Role of the third intracellular loop in the subtype-specific internalization and recycling of muscarinic M2 and M4 receptors.

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 [3H]N-methylscopolamine (NMS) binding activity. Coexpression of a dominant negative form of dynamin (DN-dynamin,dynamin K44A) greatly reduced the agonist-dependent internalization of M4 receptors but not of M2 receptors, as was reported by Vögler et al. (J Biol Chem 273, 12155-12160, 1998).

Muscarinic M4 receptor recycling requires a motif in the third intracellular loop.

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.

Ascidian arrestin (Ci-arr), the origin of the visual and nonvisual arrestins of vertebrate.

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 protein-coupled 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 beta-arrestin, which is expressed ubiquitously.