Autor(es): Radford, Jonathan C.; Terhzaz, Selim; Cabrero, Pablo; Davies, Shireen-A.; Dow, Julian A. T.

Resumo: Identification of the Anopheles gambiae leucokinin gene from the completed A. gambiae genome revealed that this insect species contains three leucokinin peptides, named Anopheles leucokinin I-III. These peptides are similar to those identified in two other mosquito species, Aedes aegypti and Culex salinarius. Additionally, Anopheles leucokinin I displays sequence similarity to Drosophila melanogaster leucokinin. Using a combination of computational and molecular approaches, a full-length cDNA for a candidate leucokinin-like receptor was isolated from A. stephensi, a close relative of A. gambiae. Alignment of the known leucokinin receptors - all G protein-coupled receptors (GPCRs) - with this receptor, identified some key conserved regions within the receptors, notably transmembrane (TM) domains I, II, III, VI and VII. The Anopheles leucokinins and receptor were shown to be a functional receptor-ligand pair. All three Anopheles leucokinins caused a dose-dependent rise in intracellular calcium ([Ca super(2+)] sub(i)) when applied to S2 cells co-expressing the receptor and an aequorin transgene, with a potency order of I>II>III. Drosophila leucokinin was also found to activate the Anopheles receptor with a similar EC sub(50) value to Anopheles leucokinin I. However, when the Anopheles peptides were applied to the Drosophila receptor, only Anopheles leucokinin I and II elicited a rise in [Ca super(2+)] sub(i). This suggests that the Anopheles receptor has a broader specificity for leucokinin ligands than the Drosophila receptor. Antisera raised against the Anopheles receptor identified a doublet of approx. 65 and 72 kDa on western blots, consistent with the presence of four N-glycosylation sites within the receptor sequence, and the known glycosylation of the receptor in DROSOPHILA: In Anopheles tubules, as in Drosophila, the receptor was localised to the stellate cells. Thus we provide the first identification of Anopheles mosquito leucokinins (Anopheles leucokinins) and a cognate leucokinin receptor, characterise their interaction and show that Dipteran leucokinin signalling is closely conserved between Drosophila and ANOPHELES:

Palavras-Chave: Genomes; Gene expression; Calcium; Specificity; Nucleotide sequence; DNA; Receptors; Peptides; Ligands; cDNA; Guanine nucleotide-binding protein; Aedes aegypti; Drosophila melanogaster; Anopheles stephensi; Culicidae; Anopheles gambiae; Culex alinarius; Brackish

Imprenta: Journal of Experimental Biology, v. 207, n. 26, p. 4573-4586, 2004.

Descritores: Aedes aegypti - Cell ; Aedes aegypti - DNA ; Aedes aegypti - Proteins

Data de publicação: 2004