IN-VIVO RECONSTITUTION OF DOPAMINE D-2S RECEPTOR-MEDIATED G-PROTEIN ACTIVATION IN BACULOVIRUS-INFECTED INSECT CELLS - PREFERRED COUPLING TOG(I1) VERSUS G(I2)

Agonist binding of the human D-2S receptor overexpressed in baculoviru s-infected Sf9 insect cells was of low affinity and GppNHp-insensitive , yet, dopaminergic agonists were able to partly inhibit forskolin-sti mulated cAMP accumulation. In order to prove full functionality of the receptor, we used an ''in vivo'' reconstitution system, which is base d on coinfection of Sf9 cells with the appropriate receptor and G prot ein encoding baculoviruses. In cells coexpressing the D-2S receptor an d either G(i1) or G(i2), the dopaminergic agonist apomorphine effectiv ely stimulated [S-35]GTP gamma S binding and GTPase activity. Agonist- stimulated [S-35]GTP gamma S binding was dependent on the ratio of G p rotein to receptor. Expression levels of receptor and G protein influe nced each other reciprocally. G protein activation could be optimized by varying the multiplicity of infection of the receptor and G protein encoding baculoviruses. Coexpression of either G(i1) or G(i2) led to the appearance of GppNHp-sensitive high-affinity agonist binding. Deta iled agonist competition binding analysis revealed that the percentage of high-affinity agonist binding sites was significantly higher in D- 2S receptor-expressing cells coinfected with G(i1) viruses than when c oinfected with G(i2) viruses. Moreover, the coexpressed Gi proteins se emed to modulate the affinity of agonists for the high-affinity form o f the receptor. In cells coexpressing G(i1), agonist high affinity was 2-4-fold higher than in cells coexpressing G(i2). Na+ increased the d issociation constant of apomorphine for the high affinity site by 2-4- fold without affecting the percentage of high-affinity sites or the pr eference for G(i1). In some dopamine competition experiments with coin fected cells, displacement data were best fit assuming three nonintera cting classes of sites in the absence and two independent classes of s ites in the presence of GppNHp. Dopamine competition curves with cells highly overexpressing the D-2S receptor or with membranes from such c ells were best fit assuming two independent classes of sites which wer e insensitive to GppNHp and might reflect abnormal compartimentalizati on and/or different states of aggregation.