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Genomic and cDNA clones encoding a muscarinic acetylcholine receptor from Drosophila melanogaster have been isolated. Sequence analysis demonstrates that this gene encodes a receptor with a high degree of amino acid identity to the mammalian muscarinic acetylcholine receptors and has three introns in the portion of the gene encoding the third putative cytoplasmic loop. A full-length cDNA clone has been placed under the control of the mouse metallothionein promotor and transfected into mouse Y1 adrenal cells. The receptor expressed in these cells exhibits the high-affinity binding for the antagonists quinuclidinyl benzilate and atropine expected of a muscarinic receptor. The Drosophila muscarinic receptor, when expressed in Y1 cells, is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism.Muscarinic acetylcholine receptors (mAcChoRs) belong to a family of receptors which are involved in the activation of various signal transduction pathways through their interaction with guanine nucleotide-binding proteins (G proteins) (1). These receptors, which include the five subtypes-of mammalian mAcChoR (2-9), the adrenergic receptors (10, 11), and the opsin pigments (12), share a predicted seven transmembrane domain structure with highly conserved amino acid sequences, especially within certain transmembrane regions. The region of least identity occurs in a proposed cytoplasmic loop located between the fifth and sixth transmembrane regions, which has been suggested to be involved in the interaction of the receptors with specific G proteins (13,14). Experiments using point and deletion mutations and chimeric receptors indicate that the membrane-proximal portions of this loop most likely determine the specificity of functional coupling (15-18).Acetylcholine is believed to be a major excitatory transmitter in the central nervous system of insects (19). Many of the components involved in cholinergic transmission in Drosophila have been studied by using genetic and molecular biological approaches. Thus, mutations in both choline acetyltransferase and acetylcholinesterase have behavioral and physiological effects and can cause lethality (20)(21)(22). The distribution of both proteins in the developing Drosophila nervous system has been determined immunocytochemically (23,24), and cDNA clones encoding both enzymes as well as nicotinic receptor subunits have been isolated (25)(26)(27)(28)(29) MATERIALS AND METHODS Isolation of Molecular Clones. A genomic library from the wild-type Oregon R strain of Drosophila melanogaster (Lambda GEM-11; Promega) was probed with a mixture consisting of a nick-translated full-length cDNA clone encoding the pig m2 mAcChoR (5) and a nick-translated 1.8-kilobase (kb) Kpn I-BamHI genomic fragment of the mouse ml mAcChoR (9). Filters containing the phage were hybridized under moderately stringent conditions (60'C, 6x SSC) and washed at 370C, lx SSC (lx SSC = 0.15 M NaCI/0.015 M sodium citrate, pH 7). DNAs from seven positive clones were isol...

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