Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase

Abstract

The PTH receptor is to our knowledge one of the first G protein-coupled receptor (GPCR) found to sustain cAMP signaling after internalization of the ligand-receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned off.

Figure 1. Effect of endosomal pH on PTHR signaling

(a) Averaged time-courses of cAMP production in response to PTH in HEK293 cells stably expressing the PTHR and the cAMP biosensor, epac-CFP/YFP. Individual cells were continuously perfused with buffer or with the ligand (100 nM) for the time indicated by the horizontal bar. Data represent mean values ± s.e.m. of five independent experiments and n = 80 cells for each condition. (b) Colocalization of PTH(1–34)^TMR and the early endosome marker, Rab5^GFP. Cells were briefly perfused (20 s) with 100 nM of PTH(1–34)^TMR and then with buffer alone for the remainder of the experiment. Data represent mean values ± s.e.m. of three independent experiments and n = 32 cells. (c) Recording pH using PTH^FITC in HEK293 cells expressing PTHR C-terminally tagged with CFP. Estimates were made using FITC fluorescence data together with the pH standard plot showed in Supplementary Fig. 2a. Data represent mean values ± s.e.m. of five independent experiments and n = 46 (control) and n = 71 (bafilomycin) cells. (d) Averaged dissociation time-courses of PTH(1–34)^TMR from PTHR N-terminally labeled with GFP. FRET recordings from HEK-293 cells treated with or without bafilomycin are shown as normalized ratio. Data represent mean values ± s.e.m. of three independent experiments and n = 68 (control) and n = 71 (bafilomycin) cells. (e) Time course of ¹²⁵I-PTH(1–34) dissociation at varying pH (n = 3). (f) Thermostability of the PTHR alone or associated with PTH(1–34) at varying pH (n = 3).

Figure 2. Negative feedback mechanism

(a) Cyclic AMP responses in osteosarcoma cells. Similar cAMP measurements as described in Fig. 1a were performed on ROS18/2.8 cells. Data represent the mean ± sem of 4 independent experiments and n = 50 (ctrl), n = 50 (bafilomycin), and n = 50 (PKA) cells. (b) Bars compare the averaged cAMP responses in ROS 17/2.8 (panel d) and HEK-293 cells (Fig. 1a) that is determined by measuring the area under the curve from 0 to 30 min of experiments. (f) Proposed model for shutting down PTHR signaling. A new regulatory mechanism of PTHR signaling where sustained cAMP signaling after receptor internalization is turned off by a negative feedback mechanism involving PKA and the v-ATPase. (c) Representative set of immunoblot of an immunoprecipitation experiment using the PKA phosphorylation substrate-specific antibody (upper) and FLAG (lower). Bars represent the mean ± s.e.m of n = 5 (^*P < 0.05). (d) Recording pH using PTH^FITC as described in Fig. 1c. Data represent the mean ± s.e.m. of five independent experiments and n = 45 (control), n = 50 (mut v-ATPase) and n = 57 (H89) cells. (e) Averaged time-courses of cAMP production in response to 100 nM PTH in live HEK293 cells stably expressing the PTHR as described in Fig. 1a. Data represent mean values ± s.e.m. of five independent experiments and n = 40 (control), n = 79 (H89), n = 124 (v-ATPase), and n = 173 (mut v-ATPase) cells.