Quantitative Protein and mRNA Profiling Shows Selective Post-Transcriptional Control of Protein Expression by Vasopressin in Kidney Cells

Sookkasem Khositseth,Trairak Pisitkun,Dane H Slentz,Guanghui Wang,Jason D Hoffert,Mark A Knepper,Ming-Jiun Yu

doi:10.1074/mcp.m110.004036

Abstract

Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: "In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?" In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells ('SILAC') coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter suggests that long-term regulatory processes may contribute to actomyosin-dependent trafficking of the water channel aquaporin-2. The results provide impetus for increased focus on translational regulation and regulation of protein degradation in physiological control in mammalian epithelial cells.

Highlights

In multicellular organisms, the phenotypes of individual cell types are specified by the subset of protein-coding genes that are expressed
Quantitative Proteomic Analysis of Long-Term Vasopressin Action—To identify proteins that change in abundance in mpkCCD clone 11 cells in response to the V2 receptorselective vasopressin analog dDAVP, we carried out quantitative proteomic analysis using stable isotopic labeling in cultured mpkCCD cells (SILAC) (Stable Isotopic Labeling by Amino acids in Cell culture) coupled with tandem mass spectrometry (LC-MS/MS)
Preliminary to the LC-MS/MS analysis, we carried out time course studies (Figs. 1B, Supplemental Fig. S2) to determine when, following the addition of dDAVP (0.1 nM), a maximal response was reached with respect to aquaporin-2 protein abundance

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Clone 11 mpkCCD cells [17] were maintained in Dulbecco’s modified Eagle’s medium:Ham’s F12 medium plus supplements and 2% fetal bovine serum at 37 °C with 5% CO2 and 95% air. To test amino acid incorporation, 50 ␮g of protein from cells grown in light and heavy media for 7 or 12 days were extracted, mixed at a 1:1 ratio, and processed for LC-MS/MS peptide identification and. To assess the possibility of arginine-to-proline conversion in the mpkCCD cells, we measured the average light to heavy peptide abundance ratios for 22 peptides containing proline and 39 peptides lacking proline from cells cultured with isotopic amino acids for 7 days (Supplemental Table S1). Multiple-Reaction Monitoring (MRM)—Cultured mpkCCD cells labeled with heavy or light amino acids as above were exposed to 0.1 nM dDAVP or vehicle for 5 days before their proteins were harvested, mixed at a 1:1 ratio, separated by one-dimensional SDS-PAGE, reduced, alkylated, trypsinized, and desalted before targeted MRM (multiple-reaction monitoring) proteomic analysis. Student’s t test was used to test the significance of change

RESULTS

DISCUSSION

Gsdmc2