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. 1995 Jan 17;92(2):512-6.
doi: 10.1073/pnas.92.2.512.

Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling

R Ashworth  1 , R Yu, E J Nelson, S Dermer, M C Gershengorn, P M Hinkle
Affiliations

Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling

R Ashworth et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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