Nucleoside diphosphate kinase associated with membranes modulates μ-opioid receptor-mediated [ [formula omitted]]GTPγS binding and agonist binding to μ-opioid receptor

Abstract

The role of nucleoside diphosphate kinase (NDKP), which converts GDP to GTP, in the coupling of μ-opioid receptors to G protein was investigated in membranes of Chinese hamster ovary cells stably transfected with the cloned rat μ-opioid receptor (rmor). Endogenous NDPK activity in membranes was determined to be 0.60±0.02 μmol/mg protein/30 min UDP (at 10 mM), a competitive substrate of NDPK for GDP with no effect on guanine nucleotide binding to G proteins, reduced basal [ 35 S ]GTPγS binding and unmasked morphine-stimulated [ 35 S ]GTPγS binding to pertussis toxin-sensitive G proteins, indicating that [ 35 S ]GTPγS binding to NDPK accounts for part of its high basal binding. UDP increased the extent of morphine-induced increase in [ 35 S ]GTPγS binding in the presence of GDP, most likely by reducing basal binding and inhibiting conversion of GDP to GTP. ATP greatly reduced morphine-induced increase in [ 35 S ]GTPγS binding, whereas AMP-PCP (adenylyl-(β,γ-methylene)-diphosphoate tetralithium salt), which cannot serve as the phosphate donor for NDPK, did not, demonstrating that effects of ATP is mediated by the NDPK product GTP. In addition, GDP and ATP increased the K d and lowered the B max of the agonist [ 3 H ]DAMGO ([ d-Ala 2, N-Me-Phe 4,Gly 5ol]-Enkephalin) for the μ-opioid receptor and GDP alone increased K d, most likely through their conversion to GTP by NDPK. Addition of exogenous NDPK enhanced the inhibitory effects of GDP and combined GDP and ATP on [ 3 H ]DAMGO binding. Thus, NDPK appears to play a role in modulating signal transduction of and agonist binding to μ-opioid receptors.