Interaction of the D 2short dopamine receptor with G proteins: analysis of receptor/G protein selectivity

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 [ 3 H ]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [ 35 S ]GTPγS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [ 35 S ]GTPγS 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 ([ 3 H ]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [ 3 H ]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [ 3 H ]NPA binding with greater potency (∼6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D 2S/Go preparation. [ 3 H ]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.