期刊
JOURNAL OF NEUROCHEMISTRY
卷 135, 期 1, 页码 76-87出版社
WILEY-BLACKWELL
DOI: 10.1111/jnc.13222
关键词
adenylyl cyclase; cell signaling; G protein-coupled receptor; immunoprecipitation; opiate opioid; regulator of G protein signaling
资金
- NRSA [102581]
- USUHS [RO75QU]
The interaction of Regulator of G protein Signaling 4 (RGS4) with the rat mu opioid receptor (MOR)/G protein complex was investigated. Solubilized MOR from rat brain membranes was immunoprecipitated in the presence of RGS4 with antibodies against the N-terminus of MOR (anti-MOR10-70). Activation of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin (DAMGO) during immunoprecipitation caused a 150% increase in Go alpha and a 50% increase in RGS4 in the pellet. When 10 mu M GTP was included with DAMGO, there was an additional 72% increase in RGS4 co-immunoprecipitating with MOR (p = 0.003). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) increased the amount of co-precipitating RGS4 by 93% (compared to DAMGO alone, p = 0.008), and the inclusion of GTP gamma S caused the ratio of MOR to RGS4 to be 1 : 1 (31 fmoles : 28 fmoles, respectively). GTP gamma S also increased the association of endogenous RGS4 with MOR. In His(6)RGS4/Ni2+-NTA agarose pull down experiments, 0.3 mu M GTP gamma S tripled the binding of Goa to His(6)RGS4, whereas the addition of 100 mu M GDP blocked this effect. Importantly, activation of solubilized MOR with DAMGO in the presence of 100 mu M GDP and 0.3 mu M GTP gamma S increased Goa binding to His(6)RGS4/Ni2+-NTA agarose (p = 0.001).
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