Mechanism and function of drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor

The capa peptide receptor, capaR (CG14575), is a G-protein coupled receptor (GPCR) for the D. melanogaster capa neuropeptides, Drm-capa-1 and -2 (capa-1 and -2). To date, the capa peptide family constitutes the only known nitridergic peptides in insects, so the mechanisms and physiological function of ligand-receptor signalling of this peptide family are of interest. Capa peptide induces calcium signaling via capaR with EC50 values for capa-1 = 3.06 nM and capa-2 = 4.32 nM. capaR undergoes rapid desensitization, with internalization via a b-arrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of capa-1. Drosophila capa peptides have a C-terminal -FPRXamide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25 and the insect -PRLamides [Drosophila pyrokinins Drm-PK-1 (capa-3), Drm-PK-2 and hugin-gamma [hugg]] against capaR were investigated. NMU-25, but not hugg nor Drm-PK-2, increases intracellular calcium ([Ca2+]i) levels via capaR. In vivo, NMU-25 increases [Ca2+]i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca2+]i and fluid transport. Finally, anti-porcine NMU-8 staining of larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. These structural and functional data suggest that vertebrate NMU is a putative functional homolog of Drm-capa-1 and -2. capaR is almost exclusively expressed in tubule principal cells; cell-specific targeted capaR RNAi significantly reduces capa-1 stimulated [Ca2+]i and fluid transport. Adult capaR RNAi transgenic flies also display resistance to desiccation. Thus, capaR acts in the key fluid-transporting tissue to regulate responses to desiccation stress in the fly

Congenital arterioportal fistula of the liver with reversal of flow in the superior mesenteric vein

We report on a newborn with a large arterioportal fistula of the liver complicated by a sudden reversal of flow in the portal and superior mesenteric vein, leading to congestion and ischemia of the small intestine. Ultrasound, duplex Doppler sonography and angiographic features are presented. Ligation of the hepatic artery led to a complete recovery

Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels\ud

Objectives: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. Methods and Results: We genotyped the control populations of two population-based studies for four AVPR2 variations: a-245c, G12E, L309L, and S331S. Rare alleles of a-245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G-V2R or 12E-V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three-fold in 12E-V2R–green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (KD) was 4.5 nm [95% confidence interval (CI) 3.6–5.4] for 12E-V2R–GFP and 16.5 nm (95% CI 10.1–22.9) for 12G-V2R–GFP. AVP-induced cAMP generation was enhanced in 12E-V2R–GFP cells. Conclusions: The 12E-V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVII

Functional rescue of mutant Vasopressin V2 Receptors in Nephrogenic Diabetes Insipidus- From molecular cause to restored phenotype.

Item does not contain fulltextMutations in G protein-coupled receptors are the cause of many inheritable disorders, including X-linked Nephrogenic Diabebetes Insipidus (NDI). Patients suffering from NDI are unable to concentrate their urine in response to the antidiuretic hormone arginine-vaspressin (AVP), which increases the risk of dehydration in these patients. In this thesis, we describe the molecular causes that underlie X-linked NDI, which is due to mutations in the vasopressin V2 receptor (V2R). These mutations cause misfolding of the V2R, which leads to its entrapment in the endoplasmic reticulum (ER) as it fails the quality control standards of this organelle. However, in overexpression studies in COS cells, we found that several mutants were able to function on the molecular level, i.e. they are able to bind AVP and activate the stimulatory G protein. Since their intracellular retention prevents them to bind AVP at the cell surface, we set out to rescue the localization and/or the function of these receptor mutants in a polarized kidney cell line that mimics the kidney collecting duct. 1)We found that several chemical chaperones were able to rescue the cell surface localization of the V2R-V206D mutant, but not of eight other V2R mutants. 2)Cell-permeable antagonists (or ?pharmacological chaperones?) of the V2R were able to stabilize of 8 out of 9 V2R mutants in the ER and promote their trafficking to the cell surface, where they could be activated to different extents. This functional rescue was a balance between the ability of the antagonist to restore cell surface expression, and its subsequent displacement by AVP. 3)Cell-permeable agonists did not induce plasma membrane trafficking, but rescued their function by activating the mutant receptors inside the cell. Since cell-permeable agonists and antagonists are available for clinical use, or may become available in the near future, they are promising candidates to relieve NDI in patients.RU Radboud Universiteit Nijmegen, 06 december 2006Promotores : Knoers, N.V.A.M., Bindels, R.J.M. Co-promotor : Deen, P.M.T.169 p

Water in health and disease: new aspects of disturbances in water metabolism.

