Agonist-induced endocytosis and recycling of the gonadotropin releasing hormone receptor:: Effect of β-arrestin on internalization kinetics

This study examined the dynamics of endocytotic and recycling events associated with the GnRH receptor, a unique G protein-coupled receptor (GPCR) without the intracellular carboxyl-terminal tail, after agonist stimulation, and investigated the role of beta-arrestin in this process. Subcellular location of fluorescently labeled epitope-tagged GnRH receptors stably expressed in HEK 293 cells was monitored by confocal microscopy, and the receptor/ligand internalization process was quantified using radioligand binding and ELISA. Agonist stimulation resulted in reversible receptor redistribution from the plasma membrane into the cytoplasmic compartment, and colocalization of internalized GnRH receptors with transferrin receptors was observed. Internalization experiments for the GnRH receptor and another GPCR possessing a carboxy-terminal tail, the TRH receptor, showed that the rate of internalization for the GnRH receptor was much slower than for the TRH receptor when expressed in both HEK 293 and COS-7 cells. TRH receptor internalization could be substantially increased by coexpression with beta-arrestin in COS-7 cells, while GnRH receptor internalization was not affected by coexpression with beta-arrestin in either cell type. Coexpression of the GnRH receptor with the dominant negative beta-arrestin (319-418) mutant did not affect its ability to internalize, and activated GnRH receptors did not induce time-dependent redistribution of beta-arrestin/green fluorescent protein to the plasma membrane. However, the beta-arrestin mutant impaired the internalization of the TRH receptor, and activated TRH receptors induced the beta-arrestin/green fluorescent protein translocation. This study demonstrates that, despite having no intracellular carboxy-terminal tail, the GnRH receptor undergoes agonist-stimulated internalization displaying distinctive characteristics described for other GPCRs that internalize via a clathrin-dependent mechanism and recycle through an acidified endosomal compartment. However, our data indicate that the GnRH receptor may utilize a beta-arrestin-independent endocytotic pathway.