title: Subcellular localization and internalization of the vasopressin V1B receptor.
abstract: Only limited information is available on agonist-dependent changes in the subcellular localization of vasopressin V1B receptors. Our radioligand binding study of membrane preparations and intact cells revealed that a large fraction of the V1B receptor is located in the cytoplasm in unstimulated CHO cells, which is in contrast to the plasma membrane localization of the V1A and V2 receptors. Moreover, when the affinity of radiolabeled arginine-vasopressin ([3H]AVP) was compared between membrane preparations and intact cells, the affinity of [3H]AVP to the cell surface V1B receptors, but not the V1A receptors, was significantly reduced. Although the number and affinity of cell surface V1B receptors decreased, they became extensively internalized upon binding with [3H]AVP. Approximately 87% of cell surface-bound [3H]AVP was internalized and became resistant to acid wash during incubation with 1 nM [3H]AVP. By contrast, less ligand (35%) was internalized in the cells expressing the V1A receptor. Extensive internalization of the V1B receptors was partially attenuated by inhibitors of cytoskeletal proteins, siRNA against β-arrestin 2, or the removal of sodium chloride from the extracellular buffer, indicating that this internalization involves clathrin-coated pits. Together, these results indicate that the mechanism that regulates the number and affinity of V1B receptors in the plasma membrane is markedly distinct from the corresponding mechanisms for the V1A and V2 receptors and plays a critical role under stress conditions, when vasopressin release is augmented.
authors: Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.
authors: Aki
authors: A
authors: Kashiwazaki
authors: Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.
authors: Yoko
authors: Y
authors: Fujiwara
authors: Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan.
authors: Hiroyoshi
authors: H
authors: Tsuchiya
authors: Department of Functional Genomics, Graduate School of Pharmaceutical Sciences, Himeji Dokkyo University, Hyogo 670-8524, Japan.
authors: Nobuya
authors: N
authors: Sakai
authors: Department of Functional Genomics, Graduate School of Pharmaceutical Sciences, Himeji Dokkyo University, Hyogo 670-8524, Japan.
authors: Katsushi
authors: K
authors: Shibata
authors: Division of Molecular Pharmacology, Department of Pharmacology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Electronic address: t_koshi@jichi.ac.jp.
authors: Taka-aki
authors: TA
authors: Koshimizu
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D001665:Binding Sites; D016466:CHO Cells; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism*; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D010902:Pituitary Gland / Q000378:metabolism*; D021381:Protein Transport; D011869:Radioligand Assay; D011921:Rats, Inbred WKY; D017483:Receptors, Vasopressin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014316:Tritium
chemical_list: D017483:Receptors, Vasopressin; D014316:Tritium; D001127:Arginine Vasopressin
keywords: Acid wash; Arginine vasopressin (PubChem CID: 644077); Internalization; Radioligand binding; Receptor trafficking; Stable expression; V(1B) vasopressin receptor