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.

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane aquaporin-2 (AQP2) in epithelial cells stably transfected with wild-type AQP2. We now show a similar effect of K44A dynamin in LLC-PK1 cells transfected with an S256 phosphorylation-deficient AQP2 mutant, AQP2(S256A), and in AQP2-transfected inner medullary collecting duct (IMCD) cells. More acute blockade of endocytosis in these cells with the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD; 10 mM) resulted in a rapid and extensive cell-surface accumulation of both wild-type AQP2 and AQP2 (S256A) within 15 min after treatment. This effect was similar to that induced by treatment of the cells with vasopressin. Blockade of endocytosis by mbetaCD was confirmed using quantitative analysis of FITC-dextran uptake and AQP2 membrane insertion was verified by cell-surface biotinylation. These data indicate that AQP2 recycles constitutively and rapidly between intracellular stores and the cell surface in LLC-PK1 and IMCD cells. The constitutive trafficking process is not dependent on phosphorylation of the serine-256 residue of AQP2, which is, however, an essential step for regulated vasopressin/cAMP-mediated translocation of AQP2. Our data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin receptor (V2R)- and phosphorylation-independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQP2.

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr325 with Phe325 prevented ligand-induced internalization of V2R determined by [3H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction.

Before the identification of aquaporin (AQP) proteins, vasopressin-regulated "water channels" were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin-coated pits on the apical surface of collecting duct principal cells. Furthermore, by using a fracture-label technique applied to LLC-PK(1) cells expressing an AQP2-c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin-coated pits and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated before internalization.

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. αA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H2O2 oxidation in an ascorbate-FeCl3-EDTA-H2O2 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type αA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H2O2. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.

Eicosanoids regulate various cellular functions that are important in physiological and pathophysiological processes. Arachidonic acid is released from membranes by phospholipase A(2) (PLA(2)) activity. Activated macrophages derived from mice lacking the 85-kDa group IV cytosolic PLA(2) (cPLA(2)) have a markedly reduced release of prostaglandin E-2 and leukotrienes B-4 and C-4. Under basal conditions and after furosemide, urinary prostaglandin E-2 excretion is reduced in cPLA(2)-knockout (cPLA(2)(-/-)) mice. Serum creatinine, Na+, K+, and Ca2+ concentrations, glomerular filtration rate, and fractional excretion of Na+ and K+ are not different in cPLA(2)(-/-) and cPLA(2)(-/-) mice. Maximal urinary concentration is lower in 48-h water-deprived cPLA(2)(-/-) mice compared with cPLA(2)(+/+) animals (1,934 +/- 324 vs. 3,541 +/- 251 mmol/kgH(2)O). Plasma osmolality is higher (337 +/- 5 vs. 319 +/- 3 mmol/kgH(2)O) in cPLA(2)(-/-) mice that lose a greater percentage of their body weight (20 +/- 2 vs. 13 +/- 1%) compared with cPLA(2)(+/+) mice after water deprivation. Vasopressin does not correct the concentrating defect. There is progressive reduction in urinary osmolality with age in cPLA(2)(-/-) mice. Membrane-associated aquaporin-1 (AQP1) expression, identified by immunocytochemical techniques, is reduced markedly in proximal tubules of older cPLA(2)(-/-) animals but is normal in thin descending limbs. However, Western blot analysis of kidney cortical samples revealed an equivalent AQP1 signal intensity in cPLA(2)(+/+) and cPLA(2)(-/-) animals. Young cPLA(2)(-/-) mice have normal proximal tubule AQP1 staining. Collecting duct AQP2, -3, and -4 were normally expressed in the cPLA(2)(-/-) mice. Thus mice lacking cPLA(2) develop an age-related defect in renal concentration that may be related to abnormal trafficking and/or folding of AQP1 in the proximal tubule, implicating cPLA(2) in these processes.

PURPOSE:To determine which component of lens alpha-crystallin is responsible for heat-induced transition, conformational change and high molecular weight (HMW) aggregation.METHODS:Recombinant alphaA- and alphaB-crystallins were used. Temperature dependent changes were probed by Trp fluorescence and circular dichroism (CD) measurements. HMW aggregates were induced by heating at 62 degrees C for 1-2 h and then cooling to room temperature. The nature of HMW aggregation was studied with fluorescent probes, 4,4'-dianilino-1, 1'-binaphthalene-5,5'-disulfonic acid (bis-ANS) and thioflavin T (ThT).RESULTS:CD and Trp fluorescence revealed that alphaB-crystallin was more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation. At temperatures greater than 70 degrees C, alphaB-crystallin precipitated but alphaA-crystallin remained soluble. Both bis-ANS and ThT probes displayed increased fluorescence intensity with HMW aggregation, but the increase for bis-ANS was greater with alphaB-crystallin than with alphaA-crystallin, while the reverse was true for ThT.CONCLUSIONS:These results indicate that alphaB-crystallin is more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation and are consistent with the notion that alphaA- and alphaB-crystallins have different biochemical and biophysical properties in spite of their high degree of homology.

In collecting duct principal cells, aquaporin 2 (AQP2) is shuttled from intracellular Vesicles to the plasma membrane upon vasopressin (VP) stimulation. VP activates adenylyl cyclase, increases intracellular cAMP, activating protein kinase A (PKA) to phosphorylate AQP2 on the COOH-terminal residue, serine 256. Using rat kidney slices and LLC-PK1 cells stably expressing AQP2 (LLC-AQP2 cells), we now show that AQP2 trafficking can be stimulated by cAMP-independent pathways. In these systems, the nitric oxide (NO) donors sodium nitroprusside (SNP) and NONOate and the NO synthase substrate L-arginine mimicked the effect of VP, stimulating relocation of AQP2 from cytoplasmic vesicles to the plasma membrane. Unlike VP, these other agents did not increase intracellular cAMP. However, SNP increased intracellular cGMP, and exogenous cGMP stimulated AQP2-membrane insertion. Atrial natriuretic factor, which signals via cGMP, also stimulated AQP2 translocation. The VP and SNP effects were blocked by the kinase inhibitor H89. SNP did not stimulate membrane insertion of AQP2 in LLC-PK1 cells expressing the phosphorylation-deficient mutant 256SerAla-AQP2, indicating that phosphorylation of Ser256 is required for signaling. Both PKA and cGMP-dependent protein kinase G phosphorylated AQP2 on this COOH-terminal residue in vitro. These results demonstrate a novel, cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion in renal epithelial cells.

There are two tryptophan residues in the lens αB‐crystallin, Trp9 and Trp60. We prepared two Trp → Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near‐ultraviolet circular dichroism (UV CD) differently. The major difference in the near‐UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near‐UV CD shows an increased intensity in the region of 270–280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a β‐strand.

There are two tryptophan residues in the lens alphaB-crystallin, Trp9 and Trp60. We prepared two Trp --> Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near-ultraviolet circular dichroism (UV CD) differently. The major difference in the near-UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near-UV CD shows an increased intensity in the region of 270-280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a beta-strand.

PURPOSE:Lens proteins underwent nonenzymatic glycation, and the advanced glycation end products (AGEs) were detected by immunological assays. One of the major AGE structures is N(epsilon)-(carboxymethyl)lysine (CML). Since the involvement of AGEs in the pathogenesis of diabetic complications is speculated, the effects of CML formation on proteins were studied.METHODS:CML adducts were generated in recombinant alphaA- and alphaB-crystallins by incubation with glyoxylic acid and NaBH3CN. SDS-PAGE and size exclusion chromatography were used to detect subunit degradation and high-molecular-weight (HMW) aggregation. Conformational change was determined by fluorescence and circular dichroism (CD) measurements. The chaperone function was studied by DTT-induced aggregation of insulin.RESULTS:Lysine modification was estimated to be 60-90% depending on the conditions of incubation. No subunit degradation or HMW aggregation was observed. Fluorescence and CD measurements detected a conformational change in CML adducts. Measurements of chaperone-like activity, however, indicated that the formation of CML increased the protein's ability to protect insulin against DTT-induced aggregation.CONCLUSIONS:Although CML adducts of alphaA- and alphaB-crystallins, the major AGE structures formed in vitro, changed protein conformation, no subunit degradation and HMW aggregation were observed. Moreover, the CML adducts increased chaperone-like activity of both alphaA- and alphaB-crystallins. The results suggest that CML formation alone may not play a major role in protein aggregation and lens opacity.

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.

Human lenses contain many photosensitizers that absorb light at wavelengths above 300 nm, most notably UVA light (320-400 nm). Kynurenine (Kyn) and 3-hydroxykynurenine (HK), two of the best-known photosensitizers in the human lens, may play a significant role in photooxidation-related changes in lens proteins, such as conformational change and aggregation. In vitro irradiation experiments with proteins indicate that the Trp residue (with maximal absorption at 295 nm) is more susceptible to photooxidation by UVB light (280-320 nm) than by UVA light, but most UVB light below 300 nm is screened by the cornea and little reaches the lens, especially the nuclear region where nuclear color develops. Therefore, if photooxidation is an important contributor to nuclear color or nuclear cataract, it must arise from a photosensitized reaction. In the present study, we use recombinant alpha A- and its Trp-deficient mutant W9F as models to study the effects of UVA irradiation in the presence of HK or Kyn and of UVB (300 nm) irradiation on alpha-crystallins. alpha A-crystallin showed a large decrease in Trp fluorescence and a large increase in non-Trp (blue) fluorescence after the HK-sensitized or 300 nm photooxidation. For the W9F mutant, a smaller decrease in protein fluorescence (lambda ex at 280 nm) and a smaller increase in blue fluorescence than for the wild-type alpha A-crystallin were observed. A decrease in the near-UV CD was also observed for both photooxidized alpha A and the W9F mutant. The effect of Kyn sensitization is smaller than that of HK sensitization. A study of chaperone-like activity indicated that only 300 nm photooxidized alpha A and the W9F mutant increased the ability to protect insulin from dithiothreitol-induced aggregation. Thus, sensitized photooxidation can occur in amino acids other than Trp by UVA in the presence of HK or Kyn with effects similar to, albeit smaller than, those of direct UVB (300 nm) photooxidation.