Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney

Abstract

Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.

African lungfish,Protopterus annectens, possess an arginine vasotocin receptor homologous to the tetrapod V2-type receptor

“…8A). The molecular mass of the immunoreactive band for lungfish V2-type receptor was similar to that for the glycosylated form (47-62 kDa) of mammalian V2 receptors (Fenton et al, 2007;Gutkowska et al, 2007). As shown in Fig.…”

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

“…Immunoblotting-The preparation of samples and immunoblotting were as described previously (25). A horseradish peroxidase-conjugated secondary antibody (DAKO P448, goat anti-rabbit IgG, or DAKO P447, goat anti-mouse IgG) was used at 1:5,000, and antibody-antigen reactions were visualized using SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Denmark).…”

Vasopressin regulates the renin-angiotensin-aldosterone system via V1a receptors in macula densa cells

“…Tissue lysates were prepared from microdissected outer medullary collecting duct (OMCD), inner medulla, and whole kidney, and Western blot analyses were performed with anti-V2R (a kind gift from Dr. Søren Nielsen, University of Aarhus, Aarhus, Denmark), anti-AQP2 (Santa Cruz Biotechnology), anti-renin, or anti-COX-2 antibody as described previously (6,8,12,23).…”