EP receptors belong to the family of G protein coupled
EP1–4 receptors belong to the family of G protein-coupled receptor (GPCR). Trafficking events such as externalization, internalization, recycling and degradation dynamically regulate GPCR cell surface density. A fast-rate of internalization and degradation reduces GPCR surface density to desensitize cell response while a fast rate of recycling increases GPCR surface density to re-sensitize cell response. We recently showed that PGE2 or EP4 agonist increased EP4 externalization in in vitro and in vivo DRG neurons (St-Jacques and Ma, 2013). This event was blocked not only by an inhibitor of anterograde trafficking from ER/Golgi complex to the cell surface, but also by an inhibitor of recycling (St-Jacques and Ma, 2013), suggesting that in addition to EP4 externalization, EP4 recycling also contributes to agonist-enhanced EP4 surface abundance and nociceptor sensitization. In this study, we attempted to test this hypothesis. The first aim was to examine the basal and agonist-stimulated EP4 internalization in cultured DRG neurons using antibody feeding-based internalization assay. Since internalized EP4 receptors could go through both recycling and degradation pathways, the second aim was to examine the co-localization of internalized EP4 with specific markers for the early endosomes (EEA1), the recycling endosomes (Rab11), the late endosomes (Rab7) and the n-acetyl-l-cysteine sale (Lamp1) to track the internalized EP4 in these organelles after internalization. Fluorescent dye-conjugated agonist has widely been used in vitro and in vivo to trace GPCR internalization (Cahill et al., 2001, Pheng et al., 2003). Alternatively, in this study we used fluorescent dye FITC-conjugated PGE2 to trace EP receptor internalization in cultured DRG neurons. Thus, the third aim was to examine FITC-PGE2-induced EP internalization and the distribution of internalized EP receptors in the early endosomes and lysosomes. The fourth aim was to examine EP4 recycling following agonist stimulation using antibody feeding-based recycling assay. As mentioned above, PGE2 stimulates the release of CGRP from primary sensory neurons and this event was mediated through EP4 (Southall and Vasko, 2001). Thus CGRP release from cultured DRG neurons evoked by EP4 agonist was used as an indicator to gauge EP4 receptor activity. Therefore our last aim was to determine whether agonist-facilitated EP4 recycling contributes to exaggerated CGRP release from cultured DRG neurons.
Results Two days after seeding, cultured DRG neurons were treated with a selective EP4 agonist 1-OH-PGE1 for 15, 30 or 60min at 37°C. When cultured cells were kept at 4°C throughout the experiment, only EP4 receptors pre-existing at the cell surface were observed (A, B). No significant difference in the surface EP4 levels was detected between vehicle and 1-OH-PGE1 treatments (A, B). Compared to the pre-existing surface EP4 revealed by surface immunostaining at 4°C, vehicle (C, E, G) or 1-OH-PGE1 (D, F, H) markedly increased EP4 internalization at 37°C. However, when cultured DRG cells were treated at 37°C, 10μM 1-OH-PGE1 significantly increased the levels of internalized EP4 (C, E, G, p<0.05–0.01) compared to vehicle treatment (0.01% ethanol in DMEM-HEPES, D, F, H). Compared to 15- and 30-min treatments (D, F, I), 1-OH-PGE1 treatment for 60min significantly increased internalized EP4 (H, I, p<0.05). No significant difference was detected between 15 and 30min of PGE2 treatment. There was also no significant difference among 15-, 30- and 60-min treatments of vehicle (C, E, G, I). Following a 60-min treatment, 10 and 50μM 1-OH-PGE1 significantly increased the density of internalized EP (J, p<0.05). However, no significant difference in the intracellular intensity of internalized EP4 was detected between 10 and 50μM 1-OH-PGE2 treatments. 1-OH-PGE1 at 1μM had no effects. To determine whether internalized EP4 undergoes the recycling and degradation pathways, co-localization of internalized EP4 with the early endosome marker EAA1, the recycling endosome marker Rab11 and the late endosome marker Rab7 and Lamp 1 was performed after 60-min 1-OH-PGE1 treatments. Following a 60-min treatment, co-localization (C, F, yellow) of internalized EP4 (A, D, green) with EEA1 (B, red) or Rab11 (E, red) were frequently seen. However, internalized EP4 receptors (G, J, green) were rarely co-localized with Lamp1 (H, red) or Rab7 (K, red) in cultured DRG neurons (I, L). Omission of N-terminal EP4 antiserum or incubation with N-terminal EP4 antiserum pre-absorbed with antigen peptides in antibody feeding resulted in no EP4-IR, but only EEA1-, Rab11-, lamp1- and Rab7-IR remained (not shown). EP4 originally existing in intracellular compartments cannot be visualized by this technical approach.