Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an int racellular carboxyl tail (C-tail), does not follow a beta -arrestin-depende nt internalization pathway. However, internalization of a chimeric GnRHR wi th the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize be ta -arrestin, Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta -arrestin-dependent i nternalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chi meric GnRHR with the catfish GnRHR C-tail is not beta -arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but no ne in the catfish GnRHR C-tail, We thus investigated a role for CKII sites in determining GPCR internalization via beta -arrestin. Sequential introduc tion of three CKII sites into the chimera with the catfish C-tail (H354D,A3 66E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta -arrestin-dependence, which only occurred when all three sites wer e introduced. Conversely, mutation of the putative CKII sites (T365A/T371A, S383A) in the C-tail of a p-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta -arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta -arre stin-dependence was observed. Visualization of beta -arrestin/GFP redistrib ution confirmed a loss or gain of beta -arrestin sensitivity for receptor m utants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta -arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites re quired for beta -arrestin-dependent internalization. Apigenin, a specific C KII inhibitor, blocked the increase in receptor internalization by beta -ar restin, thus providing further support for the involvement of CKII, This st udy presents evidence of a novel role for C-tail CKII consensus sites in ta rgeting these GPCRs to the beta -arrestin-dependent pathway.