Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other.
We have examined the trafficking of the thyrotropin-releasing hormone receptor (TRHR) and its ligand, after TRHR-TRH internalization in rat pituitary GH4C1 cells. After rapid ligand-induced receptor sequestration, the cell surface receptor pool was replenished. Replenishment was insensitive to inhibition of protein synthesis and was dependent on the duration of internalization; therefore, the replenished receptors were not newly synthesized but recycled. The total amount of recycled receptors decreased with increasing internalization time, resulting in only partial replenishment of the cell-surface receptor pool after prolonged incubation with ligand. Thus, in addition to a receptor recycling pathway, a non-cycling route exists for TRHR sorting; this route became dominant with increasing internalization periods. TRHR entry into these pathways was not determined by the affinity of the receptor-ligand interaction, because the extent of receptor recycling was similar after TRH- and methyl-TRH (MeTRH)-induced internalization. Unlike results with the TRHR, the TRH recycling pool was not depleted by the noncycling pathway. After multiple rounds of [3H]MeTRH internalization, the amount of cell-associated radioactivity increased with increasing internalization time due to accumulation of the ligand or its metabolites in a non-cycling pathway, but the absolute amount of recycled ligand remained constant after short or long internalization times. The difference in the proportion of TRHR and MeTRH that were diverted into a noncycling pathway indicated intracellular dissociation of the internalized TRHR-TRH complex. Dissociation of the internalized TRHR-TRH complex was dependent on the acidic pH in an intracellular compartment. Although extracellular acidic pH did not enhance cell-surface receptor-ligand (RL) dissociation, bafilomycin A1 inhibited both receptor and ligand recycling. We conclude that the TRHR-TRH system is unique among recycling receptors because, after RL sequestration, the TRHR-TRH complex becomes dissociated intracellularly via a bafilomycin A1-sensitive, acidic pH-dependent mechanism, and both the unoccupied TRHR and TRH recycle disassociated from each other.
Petrou
C P
CP
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA.
Tashjian
A H
AH
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018942:Macrolides; D008745:Methylation; D010902:Pituitary Gland / Q000378:metabolism; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D018942:Macrolides; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; C040929:bafilomycin A1; D003513:Cycloheximide