Vrecl M, Anderson L, Hanyaloglu A, McGregor A M, Groarke A D, Milligan G, Taylor P L, Eidne K A
MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, United Kingdom.
Mol Endocrinol. 1998 Dec;12(12):1818-29. doi: 10.1210/mend.12.12.0207.
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.
本研究检测了促性腺激素释放激素（GnRH）受体（一种独特的无细胞内羧基末端尾巴的G蛋白偶联受体（GPCR））在激动剂刺激后与内吞和再循环事件相关的动态变化，并研究了β-抑制蛋白在此过程中的作用。通过共聚焦显微镜监测稳定表达于HEK 293细胞中的荧光标记表位标签GnRH受体的亚细胞定位，并使用放射性配体结合和酶联免疫吸附测定（ELISA）对受体/配体内化过程进行定量。激动剂刺激导致受体从质膜可逆性重新分布到细胞质区室，并观察到内化的GnRH受体与转铁蛋白受体共定位。对GnRH受体和另一种具有羧基末端尾巴的GPCR（促甲状腺激素释放激素（TRH）受体）进行的内化实验表明，当在HEK 293和COS-7细胞中表达时，GnRH受体的内化速率比TRH受体慢得多。在COS-7细胞中，TRH受体与β-抑制蛋白共表达可显著增加其内化，而在这两种细胞类型中，GnRH受体与β-抑制蛋白共表达均不影响其内化。GnRH受体与显性负性β-抑制蛋白（319 - 418）突变体共表达不影响其内化能力，且活化的GnRH受体不会诱导β-抑制蛋白/绿色荧光蛋白向质膜的时间依赖性重新分布。然而，β-抑制蛋白突变体损害了TRH受体的内化，且活化的TRH受体诱导了β-抑制蛋白/绿色荧光蛋白易位。本研究表明，尽管GnRH受体没有细胞内羧基末端尾巴，但它在激动剂刺激下会发生内化，表现出与其他通过网格蛋白依赖性机制内化并通过酸化内体区室再循环的GPCR所描述的独特特征。然而，我们的数据表明，GnRH受体可能利用一种不依赖β-抑制蛋白的内吞途径。
