Nucleoside diphosphate kinase associated with membranes modulates mu-opioid receptor-mediated [S-35]GTP gamma S binding and agonist binding to mu-opioid receptor

The role of nucleoside diphosphate kinase (NDKP), which converts GDP to GTP , in the coupling of mu-opioid receptors to G protein was investigated in m embranes of Chinese hamster ovary cells stably transfected with the cloned rat mu-opioid receptor (rmor). Endogenous NDPK activity in membranes was de termined to be 0.60 +/- 0.02 mu mol/mg protein/30 min UDP (at 10 mM), a com petitive substrate of NDPK for GDP with no effect on guanine nucleotide bin ding to G proteins, reduced basal [S-35]GTP gamma S binding and unmasked mo rphine-stimulated [S-35]GTP gamma S binding to pertussis toxin-sensitive G proteins, indicating that [S-35]GTP gamma S binding to NDPK accounts for pa rt of its high basal binding. UDP increased the extent of morphine-induced increase in [S-35]GTP gamma S binding in the presence of GDP, most likely b y reducing basal binding and inhibiting conversion of GDP to GTP. ATP great ly reduced morphine-induced increase in [S-35]GTP gamma S binding, whereas AMP-PCP (adenylyl-(beta,gamma-methylene)-diphosphoate tetralithium salt), w hich cannot serve as the phosphate donor for NDPK, did not, demonstrating t hat effects of ATP is mediated by the NDPK product GTP. In addition, GDP an d ATP increased the K-d and lowered the B-max of the agonist [H-3]DAMGO ([D -Ala(2),N-Me-Phe(4),Gly(5)ol]-Enkephalin) for the mu-opioid receptor and GD P alone increased K-d, most likely through their conversion to GTP by NDPK. Addition of exogenous NDPK enhanced the inhibitory effects of GDP and comb ined GDP and ATP on [H-3]DAMGO binding. Thus, NDPK appears to play a role i n modulating signal transduction of and agonist binding to mu-opioid recept ors. (C) 1999 Elsevier Science B.V. All rights reserved.