Interaction of the D2short dopamine receptor with G proteins: analysis of receptor/G protein selectivity |
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Authors: | Nickolls Sarah A Strange Philip G |
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Institution: | School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK. |
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Abstract: | The human D(2short) (D(2S)) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using 3H]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using 35S]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of 35S]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D(2S) receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine (3H]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited 3H]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited 3H]NPA binding with greater potency ( approximately 6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D(2S)/Go preparation. 3H]Spiperone binding occurred with an increased B(max) in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D(2S) receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein. |
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Keywords: | ECL enhanced chemiluminescence GPCR G protein-coupled receptor Kl and Kh lower and higher affinity dissociation constants moi multiplicity of infection NPA 10 11-dihydroxy-N-n-propylnorapomorphine 3-PPP 3-(3-hydroxyphenyl)-N-propylpiperidine |
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