Agonist Regulation of D2 Dopamine Receptor/G Protein Interaction

The D 2 dopamine receptor has been expressed in Sf21 insect cells together with the G proteins G o and G i2 , using the baculovirus system. Expression levels of receptor and G protein (α, β, and γ subunits) in the two preparations were similar as shown by binding of [ 3 H]spiperone and quantitative Western blot, respectively. For several agonists, binding data were fitted best by a two-binding site model in either preparation, showing interaction of expressed receptor and G protein. For some agonists, binding to the higher affinity site was of higher affinity in D 2 /G o than in the D 2 /G i2 preparation. Some agonists exhibited binding data that were best fitted by a two-binding site model in D 2 /G o and a one-binding site model in D 2 /G i2 . Therefore, receptor/G protein interaction seemed to be stronger in the D 2 /G o preparation. Agonist stimulation of [ 35 S]GTPγS (guanosine 5′-3- O -(thio)triphosphate) binding in the two preparations also gave evidence for higher affinity D 2 /G o interaction. In the D 2 /G o preparation, agonist stimulation of [ 35 S]GTPγS binding occurred at higher potency for several agonists, and a higher stimulation (relative to dopamine) was achieved in D 2 /G o compared with D 2 /G i2 . Some agonists were able to stimulate [ 35 S]GTPγS binding in the D 2 /G o preparation but not in D 2 /G i2 . The extent of D 2 receptor selectivity for G o over G i2 is therefore dependent on the agonist used, and thus agonists may stabilize different conformations of the receptor with different abilities to couple to and activate G proteins.