FIGURE 2.

β-Arrestin 1 is required for angiotensin-induced internalization and functional down-regulation of TRPV4. A–C, angiotensin-induced internalization of TRPV4. A, rVSMCs were stimulated with angiotensin (Ang) (1 μm) for the indicated time period, fixed, permeabilized, and stained using goat polyclonal anti-TRPV4 antibody followed by Alexa Fluor 488-conjugated secondary antibody (green: TRPV4). B, HEK-293 cells expressing HA-AT1aR and FLAG-TRPV4 were stimulated with angiotensin for the indicated time points, and surface TRPV4 was labeled with NHS-S-S-biotin. The cells were lysed, immunoprecipitated using NeutrAvidin beads, and immunoblotted with a mouse monoclonal anti-FLAG antibody. C, densitometry analysis of angiotensin-induced TRPV4 internalization as measured by surface biotinylation (shown in panel B). Data are presented as the percentage of initial levels of TRPV4 at the cell surface and show mean ± S.E. from at 3 independent experiments. D–F, involvement of β-arrestin 1 in TRPV4 internalization. D, 4-α-PDD-induced Ca²⁺ influx measured in HEK-293 cells. The cells were prestimulated with either angiotensin or SII-angiotensin (SII-Ang), and subsequently 4-α-PDD-induced Ca²⁺ influx was measured using the Fluo-4 NW calcium assay kit. The data are presented as the percentage of control cells, i.e. cells prestimulated with saline. NS, non-stimulated. E, 4-α-PDD-induced Ca²⁺ influx measured in HEK-293 cells after depletion of β-arrestin 1 (β-arr 1) or β-arrestin 2 (β-arr 2) using siRNA. CTL is control scrambled siRNA. F, angiotensin-induced internalization was measured in HEK-293 cells by surface biotinylation after depletion of β-arrestin 1 or 2 using siRNA using mouse monoclonal anti-FLAG antibody. Representative blots are shown from at least 3 independent experiments. G, densitometry analysis of angiotensin-induced internalization of TRPV4 (shown in panel F). Data are normalized with respect to the non-treated control siRNA sample (100%). The data presented in panels D–F show the mean ± S.E. from 3–4 independent experiments, and the statistical analysis was carried out using one-way ANOVA with Bonferroni post test.