RESUMEN
In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased affinity and efficiently coupled to Galpha(q/11). Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained confined to the plasma membrane even after prolonged agonist exposure (120 min). A striking difference in receptor stimulation was observed when the OT-induced effect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative effect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory effects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.
Asunto(s)
Microdominios de Membrana/química , Receptores de Oxitocina/análisis , Animales , Células COS , Caveolina 1 , Caveolina 2 , Caveolinas/análisis , División Celular , Vesículas Cubiertas por Clatrina/química , Proteínas de Unión al GTP/fisiología , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Receptores de Oxitocina/fisiologíaRESUMEN
The aim of this study was to identify loss-of-function mutations of the V2 vasopressin receptor gene (AVPR2) in Italian patients affected by X-linked nephrogenic diabetes insipidus (NDI). Mutations were found in 15 of the 18 unrelated families investigated: nine of these mutations were previously unknown, including two affecting residues located in regions known to be important for determining the pharmacologic properties of the receptor, which were therefore functionally investigated. The first (A84D) involves a residue located near an aspartic acid (D85) that is highly conserved in all G protein-coupled receptors and that is believed to play a role in the process of their isomerization into functionally active and inactive states. The present study indicates that this mutation not only affects receptor folding in such a way as to lead to its retention inside the intracellular compartments but, as expected, also has profound effects on its binding and coupling properties. The second was a mutation of a tryptophan located at the beginning of the first extracellular loop (W99R) that greatly impaired the binding properties of the receptor and had a minor effect on its intracellular routing. Molecular analysis of the first extracellular loop bearing this mutation suggests that this residue plays a fundamental role in stabilizing the peptide/receptor interactions responsible for the high-affinity binding of agonists to the V2 receptor.