A primordial dopamine D1-like adenylyl cyclase-linked receptor from Drosophila melanogaster displaying poor affinity for benzazepines

Abstract

We report here the isolation from Drosophila melanogaster of a 2.0 kb cDNA clone encoding a 385 amino acid protein (dDA1) displaying, within putative transmembrane domains, highest amino acid sequence homology (49–53%) to members of the vertebrate dopamine D1-like receptor family. When expressed in either Sf9 or COS-7 cells, dDA1 did not bind the specific D1-like receptor antagonist [³H]SCH-23390 or numerous other dopaminergic, adrenergic or serotoninergic ligands with high affinity. However, like vertebrate dopamine D1-like receptors, dDA1 stimulated the accumulation of cAMP in response to DA (EC₅₀ ∼300 nM) and 6,7-ADTN (EC₅₀∼500 nM). The dopaminergic rank order of potency (DA > NE⪢5-HT) and the lack of stimulation by other possible neurotransmitters (octopamine, tyramine, tryptamine) or DA metabolites (e.g. N-acetyl dopamine) found in Drosophila suggests that this receptor functionally belongs to the dopamine D1-like subfamily. Benzazepines, which characteristically bind to vertebrate dopamine D1-like receptors with high affinity, were relatively poor in stimulating (SKF-38393, SKF-82526; EC₅₀ > 10 μM) dDA1-mediated accumulation of cAMP. Of the numerous compounds tested, a few dopaminergic antagonists inhibited DA-stimulated production of cAMP in a concentration-dependent manner, albeit with considerably reduced affinity, and with the rank order of potency: (+)-butaclamol(K _b∼125nM) > SCH-23390(K _b∼230nM) > α-flupenthixol (K _b ∼ 400 nM) > chlorpromazine ≥ spiperone (K _b ∼ 680 nM) ≥ clozapine In situ hybridization revealed that dDA1 receptor mRNA is expressed as a maternal transcript, and at later blastoderm stages is restricted to apical regions of the cortical peripheral cytoplasm. The generation of inter-species D1 receptor chimeras may help to identify those particular sequence-specific motifs or amino acid residues confering high affinity benzazepine receptor interactions.