AGONIST-INDUCED ENDOCYTOSIS AND RECYCLING OF THE GONADOTROPIN-RELEASING-HORMONE RECEPTOR - EFFECT OF BETA-ARRESTIN ON INTERNALIZATION KINETICS

This study examined the dynamics of endocytotic and recycling events a ssociated 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 proce ss. 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 quanti fied using radioligand binding and ELISA. Agonist stimulation resulted in reversible receptor redistribution from the plasma membrane into t he cytoplasmic compartment, and colocalization of internalized GnRH re ceptors with transferrin receptors was observed. Internalization exper iments for the GnRH receptor and another GPCR possessing a carboxy-ter minal tail, the TRH receptor, showed that the rate of internalization for the GnRH receptor was much slower than for the TRH receptor when e xpressed in both HEK 293 and COS-7 cells. TRH receptor internalization could be substantially increased by coexpression with beta-arrestin i n 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) m utant did not affect its ability to internalize, and activated GnRH re ceptors did not induce time-dependent redistribution of beta-arrestin/ green fluorescent protein to the plasma membrane. However, the beta-ar restin mutant impaired the internalization of the TRH receptor, and ac tivated TRH receptors induced the beta-arrestin/green fluorescent prot ein translocation. This study demonstrates that, despite having no int racellular carboxy-terminal tail, the GnRH receptor undergoes agonist- stimulated internalization displaying distinctive characteristics desc ribed for other GPCRs that internalize via a clathrin-dependent mechan ism and recycle through an acidified endosomal compartment. However, o ur data indicate that the GnRH receptor may utilize a beta-arrestin-in dependent endocytotic pathway.