Agonist-induced phosphorylation of the serotonin 5-HT2C receptor regulates its interaction with multiple PDZ protein 1

Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63-68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620-23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.     

The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins

The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.     

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.     

Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule.

Claudins, most of which end in valine at their COOH termini, constitute tight junction (TJ) strands, suggesting that TJ strands strongly attract PDZ-containing proteins. Indeed, ZO-1, -2, and -3, each of which contains three PDZ domains, were shown to directly bind to claudins. Using the yeast two-hybrid system, we identified ZO-1 and MUPP1 (multi-PDZ domain protein 1) as binding partners for the COOH terminus of claudin-1. MUPP1 has been identified as a protein that contains 13 PDZ domains, but it has not been well characterized. In vitro binding assays with recombinant MUPP1 confirmed the interaction between MUPP1 and claudin-1 and identified PDZ10 as the responsible domain for this interaction. A polyclonal antibody specific for MUPP1 was then generated. Immunofluorescence confocal microscopy as well as immunoelectron microscopy with this antibody revealed that in polarized epithelial cells MUPP1 was exclusively concentrated at TJs. Furthermore, in vitro binding and transfection experiments showed that junctional adhesion molecule, another TJ adhesion molecule, also bound to the PDZ9 domain of MUPP1. These findings suggested that MUPP1 is concentrated at TJs in epithelial cells through its binding to claudin and junctional adhesion molecule and that it may function as a multivalent scaffold protein that recruits various proteins to TJs.     

The coxsackievirus and adenovirus receptor interacts with the multi-PDZ domain protein-1 (MUPP-1) within the tight junction

The coxsackievirus and adenovirus receptor (CAR) is a component of the epithelial cell tight junction. In a yeast two-hybrid screen we identified the multi-PDZ domain protein MUPP1 as an interaction partner for the CAR cytoplasmic domain. CAR and MUPP1 were found to colocalize at the tight junction, to coprecipitate from epithelial cells, and to interact in vitro. The interaction was found to specifically involve the PDZ-binding motif within the CAR C terminus and MUPP1 PDZ domain 13. In transfected cells, CAR recruited MUPP1 to cell-cell contacts. The inhibition of CAR expression with small interfering RNA inhibited MUPP1 localization to the tight junction. The results indicated that CAR interacts with MUPP1 and is involved in MUPP1 recruitment to the tight junction.     

The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1

Tech is a RhoA guanine nucleotide exchange factor (GEF) that is highly enriched in hippocampal and cortical neurons. To help define its function, we have conducted studies aimed at identifying partner proteins that bind to its C-terminal PDZ ligand motif. Yeast two hybrid studies using the Tech C-terminal segment as bait identified MUPP1, a protein that contains 13 PDZ domains and has been localized to the post-synaptic compartment, as a candidate partner protein for Tech. Co-transfection of Tech and MUPP1 in human embryonic kidney 293 cells confirmed that these full-length proteins interact in a PDZ-dependent fashion. Furthermore, we confirmed that endogenous Tech co-precipitates with MUPP1, but not PSD-95, from hippocampal and cortical extracts prepared from rat brain. In addition, immunostaining of primary cortical cultures revealed co-localization of MUPP1 and Tech puncta in the vicinity of synapses. In assessing which PDZ domains of MUPP1 mediate binding to Tech, we found that Tech can bind to either PDZ domain 10 or 13 of MUPP1 as mutation of both these domains is needed to disrupt their interaction. Taken together, these findings demonstrate that Tech binds to MUPP1 and suggest that it regulates RhoA signaling pathways in the vicinity of synapses.     

Using a collection of MUPP1 domains to investigate the similarities of neurotransmitter transporters C-terminal PDZ motifs

A ubiquitous feature of neurotransmitter transporters is the presence of short C-terminal PDZ binding motifs acting as important trafficking elements. Depending on their very C-terminal sequences, PDZ binding motifs are usually divided into at least three groups; however this classification has recently been questioned. To introduce a 3D aspect into transporter’s PDZ motif similarities, we compared their interactions with the natural collection of all 13 PDZ domains of the largest PDZ binding protein MUPP1. The GABA, glycine and serotonin transporters showed unique binding preferences scattered over one or several MUPP1 domains. On the contrary, the dopamine and norepinephrine transporter PDZ motifs did not show any significant affinity to MUPP1 domains. Interestingly, despite their terminal sequence diversity all three GABA transporter PDZ motifs interacted with MUPP1 domain 7. These results indicate that similarities in binding schemes of individual transporter groups might exist. Results also suggest the existence of variable PDZ binding modes, allowing several transporters to interact with identical PDZ domains and potentially share interaction partners in vivo.     

Claudin-8 interacts with multi-PDZ domain protein 1 (MUPP1) and reduces paracellular conductance in epithelial cells.

The claudin family is a set of integral membrane proteins found at cell-cell interactions in tight junctions. To identify proteins that interact with claudin-8, we used the yeast two-hybrid system to search for binding partners. Using the C-terminal 37 amino acids of claudin-8 as bait, we screened a human kidney cDNA library and identified multi-PDZ domain protein 1 (MUPP1) as a claudin-8 binding protein. MUPP1 contains 13 PDZ domains and binds to claudin-8 though its PDZ9 domain. When MDCK cells were transfected with epitope-tagged claudin-8 or MUPP1, both molecules were concentrated at cell-cell junctions. The interaction of claudin-8 and MUPP1 in vivo was confirmed by co-immunolocalization and co-immunoprecipitation in MDCK cells. Expression of claudin-8-myc increased transepithelial electrical resistance (TER) and reduced paracellular flux using FITC-dextran as a tracer. Over-expression of FLAG-MUPP1 in MDCK cells also reduced the epithelial paracelhular conductance. Our results indicate that claudin-8 and MUPP1 interact in tight junctions of epithelial cells and are involved in the tight junction barrier function.     

