Identification of the UT-A1 Urea Channel Interactome
The urea channel Slc14a2 (or UT-A1) mediates vasopressin-regulated urea transport across the inner medullary collecting duct (IMCD). Previously, UT-A1 was found to present in a high molecular weight complex, suggesting UT-A1 is involved in certain protein-protein interactions. The present study sought to identify the proteins that interact with UT-A1 in this complex for a better understanding of how UT-A1 is regulated. Rat IMCD suspensions were treated with or without V2 receptor agonist, dDAVP, followed by in-cell crosslinking using BSOCOES and detergent solubilization. Immunoprecipitation using Dynabeads coated with UT-A1 specific antibody successfully pulled down the UT-A1 proteins. In-gel digestion protocol was carried out to prepare samples for liquid chromatographic mass spectrometry analysis of tryptic peptides using a Velos-Orbitrap mass spectrometer. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with (UTA-1 antibody/preimmune IgG) >4. A total of 128 UT-A1 interacting proteins were identified. Gene Ontology analysis maps the distribution of these proteins throughout major cell compartments: endoplasmic reticulum, Golgi, endosomes, cytosol and plasma membrane. Among them are four protein kinases (Cdc42bpb, Phkb, Camk2d, Mtor) that play roles in vasopressin-regulated phosphorylation of UT-A1. Non-label quantification was also performed to determine the stoichiometry of UT-A3 with UT-A1, the result does not support an oligomeric complex formation of UT-A1/A3. In conclusion, we have provided a refined list of UT-A1 binding proteins which can be useful for further analysis of the vasopressin signaling pathway in regulation of UT-A1 in IMCD.
Sample Processing Protocol
IMCD suspensions. Inner medullary collecting ducts were isolated from male Sprague-Dawley rats weighing 180-250 g (animal protocol H-0110R3) using the method of Stokes et al. with modifications. The suspensions were incubated at 37oC for 15 minutes with either dDAVP (a vasopressin V2 receptor agonist) at 1 nM or its vehicle. In-cell crosslinking. The crosslinking reagent bis[2-(succinimidyloxycarbonyloxy)ethyl]-sulfone (BSOCOES, spacer arm length 13.0 Å, Thermo Scientific, Rockford, IL) was added to the IMCD suspensions for 30 min according to company’s instructions. The reaction was quenched by adding 1 M Tris, pH 7.5 to a final concentration of 50 mM for 10 min. IMCDs were harvested by centrifugation (16,000 X g, 1 min). The pellet was homogenized (Potter-Elvehjem) in PBS containing 1X RIPA (1% NP-40, 0.1% SDS, 0. 5% sodium deoxycholate) and 1X HALT™ protease/phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL). The lysate was incubated at 4°C for 3 hr and spun at 100,000 Xg for 30 min to remove insoluble components prior to immunoprecipitation. Immunoprecipitation. Direct immunoprecipitation was performed using antibody-laden magnetic beads as previously described. Briefly, protein G-linked Dynabeads (Invitrogen Dynal, 9 mg per sample) were washed twice with bead wash buffer (PBS/0.02% Tween-20) before being incubated with 30 μg of affinity purified UT-A1 antibody L194 or preimmune IgG in wash buffer at 4oC for 2-3 hr. At the end of incubation, the beads were washed with 0.2 M triethanolamine (pH 8.2) and then were incubated with crosslinking reagent dimethylpimelimidate (DMP, 20 mM) in 0.2 M triethanolamine (pH 8.9) at room temperature for 45 min as described. The reaction was quenched by incubating the beads with 0.2 M triethanolamine (pH 8.2) for 5 min. The beads were subsequently incubated in 1% Triton X-100 in 0.2 M triethanolamine (pH 8.2) for 10 min to remove un-crosslinked IgG. After two washes with the bead wash buffer, the beads were incubated with the RIPA-solubilized, cross-linked IMCD proteins (600 g) prepared as above at 4°C overnight. The total reaction volume was adjusted by addition of PBS to reach a final 0.2X RIPA concentration when incubating with antibody-laden beads. After three washes with wash buffer, 50 μl of 2X Laemmli elution buffer was added to the beads and the mixture was incubated at 70oC for 20 min to elute the immunoprecipitated (IP) products. This step was repeated and the eluates were combined. The protein concentration was determined with the BCA assay (Pierce, Rockford, IL). The samples were mixed with 25 μl 1X Laemmli containing 40 mM DTT before storage at 4oC. In-gel digestion. UT-A1-specific and preimmune IP products were loaded on a 12-well 12.5% polyacrylamide gel for one-dimensional SDS-PAGE. The gel was stained with Imperial Protein Stain (Thermo Scientific, Rockford, IL) for 15 min followed by incubation in deionized H2O for 30 min. Preliminary immunoblotting with the L194 anti-UT-A antibody revealed that most of the crosslinked protein was present in at MW above 250 kDa (see Results). Two gel blocks corresponding to this >250 kDa region were isolated using a razor blade. In addition, lower molecular weight bands at 117 and 97 kDa (UT-A1) and at 44 and 66 kDa (UT-A3) were resected. Each gel piece was further sliced into 1 mm3 blocks and transferred to an Eppendorf tube. The gel pieces were dehydrated by incubating in 25 mM NH4HCO3 in 50% acetonitrile (ACN) for 10 min three times and then dried in a speed vacuum concentrator (SpeedVac, Thermo). Reduction, alkylation, and trypsinization were performed as previously described with minor modifications. Samples were reduced with 10 mM DTT in 25 mM NH4HCO3 for 1 h at 56°C and alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 for 45 min in the dark at room temperature. After washing with 25 mM NH4HCO3, the gel pieces were dehydrated in 50% ACN for 10 min twice and dried in a SpeedVac concentrator (Thermo Scientific, Rockford, IL). Enzyme digestion was carried out by adding 100 l of 12.5 ng/µl Sequencing Grade Modified Trypsin (porcine, Promega, Madison, WI) in 25 mM NH4HCO3 for 30 minutes to rehydrate gel pieces. After removal of excess trypsin solution, the gel pieces were covered with 25 mM NH4HCO3 and incubated at 37°C overnight. The digested peptides were extracted from the gel pieces twice with 50% ACN/0.5% formic acid and twice with 50% isopropanol/0.5% formic acid and dried in a SpeedVac. The peptide samples were acidified in 0.1% formic acid and desalted with C-18 spin columns (Thermo Scientific, Rockford, IL). The dried peptide samples were resuspended in 0.1% formic acid for LC/MS-MS analysis. When absolute quantification of selected peptides was performed (See ‘Absolute quantification using AQUA peptides’ below), AQUA peptides were included in 0.1% formic acid used to resuspend peptide samples.
Data Processing Protocol
LC-MS/MS analysis. The MS samples were analyzed on an Orbitrap Elite mass spectrometer (Thermo Scientific) interfaced with an Eksigent NanoLC Ultra system (Eksigent Technologies). Fragmentation of peptide ions was achieved via collision-induced dissociation. Samples were loaded onto an Agilent Zorbax 300SB-C18 trap column (0.3 mm i.d. × 5 mm length, 5-μm particle size) at a flow rate of 6 μL/min for 10 min. Reversed-phase C18 chromatographic separation of trapped peptides was carried out on a prepacked BetaBasic C18 PicoFrit column (75 μm i.d. × 25 cm length; New Objective) at 250 nL/min using the following gradient: 2–5% solvent B for 1 min; 5–35% solvent B for 40 min; 35–85% solvent B for 3 min; 85% solvent B for 5 min (solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile). Data-dependent mode was employed such that a single survey MS1 scan for precursor ions was followed by six data-dependent MS2 scans. Survey MS scans were acquired in the Orbitrap component with a resolution of 60,000, and MS2 scanning was performed in the linear ion trap. Data searching and scoring. Searches of peptides were performed using the latest version of the rat RefSeq database (National Center for Biotechnology Information) with concatenated forward and reversed sequences to allow for target-decoy analysis to control false positive. The database also contained sequences for common MS contaminants, such as human keratin and porcine trypsin. Spectra were searched with SEQUEST and InsPecT. InsPecT searches were carried out by the Biowulf Linux cluster at the National Institutes of Health (http://biowulf.nih.gov). Spectra were filtered to obtain a false discovery rate of 1%. Label-free peptide quantification was performed using NHLBI Proteomic Core’s QUOIL software. Gene Ontology and Conserved Domain Database analysis were performed using the in-house Java application, Automated BIoinformatics Extractor (ABE, http://helixweb.nih.gov/ESBL/ABE/). Gene Ontology and Network Analyses. Enrichment of individual Gene Ontology (GO) terms from the UT-A1 interactome was assessed using the Database for Annotation, Visualization and Integrated Discovery (DAVID, National Institute of Allergy and Infectious Diseases, version 6.7,http://david.abcc.ncifcrf.gov/). The complete IMCD transcriptome (http://dir.nhlbi.nih.gov/papers/lkem/imcdtr/) was used as the background data set. Functional clustering was performed in DAVID using the following databases: For Gene-Ontology, GO FAT; for Pathways: KEGG pathway and Panther pathway; for Protein Domain: InterPro, Panther family, Panther subfamily, PFAM, PIR-Superfamily, Prosite, and SMART; for Functional Categories: COG-Ontology, Swiss-Prot/Protein Information Resource Keywords, and UniProt Sequence Features. The level of enrichment was assessed by a modified Fisher’s exact test in which enriched Biological Process, Molecular Function, Cellular Component terms, and Protein Domains terms with a corrected P-value less than 0.01 were report. The Fold Enrichment for a particular term is the ratio of the number of gene symbols associated with the term in the data set to the total number gene symbols in the data set divided by the same ratio in the background data set.
Chou CL, Hwang G, Hageman DJ, Han L, Agrawal P, Pisitkun T, Knepper MA. Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct. Am J Physiol Cell Physiol. 2018 314(1):C99-C117 PubMed: 29046292