Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells.

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK(1) renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK(1) cells stained with anti-V2R antibodies and in LLC-PK(1) cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 microM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R.

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK(1) epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells.

Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca(2+)-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.

Cation channel activity of mucolipin-1: the effect of calcium.

Mucolipidosis type IV (MLIV) is a rare, neurogenetic disorder characterized by developmental abnormalities of the brain, and impaired neurological, ophthalmological, and gastric function. Considered a lysosomal disease, MLIV is characterized by the accumulation of large vacuoles in various cell types. Recent evidence indicates that MLIV is caused by mutations in MCOLN1, the gene that encodes mucolipin-1 (ML1), a 65-kDa protein showing sequence homology and topological similarities with polycystin-2 and other transient receptor potential (TRP) channels. In this report, our observations on the channel properties of ML1, and molecular pathophysiology of MLIV are reviewed and expanded. Our studies have shown that ML1 is a multiple sub-conductance, non-selective cation channel. MLIV-causing mutations result in functional differences in the channel protein. In particular, the V446L and DeltaF408 mutations retain channel function but have interesting functional differences with regards to pH dependence and Ca(2+) transport. While the wild-type protein is inhibited by Ca(2+) transport, mutant ML1 is not. Atomic force microscopy imaging of ML1 channels shows that changes in pH modify the aggregation and size of the ML1 channels, which has an impact on vesicular fusogenesis. The new evidence provides support for a novel role of ML1 cation channels in vesicular acidification and normal endosomal function.

Characterization of single channel currents from primary cilia of renal epithelial cells.

The primary cilium is a ubiquitous, non-motile microtubular organelle lacking the central pair of microtubules found in motile cilia. Primary cilia are surrounded by a membrane, which has a unique complement of membrane proteins, and may thus be functionally different from the plasma membrane. The function of the primary cilium remains largely unknown. However, primary cilia have important sensory transducer properties, including the response of renal epithelial cells to fluid flow or mechanical stimulation. Recently, renal cystic diseases have been associated with dysfunctional ciliary proteins. Although the sensory properties of renal epithelial primary cilia may be associated with functional channel activity in the organelle, information in this regard is still lacking. This may be related to the inherent difficulties in assessing electrical activity in this rather small and narrow organelle. In the present study, we provide the first direct electrophysiological evidence for the presence of single channel currents from isolated primary cilia of LLC-PK1 renal epithelial cells. Several channel phenotypes were observed, and addition of vasopressin increased cation channel activity, which suggests the regulation, by the cAMP pathway of ciliary conductance. Ion channel reconstitution of ciliary versus plasma membranes indicated a much higher channel density in cilia. At least three channel proteins, polycystin-2, TRPC1, and interestingly, the alpha-epithelial sodium channel, were immunodetected in this organelle. Ion channel activity in the primary cilium of renal cells may be an important component of its role as a sensory transducer.

Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway.

Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) "recycling" pathway. To examine this pathway, we expressed V2R-green fluorescent protein (GFP) in LLC-PK1a cells. V2R-GFP showed characteristics similar to those of wild-type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but it colocalized with the lysosomal marker Lysotracker after VP-induced internalization. Western blot analysis of V2R-GFP showed a broad 57- to 68-kDa band and a doublet at 46 and 52 kDa before VP treatment. After 4-h VP exposure, the 57- to 68-kDa band lost 50% of its intensity, whereas the lower 46-kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect, whereas lactacystin, a proteasome inhibitor, had no effect. Incubating cells at 20 degrees C to block trafficking from the trans-Golgi network reduced V2R membrane fluorescence, and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla.

Reversal of cystic fibrosis phenotype in a cultured Delta508 cystic fibrosis transmembrane conductance regulator cell line by oligonucleotide insertion.

Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5' to the deletion), accounts for approximately 75% of cases of the disease. In the present study, an oligonucleotide complex (CF4-CF6, 2'-0-methyl RNA-unmodified RNA oligonucleotide duplex, respectively) was used to restore CFTR function by insertion of missing bases in Delta508 CFTR mRNA from a cultured (Delta508) cell line. cAMP-activated whole-cell currents and Cl- transport were detected in CF4-CF6-treated, but not control Delta508, cells by patch-clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence (SPQ) quenching analyses, respectively. Further, the nucleotide addition in the deleted region of Delta508 CFTR was determined after amplification by RT-PCR. Insertion of UGU and replacement of U by C immediately 5' to the deletion site in Delta508 mRNA appear to have taken place, with phenotypic but not genotypic reversion in tissue culture of treated cells. The mechanism of insertion of nucleotides has yet to be determined.

Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells.

In this study, the presence of Na(+)-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na(+)-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an approximately 3:1 Na(+)/K(+) permeability-selectivity ratio. The Na(+)-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na(+) channel activity. Spontaneous and vasopressin-induced Na(+) channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the alphaENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although alphaENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on alphaENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na(+)-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na(+) transport mechanisms in the mammalian proximal nephron.

Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel.

Mucolipidosis type IV (MLIV) is an autosomal recessive neurogenetic disorder characterized by developmental abnormalities of the brain and impaired neurological, ophthalmologic and gastric function. Large vacuoles accumulate in various types of cells in MLIV patients. However, the pathophysiology of the disease at the cellular level is still unknown. MLIV is caused by mutations in a recently described gene, MCOLN1, encoding mucolipin-1 (ML1), a 65 kDa protein whose function is also unknown. ML1 shows sequence homology and topological similarities with polycystin-2 and other transient receptor potential (Trp) channels. In this study, we assessed both, whether ML1 has ion channel properties, and whether disease-causing mutations in MCOLN1 have functional differences with the wild-type (WT) protein. ML1 channel function was assessed from endosomal vesicles of null (MCOLN1(-/-)) and ML1 over-expressing cells, and liposomes containing the in vitro translated protein. Evidence from both preparations indicated that WT ML1 is a multiple subconductance non-selective cation channel whose function is inhibited by a reduction of pH. The V446L and DeltaF408 MLIV causing mutations retain channel function but not the sharp inhibition by lowering pH. Atomic force imaging of ML1 channels indicated that changes in pH modified the aggregation of unitary channels. Mutant-ML1 did not change in size on reduction of pH. The data indicate that ML1 channel activity is regulated by a pH-dependent mechanism that is deficient in some MLIV causing mutations of the gene. The evidence also supports a novel role for cation channels in the acidification and normal endosomal function.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation.

Smad4, the common Smad, is central for transforming growth factor (TGF)-beta superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-beta and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-beta-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

Human fast skeletal myosin light chain 2 cDNA: isolation, tissue specific expression of the single copy gene, comparative sequence analysis of isoforms and evolutionary relationships.

A cDNA clone encoding human fast skeletal myosin regulatory light chain (HSRLC) has been isolated and characterized from a fetal muscle cDNA library. The cDNA contains the coding sequence of 170 amino acids (aa) and 58 and 91 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. HSRLC is encoded by a single copy gene in the human genome and shows a tissue-specific pattern of expression in skeletal muscle. Comparison of derived amino acid sequence of HSRLC with database sequences reveals highly conserved 12 amino acid residues in a putative calcium-binding region. HSRLC is unique among all RLC sequences in having three consecutive potential phosphorylatable serine residues. The Cys-129 of HSRLC corresponds to the critical Gly-117 of scallop RLC that is essential for its regulatory function. The clusters of hydrophobic residues that are believed to stabilize the binding of NH2-terminal of RLC with myosin heavy chain show high sequence conservation in RLCs. Besides identifying specific targets for functional studies of HSRLC by mutagenesis, the results support the concept of an ancestral gene from which the RLC genes have evolved.