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

• Published in: Biomedical Research Vol. 35; no. 3; pp. 185 - 192
• Main Authors: YOSHIDA, Norihiro, JOJIMA, Eri, SAITO, Hiroyuki, HAGA, Tatsuya
• Format: Journal Article
• Language: English
• Published: Japan Biomedical Research Press 2014; Japan Science and Technology Agency
• Subjects: Carbachol - pharmacology; Cells, Cultured; Dynamins - genetics; Dynamins - metabolism; G-Protein-Coupled Receptor Kinase 2 - genetics; G-Protein-Coupled Receptor Kinase 2 - metabolism; Gene Expression; HEK293 Cells; Humans; Kinetics; Protein Binding; Protein Interaction Domains and Motifs - genetics; Receptor, Muscarinic M2 - agonists; Receptor, Muscarinic M2 - chemistry; Receptor, Muscarinic M2 - genetics; Receptor, Muscarinic M2 - metabolism; Receptor, Muscarinic M4 - agonists; Receptor, Muscarinic M4 - chemistry; Receptor, Muscarinic M4 - genetics; Receptor, Muscarinic M4 - metabolism; Time Factors
• ISSN: 0388-6107; 1880-313X
• DOI: 10.2220/biomedres.35.185

Muscarinic M2, M4, and M2-M4 chimera receptors were transiently expressed in HEK-293tsA201 cells, and agonist-dependent internalization of these receptors and recycling of internalizedreceptors 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 (DNdynamin,dynamin K44A) greatly reduced the agonist-dependent internalization of M4 receptorsbut not of M2 receptors, as was reported by Vögler et al. (J Biol Chem 273, 12155–12160, 1998).The agonist-dependent internalization of M2/M4-i3/M2 chimera receptors (M2 receptors with thei3 loop replaced by that of M4 receptors) was greatly reduced by co-expression of DN-dynaminas was the case for M4 receptors, whereas the agonist-dependent internalization of M4/M2-i3/M4chimera receptors was hardly affected by co-expression of DN-dynamin as was the case for M2 receptors.Internalized M2/M4-i3/M2 receptors as well as internalized M4 receptors were shown tobe recycled back to the cell surface after removal of agonists, whereas no recycling was observedfor M4/M2-i3/M4 receptors as well as M2 receptors. These results indicate that the i3 loops ofM2 and M4 receptors take a major role in their agonist-dependent internalization and recycling.