INVESTIGADORES
GIRONACCI Mariela Mercedes
congresos y reuniones científicas
Título:
Angiotensin-(1-7) induces Mas-YFP receptor redistribution in HEK293 cells
Autor/es:
N FERNANDEZ, OA CARRETERO, RAS SANTOS, P ORTIZ, HP ADAMO, MM GIRONACCI
Lugar:
Belo Horizonte, Brasil
Reunión:
Congreso; XVIIIth Scientific Sessions of the Inter-American Society of Hypertension; 2009
Resumen:
<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Signal transduction coupled to G-protein coupled receptors (GPCRs) activation is carefully controlled by: desensitization, internalization of the receptor-ligand complex, loss of extracellular ligand-binding sites or modulation of receptor affinity. The Mas receptor, a member of the GPCR family, mediates most of the actions of angiotensin-(1-7) (Ang-(1-7)). Mas receptors have been cloned but the mechanism by which its activation is controlled is unknown. In this study we examined the functional heterologous expression of the Mas receptor in HEK 293 cells. The yellow fluorescent protein (YFP) was attached to the carboxy-terminus end of the Mas receptor by the megaprimer method and cloned into XhoI-ApaI sites of pEYFP-N1 (Clontech) plasmid. The sequence of the construct was confirmed by enzymatic digestion and sequence analysis. HEK293 cells were transiently transfected with the Mas-YFP coding vector using lipofectamine 2000 (Amersham). Confocal fluorescence microscopy showed that a large proportion of the cells expressed the fluorescent construction 48 h after the transfection. To determine whether the fusion of Mas receptor to YFP alter Ang-(1-7) binding we studied 125I-Ang-(1-7) binding to transiently transfected HEK293 cells at 0 oC during 60 min in the presence of increasing concentrations of unlabeled Ang-(1-7). Ang-(1-7) displaced the binding of 125I-Ang-(1-7) to cells (IC50: 8.66+5.04 x10-8 M). In addition, the Mas receptor antagonist D-Ala7-Ang-(1-7) or the AT2 receptor antagonist PD123319, but not the AT1 receptor antagonist losartan, displaced the binding of 125I-Ang-(1-7). To begin studying whether Ang-(1-7) may induce Mas receptor internalization, transiently transfected HEK293 cells were incubated with 1 mM Ang-(1-7) for 5, 10, 15, 30 and 60 min, then cooled and fixed. The relative cellular distribution of Mas-YFP was observed by confocal microscopy. The obtained fluorescent images suggest that a larger proportion of intracellular Mas-YFP is found in cells treated with Ang-(1-7), suggesting internalization of the Mas receptor after 5 min. We conclude that fusion of Mas with YFP results in plasma membrane expression of Mas receptor. Our data suggest that Mas receptor internalization may be involved in desensitization during prolonged stimulation with Ang-(1-7).