Thyrotropin-releasing hormone receptor processing: role of ubiquitination and proteasomal degradation.
These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.
Cook
Laurie B
LB
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Zhu
Chang-Cheng
CC
Hinkle
Patricia M
PM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006031:Glycosylation; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D009097:Multienzyme Complexes; D018025:Receptors, Thyrotropin-Releasing Hormone; D025801:Ubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex