Agonist regulation of D2 dopamine receptor/G protein interaction -: Evidence for agonist selection of G protein subtype

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 (alpha, beta, and gamma subunits) in the two preparations were similar as shown by binding of [H-3]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 [S-35]GTP gammaS (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 [S-35]GTP gammaS binding occurred at higher potency for several agonists, and a higher stimulation (relative to dopamine) was achieved in D-2/G(o) compared with D2/Gi2, Some agonists were able to stimulate [S-35]GTP gammaS binding in the D-2/G(o) preparation but not in D2/Gi2. The extent of D2 receptor selectivity for Go over Gi2 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.