CONSTITUTIVE ACTIVITY OF A CHIMERIC D-2 D-1 DOPAMINE-RECEPTOR/

Chimeric D-1/D-2 receptors were constructed to identify structural det erminants of drug affinity and efficacy. We previously reported that c himeras that had D-1 receptor transmembrane domain VII together with a mino-terminal sequence from the D-2 receptor were nonfunctional. D-2/D -1 chimeras were constructed that contained D-2 receptor sequence at t he amino-and carboxyl-terminal ends and D-1 receptor sequence in the i ntervening region. Chimeric receptors with D-2 sequence from transmemb rane domain 7 to the carboxyl terminus together with D-2 receptor sequ ence from the amino terminus through transmembrane helix 4 (D-2[1-4,D- 7]) and 5 (D-2[1-5,D-7]) bound [H-3]spiperone with high affinity, cons istent with the hypothesis that D-2 receptor transmembrane domain I or II is incompatible with D-1 receptor transmembrane domain VII. D-2[1- 4,D-7] and D-2[1-5,D-7] had affinities similar to D-1 and D-2 receptor s for most nonselective dopamine antagonists and had affinities for mo st of the selective antagonists that were intermediate between those o f the parent receptors. D-2[1-4,D-7] and D-2[1-5,D-7] mediated dopamin e receptor agonist-induced stimulation and inhibition, respectively, o f cAMP accumulation, The more efficient coupling of D-2[1-5,D-7] to in hibition of cAMP accumulation, compared with the coupling of D2[5-7] a nd D2[3-7], supports the view that multiple D-2 receptor cytoplasmic d omains acting in concert are necessary for receptor activation of G(i) . In contrast, D-2[1-4,D-7], which contains only one cytoplasmic loop (the third) from the D-1 receptor, is capable of activating G(s). D-2[ 1-4,D-7] exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP ac cumulation, high affinity for agonists even in the presence of GTP, an d blunted agonist-stimulated cAMP accumulation. A number of dopamine r eceptor antagonists were inverse agonists at D-2[1-4,D-7], inhibiting basal cAMP accumulation, Some of these drugs were also inverse agonist s at the D-1 receptor. Interestingly, several antagonists also potenti ated forskolin-stimulated cAMP accumulation via D-2[1-5,D-7] and via t he D-2 receptor, which could reflect inverse agonist inhibition of nat ive constitutive activity of this receptor.