Fig. 3.

TRPV1 activation induces nuclear translocation of GRK5, the Ca²⁺/CaM-binding kinase responsible for MOR1 phosphorylation. (A) Western blot of HEK293 cells coexpressing HA-MOR1 and myc-TRPV1 treated combinatorially with capsaicin (1 µM) for 5 min before and after 15 min of DAMGO. (B) Western blot of membrane fractions collected from HEK293 cells coexpressing myc-TRPV1, HA-MOR1-WT, and FLAG-GRK5. Before membrane isolation, cells were treated combinatorially with RTX (1 µM) for 5 min, followed by DAMGO (1 µM) with or without EGTA (5 mM) for 15 min. (C) Immunofluorescence of HEK293 cells coexpressing myc-empty vector or myc-TRPV1 with FLAG-GRK5 with or without RTX (1 µM) for 5 min. (D and E) Calmodulin-Sepharose pulldown with lysate from HEK293 cells containing transiently overexpressed FLAG-GRK5 WT or FLAG-EV (D) or truncated FLAG-GRK5 Δ20–39 (E) in the presence (2 mM CaCl₂) or absence (5 mM EGTA) of calcium. (F) Western blot of HEK293 cells coexpressing myc-TRPV1, HA-MOR1-WT, and FLAG-GRK5 (WT, 1–540, Δ20–39). Cells treated with capsaicin or ionomycin (1 µM) for 5 min, followed by DAMGO (1 µM) for 15 min.