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 GH(4)C(1) cells. After rapid ligand-induced receptor sequest ration, the cell surface receptor pool was replenished. Replenishment was insensitive to inhibition of protein synthesis and was dependent o n the duration of internalization; therefore, the replenished receptor s were not newly synthesized but recycled. The total amount of recycle d 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 recy cling pathway, a non-cycling route exists for TRHR sorting; this route became dominant with increasing internalization periods. TRHR entry i nto these pathways was not determined by the affinity of the receptor- ligand interaction, because the extent of receptor recycling was simil ar after TRH- and methyl-TRH (MeTRH)-induced internalization. Unlike r esults with the TRHR, the TRH recycling pool was not depleted by the n oncycling pathway. After multiple rounds of[H-3]MeTRH internalization, the amount of cell-associated radioactivity increased with increasing internalization time due to accumulation of the ligand or its metabol ites in a non-cycling pathway, but the absolute amount of recycled lig and remained constant after short or long internalization times. The d ifference in the proportion of TRHR and MeTRH that were diverted into a noncycling pathway indicated intracellular dissociation of the inter nalized TRHR-TRH complex. Dissociation of the internalized TRHR-TRH co mplex was dependent on the acidic pH in an intracellular compartment. Although extracellular acidic pH did not enhance cell-surface receptor -ligand (RL) dissociation, bafilomycin Al 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 com plex becomes dissociated intracellularly via a bafilomycin Al-sensitiv e, acidic pH-dependent mechanism, and both the unoccupied TRHR and TRH recycle disassociated from each other.