Subcellular trafficking of the TRH receptor: effect of phosphorylation.
Activation of the G protein-coupled TRH receptor leads to its phosphorylation and internalization. These studies addressed the fundamental question of whether phosphorylation regulates receptor trafficking or endosomal localization regulates the phosphorylation state of the receptor. Trafficking of phosphorylated and dephosphorylated TRH receptors was characterized using phosphosite-specific antibody after labeling surface receptors with antibody to an extracellular epitope tag. Rab5 and phosphoreceptor did not colocalize at the plasma membrane immediately after TRH addition but overlapped extensively by 15 min. Dominant-negative Rab5-S34N inhibited receptor internalization. Later, phosphoreceptor was in endosomes containing Rab5 and Rab4. Dephosphorylated receptor colocalized with Rab4 but not with Rab5. Dominant-negative Rab4, -5, or -11 did not affect receptor phosphorylation or dephosphorylation, showing that phosphorylation determines localization in Rab4(+)/Rab5(-) vesicles and not vice versa. No receptor colocalized with Rab7; a small amount of phosphoreceptor colocalized with Rab11. To characterize recycling, surface receptors were tagged with antibody, or surface receptors containing an N-terminal biotin ligase acceptor sequence were labeled with biotin. Most recycling receptors did not return to the plasma membrane for more than 2 h after TRH was removed, whereas the total cell surface receptor density was largely restored in less than 1 h, indicating that recruited receptors contribute heavily to early repopulation of the plasma membrane.
Jones
Brian W
BW
Department of Pharmacology, University of Rochester Medical Center, Box 711, Rochester, New York 14642, USA.
Hinkle
Patricia M
PM
D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D010766:Phosphorylation; D021381:Protein Transport; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D018025:Receptors, Thyrotropin-Releasing Hormone; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins