HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Figures and Tables All Versions of this Article: 295/3/C661    most recent 90650.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Yu, M.-J. Articles by Knepper, M. A. Search for Related Content PubMed PubMed Citation Articles by Yu, M.-J. Articles by Knepper, M. A.

PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct

¹Laboratory of Kidney and Electrolyte Metabolism and ²Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and ³Universidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas, Cantoblanco, Madrid, Spain

Submitted 26 December 2007 ; accepted in final form 2 July 2008

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.

aquaporin-2; vasopressin; membrane rafts; mass spectrometry; proteomics

This article has been cited by other articles:

				X. Feng, H. Huang, Y. Yang, O. Frohlich, J. D. Klein, J. M. Sands, and G. Chen Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane Am J Physiol Renal Physiol, June 1, 2009; 296(6): F1514 - F1520. [Abstract] [Full Text] [PDF]

				A. N. Sachs, T. Pisitkun, J. D. Hoffert, M.-J. Yu, and M. A. Knepper LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1799 - F1806. [Abstract] [Full Text] [PDF]