Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other

Abstract

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.

Differential Internalization of Somatostatin in COS-7 Cells Transfected with SST1and SST2Receptor Subtypes: A Confocal Microscopic Study Using Novel Fluorescent Somatostatin Derivatives1

“…These endosome-like particles increased in size with time, presumably due to their conglomeration and/or fusion given the trend toward their concurrent decrease in number. Whether, as demonstrated for other peptides (19,29,53), this internalization process results in a recycling of the receptors and/or provides for their down-regulation remains to be established. This pattern of internalization is congruent with what has now been described for a variety of peptide ligands both in vitro and in vivo and shown to correspond, at least in some systems, to sequestration of receptor-ligand complexes into clathrincoated pits, leading to their transfer into early endosomes, late endosomes, multivesicular bodies, and lysosomes (16).…”

Recruitment of Activated G Protein-coupled Receptors to Pre-existing Clathrin-coated Pits in Living Cells

“…In this system the endocytosis rate of the TRHR (t1 ⁄2 of ϳ10 min, Fig. 1a) is similar to that obtained in rat pituitary GH 4 C 1 cells expressing endogenous TRHR (38). The cellular distribution of TRHR and ␤-arrestin2 was analyzed by immunofluorescence: VSV-TRHR was detected using the P5D4 monoclonal antibody specific to the VSV-tag, and ␤-arrestin2-GFP was directly visualized by the green fluorescence emitted by GFP.…”

Expression analysis of the thyrotropin‐releasing hormone receptor (TRHR) in the immune system using agonist anti‐TRHR monoclonal antibodies

“…The location of staining at the cell periphery and plasma membrane conforms with the location expected for a membrane receptor. Intracytoplasmic staining may indicate intracellular receptor pools in the processes of synthesis, endocytosis and trafficking [40–42]. Electron microscopy studies will be necessary to elucidate the precise location of these TRHR pools.…”