Basolateral polarity of thyrotropin-releasing hormone receptors in transfected mdck cells is established independently of endocytosis signals and g-protein coupling. Yeaman, Charles, Marcos Heinflink, Erik Falck-Pedersen, Enrique Rodriguez-Boulan, and Marvin C. Gershengorn. Department of Cell Biology and Anatomy, Division of Molecular Medicine, Department of Medicine, and Department of Microbiology, Cornell University Medical College and The New York Hospital, 1300 York Avenue, New York, N.Y. 10021
APStracts 2:0340C, 1995.
Information concerning the molecular sorting of G-protein-coupled receptors in polarized epithelial cells is limited. Therefore we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (&GT 80 %) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80 % are internalized in 15 minutes. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (&LT 10 %) but is nevertheless basolaterally expressed (&GT 85 %). A second mutant TRH receptor, D218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins upon binding agonist. This receptor internalizes TRH approximately half as efficiently as wild type TRH receptors, but is nevertheless strongly polarized to the basolateral surface (&GT 90 %). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins. 

Received 27 June 1995; accepted in final form 8 September 1995.
APS Manuscript Number C379-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 September 1995.