kappa 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 G(i)/G(o) proteins.
We previously showed that Na(+)/H(+)-exchanger regulatory factor-1/Ezrin-radixin-moesin-binding phosphoprotein-50 (NHERF-1/EBP50) co-immunoprecipitated with the human kappa opioid receptor (hKOR) and that its overexpression blocked the kappa 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 micro or delta 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 G(i)/G(o) proteins. In OKH cells, a subclone of OK cells expressing a much lower level of NHERF-1/EBP50, U50,488H had no effect on Na(+)/H(+) exchange, although it enhanced p44/42 mitogen-activated protein kinase phosphorylation via G(i)/G(o) 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.
Huang
Peng
P
Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Steplock
Deborah
D
Weinman
Edward J
EJ
Hall
Randy A
RA
Ding
Zhe
Z
Li
Jianguo
J
Wang
Yulin
Y
Liu-Chen
Lee-Yuan
LY
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009270:Naloxone / Q000494:pharmacology; D009893:Opossums; D037342:Pertussis Toxin / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010766:Phosphorylation; D017473:Receptors, Opioid, kappa / Q000737:chemistry / Q000502:physiology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D002352:Carrier Proteins; D010750:Phosphoproteins; D017473:Receptors, Opioid, kappa; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D009270:Naloxone; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D037342:Pertussis Toxin; D020928:Mitogen-Activated Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go