Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling.
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 (>80%) on the basolateral cell surface. Receptors undergo rapid 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, delta218-263, lacks essentially 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 strongly polarized to the basolateral surface (>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.
Yeaman
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C
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021, USA.
Heinflink
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M
Falck-Pedersen
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Rodriguez-Boulan
E
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Gershengorn
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004285:Dogs; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000502:physiology*; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D014316:Tritium; D013973:Thyrotropin-Releasing Hormone; D019204:GTP-Binding Proteins