The activation of G-protein gated inwardly rectifying K+ channels by a cloned Drosophila melanogaster neuropeptide F-like receptor.

A Drosophila melanogaster G-protein-coupled receptor (NPFR76F) that is activated by neuropeptide F-like peptides has been expressed in Xenopus oocytes to determine its ability to regulate heterologously expressed G-protein-coupled inwardly rectifying potassium channels. The activated receptor produced inwardly rectifying potassium currents by a pertussis toxin-sensitive G-protein-mediated pathway and the effects were reduced in the presence of proteins, such as the betaARK 1 carboxy-tail fragment and alpha-transducin, which bind G-protein betagamma-subunits. Short Drosophila NPF-like peptides were more potent than long NPF-like peptides at coupling the receptor to the activation of inwardly rectifying potassium channels. The putative endogenous short Drosophila NPF-like peptides showed agonist-specific coupling depending on whether their actions were assessed as the activation of the inwardly rectifying potassium channels or as the activation of endogenous inward chloride channels through a co-expressed promiscuous G-protein, Galpha16. As inwardly rectifying potassium channels are known to be encoded in the Drosophila genome and the NPFR76F receptor is widely expressed in the Drosophila nervous system, the receptor could function to control neuronal excitability or slow wave potential generation in the Drosophila nervous system.

Site-directed mutagenesis studies on the Drosophila octopamine/tyramine receptor.

The cloned Drosophila octopamine/tyramine receptor can be coupled to second messenger pathways in an agonist-specific fashion by the endogenously occurring biogenic amines, octopamine and tyramine, when expressed in Chinese hamster ovary cells. We have mutated to alanine a range of receptor amino acids that could potentially form hydrogen bonds with the beta-hydroxyl group of octopamine based on homologies with alpha- and beta-adrenergic receptor subtypes. After stable expression of the mutant receptors in CHO cells we have compared the ability of octopamine and tyramine to displace [(3)H]yohimbine binding to membrane fractions from the mutant cell lines with their ability to modulate adenylyl cyclase activity in intact cells. The results suggest that none of the mutated amino acids residues, at least in isolation, are likely to be involved in interactions with the beta-hydroxyl group of the octopamine side chain. It is possible that amino acids not mutated in the present study are somehow involved in this interaction. Alternatively, it is also possible that the beta-hydroxyl group of the octopamine side chain is capable of interacting with more than one of the amino acids mutated in the present study.

Analysis of the CoIE1 stability determinant Rcd.

Multimer formation is an important cause of instability for many multicopy plasmids. Plasmid CoIE1 is maintained stably because multimers are converted to monomers by Xer-mediated site-specific recombination at the cer site. However, multimer resolution is not the whole story; inactivation of a promoter (Pcer) within cer causes plasmid instability even though recombination is unaffected. The promoter directs the synthesis of a short transcript (Rcd) which is proposed to delay the division of multimer-containing cells. Mapping of the 5' terminus of Rcd confirms that transcription initiates from Pcer. The 3' terminus shows considerable heterogeneity, consistent with a primary transcript of 95 nt being degraded via intermediates of 79 and 70 nt. Secondary structure predictions for Rcd are presented. Of four mutations which abolish Rcd-mediated growth inhibition, one reduces the activity of Pcer while the other three map to the rcd coding sequence and reduce the steady-state level of the transcript. RNA folding analysis suggests that these three mutant transcripts adopt a common secondary structure in which the major stem-loop differs from that of wild-type Rcd. A survey of 24 cer-like multimer resolution sites revealed six which contain Pcer-like sequences. The putative transcripts from these sites have similar predicted secondary structures to Rcd and contain a highly conserved 15 base sequence. To test the hypothesis that Rcd acts as an anti-sense RNA, interacting with its target gene(s) through the 15 nt sequence, we used DNA hybridization and sequence analysis to find matches to this sequence in the Escherichia coli chromosome. Our failure to find plausible anti-sense targets has led to the suggestion that Rcd may interact directly with a protein target.