IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
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:
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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).