ESPE2015 Poster Category 3 Late Breaking Posters (15 abstracts)
Profiling of a Novel NSIAD-Causing Mutation of Vasopressin Receptor 2 and its Differential Effect on Receptor Trafficking Compared to Previously Identified Mutations
Anatoly Tiulpakov a , Carl W White b, , Rekhati Abhayawardana b, , Natalia Zubkova a , Ruth M Seeber b, , Heng B See b, & Kevin D G Pfleger b,
aEndocrinology Research Centre, Moscow, Russia; bHarry Perkins Institute of Medical Research, Nedlands, Western Australia, Australia; cThe University of Western Australia, Crawley, Western Australia, Australia
Background: Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) results from gain-of-function mutations in the AVPR2 gene coding for vasopressin receptor 2 (V2R). In contrast, nephrogenic diabetes insipidus (NDI) is caused by loss-of-function mutations in AVPR2. Here we describe and functionally characterize a novel mutation located in the seventh transmembrane domain of V2R. This mutation was identified in a boy suffering from water-induced hyponatremic seizures at the age of 5.8 years.
Objective and hypotheses: The objective of this study was to profile a novel mutation of V2R found in a boy exhibiting symptoms of NSIAD, with the hypothesis that it was disease-causing. A further objective was to apply a cutting-edge biophysical approach to compare the real-time intracellular trafficking profile of this mutant receptor compared with a number of other NSIAD- and NDI-associated mutant receptors.
Method: cAMP and inositol phosphate signalling were assessed using homogeneous time-resolved fluorescence (HTRF) assays. Arrestin recruitment and proximity to cellular compartment markers was assessed using bioluminescence resonance energy transfer (BRET) in real-time, with live HEK293FT cells transiently transfected with appropriate cDNA constructs.
Results: The novel mutation resulted in substantial constitutive cAMP signalling. Furthermore, the trafficking profile differed from that observed with other mutant receptors.
Conclusion: The observed constitutive cAMP signalling is consistent with the NSIAD phenotype, indicating that this is indeed a novel NSIAD-causing mutation. Moreover, NSIAD and NDI can be caused by mutations that have a variety of effects on V2R function.
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