immature subunits. The presence of immature channel complexes at the plasma membrane provides epithelial cells with a reserve of poorly functional channels that can be activated by proteases in post-Golgi compartments.
Animals↗
Epithelial sodium channels are activated by furin-dependent proteolysis.
Epithelial Na(+) channels (ENaCs) are activated by extracellular trypsin or by co-expression with channel-activating proteases, although there is no direct evidence that these proteases activate ENaC by cleaving the channel. We previously demonstrated that the alpha and gamma subunits of ENaC are cleaved during maturation near consensus sites for furin cleavage. Using site-specific mutagenesis of channel subunits, ENaC expression in furin-deficient cells, and furin-specific inhibitors, we now report that ENaC cleavage correlates with channel activity. Channel activity in furin-deficient cells was rescued by expression of furin. Our data provide the first example of a vertebrate ion channel that is a substrate for furin and whose activity is dependent on its proteolysis.
Amino Acid Substitution↗
Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits.
The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.
Animals↗
Multiple epithelial Na+ channel domains participate in subunit assembly.
Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus laevis oocyte expression system was used to identify regions within the ENaC alpha-subunit that confer a dominant negative phenotype on functional expression of alphabetagamma-ENaC to define domains that have a role in subunit-subunit interactions. Coexpression of full-length mouse alphabetagamma-ENaC with either 1) the alpha-subunit first membrane-spanning domain and short downstream hydrophobic domain (alpha-M1H1); 2) alpha-M1H1 and its downstream hydrophilic extracellular loop (alpha-M1H1-ECL); 3) the membrane-spanning domain of a control type 2 transmembrane protein (glutamyl transpeptidase; gamma-GT) fused to the alpha-ECL (gamma-GT-alpha-ECL); 4) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the alpha-subunit second membrane-spanning domain and short upstream hydrophobic domain (Tac-alpha-H2M2); or 5) the alpha-subunit cytoplasmic COOH terminus (alpha-Ct) significantly reduced amiloride-sensitive Na+ currents in X. laevis oocytes. Functional expression of Na+ channels was not inhibited when full-length alphabetagamma-ENaC was coexpressed with either 1) the alpha-ECL lacking a signal-anchor sequence, 2) alpha-M1H1 and alpha-Ct expressed as a fusion protein, 3) full-length gamma-GT, or 4) full-length Tac. Furthermore, the expression of ROMK channels was not inhibited when full-length ROMK was coexpressed with either alpha-M1H1-ECL or alpha-Ct. Full-length FLAG-tagged alpha-, beta-, or gamma-ENaC coimmunoprecipitated with myc-tagged alpha-M1H1-ECL, whereas wild-type gamma-GT did not. These data suggest that multiple sites within the alpha-subunit participate in subunit-subunit interactions that are required for proper assembly of the heterooligomeric ENaC complex.
Amiloride↗