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First published online December 3, 2004
Journal of Experimental Biology 207, 4573-4586 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01317

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Functional characterisation of the Anopheles leucokinins and their cognate G-protein coupled receptor

Jonathan C. Radford, Selim Terhzaz, Pablo Cabrero, Shireen-A. Davies and Julian A. T. Dow^*

Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK

^* Author for correspondence (e-mail: j.a.t.dow{at}bio.gla.ac.uk)

Accepted 29 September 2004

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 ([Ca²⁺]_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 EC₅₀ 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 [Ca²⁺]_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.

Key words: mosquito, Anopheles gambiae, Anopheles stephensi, aequorin, calcium, leucokinin

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