Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct.
ABSTRACT: In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n=22) or immunoblotting (n=6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.
Project description:The final adjustment of urine volume occurs in the inner medullary collecting duct (IMCD), chiefly mediated by the water channel aquaporin 2 (AQP2). With vasopressin stimulation, AQP2 accumulation in the apical plasma membrane of principal cells allows water reabsorption from the lumen. We report that FXYD1 (phospholemman), better known as a regulator of Na,K-ATPase, has a role in AQP2 trafficking. Daytime urine of Fxyd1 knockout mice was more dilute than WT despite similar serum vasopressin, but both genotypes could concentrate urine during water deprivation. FXYD1 was found in IMCD. In WT mice, phosphorylated FXYD1 was detected intracellularly, and vasopressin induced its dephosphorylation. We tested the hypothesis that the dilute urine in knockouts was caused by alteration of AQP2 trafficking. In WT mice at baseline, FXYD1 and AQP2 were not strongly co-localized, but elevation of vasopressin produced translocation of both FXYD1 and AQP2 to the apical plasma membrane. In kidney slices, baseline AQP2 distribution was more scattered in the Fxyd1 knockout than in WT. Apical recruitment of AQP2 occurred in vasopressin-treated Fxyd1 knockout slices, but upon vasopressin washout, there was more rapid reversal of apical AQP2 localization and more heterogeneous cytoplasmic distribution of AQP2. Notably, in sucrose gradients, AQP2 was present in a detergent-resistant membrane domain that had lower sedimentation density in the knockout than in WT, and vasopressin treatment normalized its density. We propose that FXYD1 plays a role in regulating AQP2 retention in apical membrane, and that this involves transfers between raft-like membrane domains in endosomes and plasma membranes.
Project description:We carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000-g fraction to the 200,000-g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000-g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000-g fractions. LC-MS/MS analysis showed that markers of "intracellular vesicles," chiefly endosomal markers, were present in both the 17,000- and the 200,000-g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000-g fractions. Proteins associated with several multiprotein complexes (e.g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000-g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000- and 200,000-g fractions are highly heterogeneous and cannot be equated with "plasma membrane" and "intracellular vesicle" fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.
Project description:The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of ?-catenin, a multifunctional protein that acts as a transcriptional coregulator in the nucleus. The purpose of this study was to identify ?-catenin-interacting proteins that might be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells, using experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 ?-catenin-binding proteins, including several known ?-catenin-binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj, and several histones). Many of the identified ?-catenin-binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II preinitiation complex. To identify sequence-specific TFs that might interact with ?-catenin, Bayes' theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with ?-catenin in the regulation of Aqp2 gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1, and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.
Project description:Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.
Project description:Large-scale phosphoproteomic analysis employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often requires a significant amount of manual manipulation of phosphopeptide datasets in the post-acquisition phase. To assist in this process, we have created software, PhosphoPIC (PhosphoPeptide Identification and Compilation), which can perform a variety of useful functions including automated selection and compilation of phosphopeptide identifications from multiple MS levels, estimation of dataset false discovery rate, and application of appropriate cross-correlation (XCorr) filters. In addition, the output files generated by this program are compatible with downstream phosphorylation site assignment using the Ascore algorithm, as well as phosphopeptide quantification via QUOIL. In this report, we utilized this software to analyze phosphoproteins from short-term vasopressin-treated rat kidney inner medullary collecting duct (IMCD). A total of 925 phosphopeptides representing 173 unique proteins were identified from membrane-enriched fractions of IMCD with a false discovery rate of 1.5%. Of these proteins, 106 were found only in the membrane-enriched fraction of IMCD cells and not in whole IMCD cell lysates. These identifications included a number of well-studied ion and solute transporters including ClC-1, LAT4, MCT2, NBC3, and NHE1, all of which contained novel phosphorylation sites. Using a label-free quantification approach, we identified phosphoproteins that changed in abundance with vasopressin exposure including aquaporin-2 (AQP2), Hnrpa3, IP3 receptor 3, and pur-beta.
Project description:Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 (Aqp2) and aquaporin-3 (Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool (ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as "transcription factor" proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5'-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5'-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS(3) technique. The newly identified proteins have been incorporated into the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).
Project description:By phosphoproteome analysis, we identified a phosphorylation site, serine 264 (pS264), in the COOH terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). In this study, we examined the regulation of AQP2 phosphorylated at serine 264 (pS264-AQP2) by vasopressin, using a phospho-specific antibody (anti-pS264). Immunohistochemical analysis showed pS264-AQP2 labeling of inner medullary collecting duct (IMCD) from control mice, whereas AQP2 knockout mice showed a complete absence of labeling. In rat and mouse, pS264-AQP2 was present throughout the collecting duct system, from the connecting tubule to the terminal IMCD. Immunogold electron microscopy, combined with double-labeling confocal immunofluorescence microscopy with organelle-specific markers, determined that the majority of pS264 resides in compartments associated with the plasma membrane and early endocytic pathways. In Brattleboro rats treated with [deamino-Cys-1, d-Arg-8]vasopressin (dDAVP), the abundance of pS264-AQP2 increased 4-fold over controls. Additionally, dDAVP treatment resulted in a time-dependent change in the distribution of pS264 from predominantly intracellular vesicles, to both the basolateral and apical plasma membranes. Sixty minutes after dDAVP exposure, a proportion of pS264-AQP2 was observed in clathrin-coated vesicles, early endosomal compartments, and recycling compartments, but not lysosomes. Overall, our results are consistent with a dynamic effect of AVP on the phosphorylation and subcellular distribution of AQP2.
Project description:The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation.
Project description:Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.
Project description:Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.