Casein Kinase II Sites in the Intracellular C-terminal Domain of the Thyrotropin-releasing Hormone Receptor and Chimeric Gonadotropin-releasing Hormone Receptors Contribute to β-Arrestin-dependent Internalization

Abstract

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a β -arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize β-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring β-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not β-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via β-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and β-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a β-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of β-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of β-arrestin-dependence was observed. Visualization of β-arrestin/GFP redistribution confirmed a loss or gain of β-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent β-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for β-arrestin-dependent inter. nalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by β-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the β-arrestin-dependent pathway.