Abstract

Chimeric D₁/D₂ receptors were constructed to identify structural determinants of drug affinity and efficacy. We previously reported that chimeras that had D₁ receptor transmembrane domain VII together with amino-terminal sequence from the D₂ receptor were nonfunctional. D₂/D₁ chimeras were constructed that contained D₂ receptor sequence at the amino- and carboxyl-terminal ends and D₁ receptor sequence in the intervening region. Chimeric receptors with D₂ sequence from transmembrane domain 7 to the carboxyl terminus together with D₂ receptor sequence from the amino terminus through transmembrane helix 4 (D_2[1–4,7]) and 5 (D_2[1–5,7]) bound [³H]spiperone with high affinity, consistent with the hypothesis that D₂ receptor transmembrane domain I or II is incompatible with D₁ receptor transmembrane domain VII. D_2[1–4,7] and D_2[1–5,7] had affinities similar to D₁ and D₂ receptors for most nonselective dopamine antagonists and had affinities for most of the selective antagonists that were intermediate between those of the parent receptors. D_2[1–4,7] and D_2[1–5,7]mediated dopamine receptor agonist-induced stimulation and inhibition, respectively, of cAMP accumulation. The more efficient coupling of D_2[1–5,7] to inhibition of cAMP accumulation, compared with the coupling of D_2[5–7] and D_2[3–7], supports the view that multiple D₂ receptor cytoplasmic domains acting in concert are necessary for receptor activation of G_i. In contrast, D_2[1–4,7], which contains only one cytoplasmic loop (the third) from the D₁ receptor, is capable of activating G_s. D_2[1–4,7]exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP accumulation, high affinity for agonists even in the presence of GTP, and blunted agonist-stimulated cAMP accumulation. A number of dopamine receptor antagonists were inverse agonists at D_2[1–4,7], inhibiting basal cAMP accumulation. Some of these drugs were also inverse agonists at the D₁ receptor. Interestingly, several antagonists also potentiated forskolin-stimulated cAMP accumulation via D_2[1–5,7] and via the D₂ receptor, which could reflect inverse agonist inhibition of native constitutive activity of this receptor.