κ opioid receptor interacts with Na+/H +-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) to stimulate Na+/H+ exchange independent of Gi/Go proteins

Abstract

We previously showed that Na+/H+-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) co-immunoprecipitated with the human κ opioid receptor (hKOR) and that its over-expression blocked the κ agonist U50, 488H-induced hKOR down-regulation by enhancing recycling. Here, we show that glutathione S-transferase (GST)-hKOR C-tail interacted with purified NHERF-1/EBP50, whereas GST or GST-C-tails of μ or δ opioid receptors did not. GST-hKOR C-tail, but not GST, bound HA-NHERF-1/EBP50 transfected into Chinese hamster ovary cells and endogenous NHERF-1/EBP50 in opossum kidney proximal tubule epithelial cells (OK cells). The PDZ domain I, but not II, of NHERF-1/EBP50 was involved in the interaction. Association of NHERF-1/EBP50 with HKOR C-tail enhanced oligomerization of NHERF-1/EBP50. NHERF-1/EBP50 was previously shown to regulate Na+/H+-exchanger 3 (NHE3) activities in OK cells. We found stimulation of OK cells with U50,488H significantly enhanced Na+/H+ exchange, which was blocked by naloxone but not by pertussis toxin pretreatment, indicating it is mediated by KORs but independent of Gi/Go proteins. In OKH cells, a subclone of OK cells expressing a much lower level of N-HERF-1/EBP50, U50, 488H had no effect on Na+/H+ exchange, although it enhanced p44/42 mitogen-activated protein kinase phosphorylation via G i/Go proteins similar to that in OK cells. Stable transfection of NHERF-1/EBP50 into OKH cells restored the stimulatory effect of U50,488H upon Na+/H+ exchange. Thus, NHERF-1/EBP50 binds directly to KOR, and this association plays an important role in accelerating Na+/H+ exchange. We hypothesize that binding of the KOR to NHERF-1/EBP50 facilitates oligomerization of NHERF-1/EBP50, leading to stimulation of NHE3. This study provides the first direct evidence that a G protein-coupled receptor through association with NHERF-1/EBP-50 stimulates NHE3.