A PRIMORDIAL DOPAMINE D1-LIKE ADENYLYL CYCLASE-LINKED RECEPTOR FROM DROSOPHILA-MELANOGASTER DISPLAYING POOR AFFINITY FOR BENZAZEPINES

We report here the isolation from DI 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 [H-3]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 (EC(50) similar to 300 nM) and 6,7-ADTN (EC(50) similar to 500 nM), The dopaminergic rank order of potency (DA > NE much greater than 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; EC(50) > 10 mu M) 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 similar to 125M) > SCH-23390(K-b similar to 230nM) > alpha-flupenthixol (K-b similar to 400 nM) > chlorpromazine greater than or equal to spiperone (K-b similar to 680 nM) greater than or equal to 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.