Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule

The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pen tamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tpr], had been previously determined as the minimum fragment able to eli cit a functional response. The receptor(s) for these insect neuropeptides h as not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes a egypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bp a-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity r elationships for leucokinins. LPA caused depolarization of the transepithel ial voltage (TEV) in female Malpighian tubule, confirming the activity of t he peptide. The effective concentration to give half the maximum depolariza tion (EC50) was 17 nM. The I-125-LPA was then used to characterize leucokin in binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/aut oradiography and by competition experiments with excess unlabeled leucokini n analogues. I-125-LPA bound to the 54 kDa protein(s) with a K-d value of 1 3+/-3 nM in agreement with the EC50 for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors. Published b y Elsevier Science Ltd.