Arrestin is one of the key proteins for the termination of G protein signaling. Activated G protein-coupled receptors (GPCRs) are specifically phosphorylated by G proteincoupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Vertebrates possess two types of arrestin; visual arrestin expressed exclusively in photoreceptor cells in retinae and pineal organs, and b-arrestin, which is expressed ubiquitously. Unlike visual arrestin, b-arrestin contains the clathrinbinding domain at the C-terminus, responsible for the agonist-induced internalization of GPCRs. Here, we isolated a novel arrestin gene (Ci-arr) from the primitive chordate, the ascidian Ciona intestinalis larvae. The deduced amino acid sequence suggests that Ci-Arr be closely related to vertebrate arrestins. Interestingly, this arrestin has the feature of both visual and b-arrestin. Whereas the expression of Ci-arr was restricted to the photoreceptors in the larvae similarly to visual arrestin, the gene product, containing the clathrin-binding domain, promoted the GPCR internalization in HEK293tsA201 cells similarly to b-arrestin. The phylogenetic tree shows that Ci-Arr is branched from a common root of visual and b-arrestins. Southern analysis suggests that the Ciona genome contains only one gene for the arrestin family. These results suggest that the visual and b-arrestin genes were generated by the duplication of the prototypical arrestin gene like Ci-arr in the early evolution of vertebrates.

The present study was performed to identify sequence(s) in the third intracellular loop (i3) of the muscarinic acetylcholine receptor M4 subtype (M4 receptor) involved in its internalization and recycling. In transiently transfected human embryonic kidney 293-tsA201 cells, 40 to 50% of cell-surface M4 receptors are internalized in an agonist-dependent manner, and approximately 65% of internalized receptors are recycled back to the cell surface after removal of the agonist. We examined the internalization and recycling of M4 receptor mutants with partial deletion in i3 and found that various mutants (M4del-K 235 -K 240 , M4del-T 241 -K 271 , and M4del-W 339 -N 372 ) showed internalization and cell-surface recycling in a similar manner to the M4 receptor. We also found that the mutant M4del-L 272 -R 338 was internalized to only half the extent of the M4 receptor and was recycled after agonist removal, and the mutant M4del-V 373 -A 393 was also internalized to half the extent of the wild type but was not recycled back to the cell surface after agonist removal. When the sequence corresponding to Val 373 -Ala 393 was grafted onto the i3 portion of a recycling-negative mutant of muscarinic M2 receptor with deletion of almost the whole of the i3 sequence, approximately 40% of the chimeric receptor on the cell surface was internalized, and more than 65% of the internalized receptors were recycled back to the cell surface.

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Muscarinic M2, M4, and M2-M4 chimera receptors were transiently expressed in HEK-293 tsA201 cells, and agonist-dependent internalization of these receptors and recycling of internalized receptors were examined by measuring the amount of cell-surface receptors as [

No abstract