Contains fulltext : 53130.pdf (publisher's version ) (Open Access)Vasopressin is a critical regulator of water homeostasis. There are two major receptors for vasopressin: V1 and V2 receptors. Disturbances in water balance are commonly encountered in clinical practice and can be divided into disorders of urinary dilution and concentration. The major representatives of such disorders are diabetes insipidus and the syndrome of inappropriate secretion of antidiuretic hormone (SI ADH). Recent studies show that genetic forms of nephrogenic diabetes insipidus are due to mutations in the genes coding for the vasopressin V2 receptor (V2R) or aquaporin-2 (AQP2). Identification of the genes involved and analysis of the cellular fate of the V2R and AQP2 mutants are relevant for understanding the functioning of the V2R and AQP2 protein. These developments also have implications for future therapeutic options. The development of nonpeptide vasopressin receptor antagonists (VRAs) offers prospects for the treatment of euvolaemic (SI ADH) or hypervolaemic hyponatraemia (congestive heart failure or cirrhosis). Several nonpeptide VRAs are now in various stages of clinical trials. At present, only conivaptan is registered by the FD A for intravenous treatment of euvolaemic and hypervolaemic hyponatremia. A recent long-term study comparing tolvaptan with placebo in patients with chronic heart failure showed no reduction in risk of death and hospitalisation

Pharmacological chaperones in nephrogenic diabetes insipidus: possibilities for clinical application.

Contains fulltext : 53582.pdf (publisher's version ) (Closed access)The antidiuretic hormone arginine-vasopressin regulates water homeostasis in the human body by binding to its vasopressin type 2 receptor (V2R). Mutations in AVPR2, the gene encoding V2R, lead to the X-linked congenital form of nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine in response to vasopressin; often this involves missense mutations or deletion of one or a few amino acids. In vitro V2R expression studies revealed that the function of most of these receptors is not disturbed, but due to their misfolding, the quality control mechanism of the endoplasmic reticulum (ER) retains these receptors inside the cell, thereby preventing their functioning at the plasma membrane. This review summarizes our current knowledge on ER retention of V2R mutants, and describes the different approaches that have been undertaken to restore the plasma membrane expression and function of V2R mutants in NDI in vitro and in vivo. The use of cell permeable receptor ligands (called 'pharmacological chaperones') appears promising for the treatment of NDI in a subset of patients

Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus

In the renal collecting duct, water reabsorption is regulated by the antidiuretic hormone vasopressin (AVP). Binding of this hormone to the vasopressin V2 receptor (V2R) leads to insertion of aquaporin-2 (AQP2) water channels in the apical membrane, thereby allowing water reabsorption from the pro-urine to the interstitium. The disorder nephrogenic diabetes insipidus (NDI) is characterized by the kidney's inability to concentrate pro-urine in response to AVP, which is mostly acquired due to electrolyte disturbances or lithium therapy. Alternatively, NDI is inherited in an X-linked or autosomal fashion due to mutations in the genes encoding V2R or AQP2, respectively. This review describes the current knowledge of the cell biological causes of NDI and how these defects may explain the patients' phenotypes. Also, the increased understanding of these cellular defects in NDI has opened exciting initiatives in the development of novel therapies for NDI, which are extensively discussed in this review

Characterization of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus in a polarized cell model

Contains fulltext : 48643.pdf (publisher's version ) (Open Access)X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the gene encoding the vasopressin V2 receptor (V2R). For the development of a tailored therapy for NDI, knowledge of the cellular fate of V2R mutants is needed. It would be useful when this fate could be predicted from the location and type of mutation. To identify similarities and differences in localization, maturation, stability, and degradation of COOH-terminal GFP-tagged V2R mutants, we stably expressed nine mutants in polarized Madin-Darby canine kidney cells. The mutants V2R-L44P, -Delta62-64, -I130F, -S167T, -S167L, and -V206D were mainly expressed in the endoplasmic reticulum (ER) as immature proteins. These mutants had relatively short half-lives due to proteasomal degradation, except for V2R-Delta62-64. In contrast, V2R-R113W, -G201D, and -T204N were expressed in the ER and in the basolateral membrane as immature, high-mannose glycosylated, and mature complex-glycosylated proteins. The immature forms of V2R-R113W and -T204N, but not V2R-G201D, were rapidly degraded. The mature forms varied extensively in their stability and were degraded by only lysosomes (V2R-T204N and wild-type V2R) or lysosomes and proteasomes (V2R-G201D, -R113W). These data reveal that most missense V2R mutations lead to retention in the ER and suggest that mutations that likely distort a transmembrane domain or introduce a charged amino acid close to it make a V2R mutant more prone to ER retention. Because six of the mutants tested showed significant increases in intracellular cAMP levels on transient expression in COS cells, activation of these six receptors following rescue of cell-surface expression might provide a cure for NDI patients