Systematic G-protein-coupled Receptor Analysis inDrosophila melanogaster Identifies a Leucokinin Receptor with Novel Roles

Abstract

Leucokinins are insect neuropeptides that stimulate hindgut motility and renal fluid secretion. Drosophila has a single leucokinin gene, pp, encoding the longest known leucokinin, Drosokinin. To identify its receptor, a genome-wide scan for G-protein-coupled receptors was performed in silico and candidate receptors identified by similarity to known tachykinin receptors. The deduced peptides were expressed, with a transgene for the calcium reporter aequorin, in S2 cells and only one gene (CG10626) encoded a protein that responded to Drosokinin. The properties of the heterologously expressed receptor (action through intracellular calcium with an EC 50 of 4 ؋ 10 ؊11 M and a t1 ⁄2 <1 s) match closely those reported for the action of Drosokinin on Malpighian (renal) tubules. Antibodies raised against the receptor identified known sites of leucokinin action: stellate cells of the Malpighian tubule, two triplets of cells in the pars intercerebralis of the adult central nervous system, and additional cells in larval central nervous system. Western blots and reverse transcription-PCR confirmed these locations, but also identified expression in male and female gonads. These tissues also displayed elevated calcium in response to Drosokinin, demonstrating novel roles for leucokinin. A functional genomic approach has thus yielded the first complete characterization of a leucokinin receptor in an insect.

Resolution of the insect ouabain paradox

“…Fig. 2A shows that this is the case: taurocholate at 6 ϫ 10 Ϫ4 M [chosen as the highest concentration that does not affect secretion: (31)] unmasks Malpighian tubule sensitivity to ouabain. [It should be noted that, in these and other results, ouabain reduced fluid secretion by 50-60%, but not by 100%; the ouabain-insensitive component is believed to reflect K ϩ entry through basolateral potassium channels and Na ϩ ͞K ϩ ͞ 2Cl Ϫ cotransporters (9,32,33).…”

Leucokinin mimetic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron

“…Drosophila kinin is named "drosokinin" or "leucokinin" (lk) (22). Three pairs of lk subesophageal neurons (SELKs) receive projections from gustatory receptor neurons in the head (23, 24).…”

Serotonin and Downstream Leucokinin Neurons Modulate Larval Turning Behavior inDrosophila

“…The preparation was then fixed and stained as described previously (Nose et al, 1997). We used rabbit anti-GFP (Invitrogen; 1:1000), mouse anti-GFP (Sigma; 1:100), rabbit anti-5-HT (Sigma; 1:200), rabbit anti-leucokinin (Al-Anzi et al, 2010), rabbit antileucokinin receptor (Radford et al, 2002), rat anti-elav (Developmental Studies Hybridoma Bank), and mouse anti-V5 (Invitrogen; 1:500) as primary antibodies. We used goat anti-rabbit IgG conjugated with Alexa 488 (Invitrogen; 1:300), goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen; 1:300), goat anti-rabbit IgG conjugated with Cy3 (Invitrogen; 1:300), and goat anti-mouse IgG conjugated with Alexa 555 (Invitrogen; 1:300) as secondary antibodies.…”