RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter.
The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl-releasing protein 1 (RasGRP1), triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression (Ko B, Joshi LM, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. Proc Natl Acad Sci USA 104: 20120-20125, 2007). Little is known about the mechanisms which underlie these effects on NCC activity. Regulation of NCC via changes in NCC surface expression has been reported, but endocytosis of NCC has not been demonstrated. In this study, utilizing biotinylation, internalization assays, and a dynamin dominant-negative construct, we demonstrate that the regulation of NCC by PE occurs via an enhancement in internalization of NCC and is dynamin dependent. In addition, immunoprecipitation of NCC and subsequent immunoblotting for ubiquitin showed increased ubiquitination of NCC with phorbol ester treatment. MEK1/2 inhibitors and gene silencing of RasGRP1 indicated that this effect was dependent on RasGRP1 and ERK1/2 activation. Inhibition of ubiquitination prevents any PE-mediated decrease in NCC surface expression as measured by biotinylation or NCC activity as measured by radiotracer uptake. These findings confirmed that the PE effect on NCC is mediated by endocytosis of NCC. Furthermore, ubiquitination of NCC is essential for this process and this ubiquitination is dependent upon RasGRP1-mediated ERK1/2 activation.
Ko
Benjamin
B
Department of Medicine, University of Chicago, Illinois, USA.
Kamsteeg
Erik-Jan
EJ
Cooke
Leslie L
LL
Moddes
Lauren N
LN
Deen
Peter M T
PM
Hoover
Robert S
RS
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D007686:Kidney Tubules, Distal / Q000187:drug effects / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D011955:Receptors, Drug / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D054875:Ubiquitination; D015854:Up-Regulation
D020536:Enzyme Activators; D020662:Guanine Nucleotide Exchange Factors; D047428:Protein Kinase Inhibitors; C112278:Rasgrp1 protein, mouse; D011955:Receptors, Drug; C578875:Slc12a3 protein, mouse; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D034281:Dynamins; D044764:Ubiquitin-Activating Enzymes; D013755:Tetradecanoylphorbol Acetate
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