| G protein-coupled receptor function is influenced by protein-protein interactions and post-translational modifications. These studies were designed to both identify potential thyrotropin-releasing hormone (TRH) receptor interactors and investigate the function of TRH receptor phosphorylation, ubiquitination and palmitoylation. Yeast interaction traps were performed using the third intracellular loop and C-tail regions of the TRH receptor. Several positive clones were isolated; these include ubiquitin specific protease 11, dynein, adaptor protein-2 mu2 subunit, and cyclophilin A. Agonist-activated TRH receptors were phosphorylated. TRH receptor phosphorylation did not require phospholipase C, protein kinase C, casein kinase II or G protein-coupled receptor kinase (GRK) 2, suggesting that receptors are phosphorylated by other GRKs. TRH receptors were shown to be ubiquitinated. The role of the ubiquitin-proteasome pathway in TRH receptor internalization and trafficking was investigated. TRH receptor ubiquitination was independent of agonist. Inhibition of the proteasome resulted in accumulation of newly synthesized, ubiquitinated receptors localized to endoplasmic reticulum (ER) and Golgi fractions and cytosol. Surface receptor levels remained unchanged. Mislocalized TRH receptors were ubiquitinated. These data suggest that the ubiquitin-proteasome pathway participates in receptor quality control through the ER-degradation pathway. TRH receptors internalized normally and produced calcium signals in the absence of a functional ubiquitin-proteasome pathway. A C-terminally truncated TRH receptor that cannot internalize was ubiquitinated, therefore ubiquitination is not primarily localized to the C-terminal region. Ubiquitinated TRH receptors were glycosylated, therefore they had been to the Golgi apparatus, however none were detected at the cell surface. In addition to TRH receptor quality control, ubiquitin may function at the Golgi or a post-Golgi compartment to prevent TRH receptor trafficking to the plasma membrane. Upon activation of TRH receptors, a signal may be sent to this reserve of ubiquitinated receptors resulting in their de-ubiquitination and plasma membrane localization.; TRH receptor palmitoylation was undetectable. A TRH receptor missing two putative palmitoylation sites and a TRH receptor with a mutated calmodulin-binding domain were studied. Both receptors produced normal calcium signals and desensitized, but beta-arrestin translocation was slightly inhibited and receptor internalization was sub-maximal, suggesting that palmitoylation and calmodulin-binding may play a role in regulating receptor internalization. |
