Differential modulation of mu- and delta-opioid receptor agonists by endogenous RGS4 protein in SH-SY5Y cells.
Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express mu- and delta-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by approximately 90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of delta-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the mu-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the delta-opioid receptor but not the mu-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the delta- and mu-opioid receptors, the C-tail and third intracellular domain of the delta-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating delta-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.
Wang
Qin
Q
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Liu-Chen
Lee-Yuan
LY
Traynor
John R
JR
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D005910:Glioma; D006031:Glycosylation; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009020:Morphine / Q000494:pharmacology; D009447:Neuroblastoma; D010879:Piperazines / Q000494:pharmacology; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D000701:Analgesics, Opioid; D001549:Benzamides; D010879:Piperazines; D020710:RGS Proteins; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C088464:4-(alpha-(4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N,N-diethylbenzamide; C098799:RGS4 protein; D005576:Colforsin; D009020:Morphine; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases