Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin.
Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of G(q)- and G(i)-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.
Huang
Ying
Y
Department of Biology, Syracuse University, Syracuse, New York 13244, USA.
Niwa
Jun-ichi
J
Sobue
Gen
G
Breitwieser
Gerda E
GE
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D047468:Immunoprecipitation; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007976:Leupeptins; D009687:Nuclear Proteins; D010455:Peptides; D044169:Receptors, Calcium-Sensing; D025801:Ubiquitin; C046951:valosin; D014415:Tunicamycin; C423571:RNF19A protein, human; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde