Agonist-Induced Endocytosis and Recycling of the Gonadotropin-Releasing Hormone Receptor: Effect of -Arrestin on Internalization Kinetics

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Abstract
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 b-arrestin in this process. Subcellular loca- tion of fluorescently labeled epitope-tagged GnRH receptors stably expressed in HEK 293 cells was monitored by confocal microscopy, and the recep- tor/ligand internalization process was quantified using radioligand binding and ELISA. Agonist stimulation resulted in reversible receptor redis- tribution from the plasma membrane into the cytoplasmic compartment, and colocalization of internalized GnRH receptors with transferrin re- ceptors was observed. Internalization experi- ments for the GnRH receptor and another GPCR possessing a carboxy-terminal tail, the TRH re- ceptor, 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 coexpres- sion with b-arrestin in COS-7 cells, while GnRH receptor internalization was not affected by co- expression with b-arrestin in either cell type. Co- expression of the GnRH receptor with the domi- nant negative b-arrestin (319-418) mutant did not affect its ability to internalize, and activated GnRH receptors did not induce time-dependent

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