Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling.
Endocytosis and recycling of both thyrotropin-releasing hormone (TRH) and its G-protein-coupled receptor were visualized by conventional and confocal fluorescence microscopy in pituitary cells using a rhodamine-labeled TRH analog (Rhod-TRH) and indirect immunofluorescent staining of cells stably transfected with an epitope-tagged TRH receptor (TRHR). The epitope-tagged TRHR was confined to the cell surface prior to agonist treatment. Both Rhod-TRH and TRHR were also localized on the plasma membrane after agonist binding at 0 degrees C. Ligand binding at 37 degrees C resulted in rapid endocytosis, and both Rhod-TRH and the epitope-tagged TRHR appeared in cytoplasmic vesicles within 5 min. Fluorescently labeled TRH and transferrin colocalized in the same endocytotic vesicles, and internalization of Rhod-TRH and TRHR was inhibited by hypertonic medium, suggesting that endocytosis occurred by a clathrin-dependent mechanism. Internalized TRHRs returned to the membrane within 20 min after removal of TRH, and cycloheximide did not block receptor recycling. A mutant TRHR truncated at Cys335 signaled but did not internalize Rhod-TRH, confirming the importance of the carboxyl terminus of the TRHR in receptor-mediated endocytosis. Thus, the TRH-TRHR complex is endocytosed via clathrin-coated vesicles and the receptor is recycled to the plasma membrane.
Ashworth
R
R
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, NY 14642.
Yu
R
R
Nelson
E J
EJ
Dermer
S
S
Gershengorn
M C
MC
Hinkle
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D004705:Endocytosis / Q000502:physiology*; D007150:Immunohistochemistry; D008024:Ligands; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012235:Rhodamines; D012377:Rodentia; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D008024:Ligands; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D012235:Rhodamines; D013973:Thyrotropin-Releasing Hormone