大鼠MUPP1蛋白PDZ8结构域的生化及晶体结构特性

多重PDZ结构域蛋白1型（MUPP1）是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明: MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 kD.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I 型PDZ 结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.     

Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain

hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ- protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2- mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.     

Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.     

The PDZ protein mupp1 promotes Gi coupling and signaling of the Mt1 melatonin receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.     

PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor

Taking advantage of three cellular systems, we established that 5-HT(2B) receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line and Mastomys natalensis carcinoid cells, which naturally express the 5-HT(2B) receptor, as well as in transfected LMTK(-) fibroblasts, stimulation of the 5-HT(2B) receptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT(2B) receptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT(2B) receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT(2B) peptide in the three cell types but also in LMTK(-) fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT(2B)/iNOS coupling mechanisms appear more complex because neutralization of endogenous Galpha(13) by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT(2B) receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor.     

Molecular basis for the recognition of adenomatous polyposis coli by the Discs Large 1 protein

The human Discs Large 1 (DLG1) protein uses two of its three PDZ domains to interact with the C-terminal peptide of the Adenomatous Polyposis Coli (APC) tumor suppressor protein. The DLG1/APC complex inhibits the cell cycle progression from the G0/G1 to the S phase, regulates epithelial cell migration and morphogenesis, and is required for polarization of the microtubule cytoskeleton. However, the molecular details of how DLG1 recognizes APC is not clear. In this study, we performed biochemical and biophysical assays to investigate the interactions between PDZ domains of DLG1 and the C-terminal peptide of APC. In addition, we determined the crystal structures of the PDZ1 and PDZ2 domains of DLG1 each in complex with the C-terminal 11-residue peptide of APC. Our biochemical, biophysical, and structural results revealed structural elements and residues on PDZ1 and PDZ2 domains of DLG1 and on APC crucial for their mutual interaction. In particular, our results show that the β2/β3 loops of PDZ1 and PDZ2 play important roles in contributing to the binding affinities between PDZ domains and APC, through interacting with the residues upstream of the canonical PDZ-binding S/T-X-V motif. The results provide new insights into the binding mode of a defined C-terminal segment of APC by the PDZ domains of DLG1.     

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.     

An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for understanding the interaction networks they establish. We determined the binding preference of the five PDZ domains in the protein tyrosine phosphatase PTP-BL by screening a random C-terminal peptide lambda phage display library. Interestingly, the potential of PDZ2 to interact with class III-type ligands was found to be modulated by the presence of PDZ1. Structural studies revealed a direct and specific interaction of PDZ1 with a surface on PDZ2 that is opposite the peptide binding groove. Long-range allosteric effects that cause structural changes in the PDZ2 peptide binding groove thus explain the altered PDZ2 binding preference. Our results experimentally corroborate that the molecular embedding of PDZ domains is an important determinant of their ligand binding specificity.     

Outer membrane protein 25-a mitochondrial anchor and inhibitor of stress-activated protein kinase-3

Stress-activated protein kinase-3 (SAPK3) is unique amongst the mitogen-activated protein kinase (MAPK) family with its C-terminal 5 amino acids directing interaction with the PDZ domain-containing substrates alpha1-Syntrophin and SAP90/PSD95. Here, we identify three additional PDZ domain-containing binding partners, Lin-7C, Scribble, and outer membrane protein 25 (OMP25). This latter protein is localised together with SAPK3 at the mitochondria but it is not a SAPK3 substrate. Instead, OMP25 inhibits SAPK3 activity towards PDZ domain-containing substrates such as alpha1-Syntrophin and substrates without PDZ domains such as the mitochondrial protein Sab. This is a new mechanism for the regulation of SAPK3 and suggests that its intracellular activity should not be solely assessed by its phosphorylation status.     

Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells

To determine the intracellular signaling mechanism of the 5-HT(2C) receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.     

Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells

We previously reported a 120-kDa phosphoprotein that translocated from cytosol to the apical membrane of gastric parietal cells in association with stimulation of HCl secretion. To determine the molecular identity of the protein, we performed molecular cloning and expression of the protein. Immunoblot analysis showed that this protein was highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. We named this protein "parchorin" based on its highest enrichment in parietal cells and choroid plexus. We obtained cDNA for parchorin from rabbit choroid plexus coding a protein consisting of 637 amino acids with a predicted molecular mass of 65 kDa. The discrepancy in size on 6% SDS-polyacrylamide gel electrophoresis is considered to be due to its highly acidic nature (pI = 4.18), because COS-7 cells transfected with parchorin cDNA produced a protein with apparent molecular mass of 120 kDa on 6% SDS-polyacrylamide gel electrophoresis. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels (CLIC), especially the chloride channel from bovine kidney, p64, in the C-terminal 235 amino acids. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, existed mainly in the cytosol. Furthermore, when Cl(-) efflux from the cell was elicited, GFP-parchorin translocated to the plasma membrane. These results suggest that parchorin generally plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport.