POLARITY OF TRH RECEPTORS IN TRANSFECTED MDCK CELLS IS INDEPENDENT OFENDOCYTOSIS SIGNALS AND G-PROTEIN COUPLING

Information concerning the molecular sorting of G protein-coupled rece ptors in polarized epithelial cells is limited. Therefore, we have exp ressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-D arby canine kidney (MDCK) cells by adenovirus-mediated gene transfer t o determine its distribution in a model cell system and to begin analy zing the molecular information responsible for its distribution. Equil ibrium binding of [methyl-H-3]TRH to apical and basolateral surfaces o f polarized MDCK cells reveals that TRH receptors are expressed predom inantly (>80%) on the basolateral cell surface. Receptors undergo rapi d endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, Delta 218-263, lacks essentiall y all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongl y polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of T RH receptors can be segregated from signals for ligand-induced recepto r endocytosis and coupling to heterotrimeric G proteins.