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Background: Neuropeptides are examples of small, flexible molecules that bind to receptors and induce signal transduction, thereby eliciting biological activity. The multifunctional insect kinin neuropeptides retain full activity when reduced to only their carboxy-terminal pentapeptide (Phe 1 -X 2 -X 3 -Trp 4 -Gly 5 -NH 2 ), thereby allowing extensive structure-function studies and conformational analysis. Results: A combined experimental and theoretical analysis of the insect kinin carboxy-terminal pentapeptide was used to probe the role of each residue, define the bioactive conformation, and design a constrained bioactive analog. Coupling receptor-binding data with two biological activity assays allowed receptor binding and signal transduction to be differentiated. A preferred β-turn conformation, found for residues 1–4 by molecular dynamics simulations, was tested by designing a conformationally restricted cyclic hexapeptide. This cyclic analog showed a preference for the β-turn conformation, as shown by a conformational search and nuclear magnetic resonance spectroscopy, and it showed stronger receptor binding but decreased activity relative to highly active linear analogs. Conclusions: Each residue of the insect kinin carboxy-terminal pentapeptide has a distinct role in conformational preference, specific receptor interactions or signal transduction. The (β-turn preference of residues Phe 1 -X 2 -X 3 -Trp 4 implicates this as the bioactive conformation. The amidated carboxyl terminus, required for activity in many neuropeptide families, may be generally important for signal transduction and its inclusion may therefore be essential for agonist design.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

Previously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-duca-DH) has been isolated from Manduca sexta and shown to stimulate fluid excretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering postfeeding Manduca sexta larvae. However, Manduca-DH was reported to have no effect on Malpighian tubules in vitro. Manduca-DH and [Nle2,11]-Manduca-DH were synthesized in Texas and assayed in London on isolated Malpighian tubules of Acheta domesticus. Man-duca-DH stimulated fluid secretion by about 60% of the maximum response achievable with extracts of corpora cardiaca and increased the production of cyclic AMP. In combination with 10−4moll−1 3-isobutyl-l-methyl xanthine (IBMX), Manduca-DH stimulated maximal secretion. A number of CRF-related peptides also stimulated fluid secretion and cyclic AMP production in cricket tubules, and the CRF antagonist α-helical-CRF[9–41] blocked the stimulation of fluid secretion by Manduca-DH. [Nle2,11]-Manduca-DH was more active than Manduca-DH in both assays, suggesting that methionine residues in the natural peptide may become oxidized. Taken in conjunction with previous in vivo studies, the present findings suggest that a Manduca-DH-like diuretic peptide is the hormone controlling post-eclosion diuresis in butterflies, and Manduca-DH was shown to stimulate both fluid secretion and cyclic AMP production in Malpighian tubules from 1–12 h posteclosion Pieris rapae adults. The function of the peptide in Manduca sexta is discussed.

A biologically active 125 I-labeled analogue of AK-II (3′-hydroxyphenyl propionic-Gly-Gly-Gly-Phe-Ser-Pro-Trp-Gly-NH 2 ) was used to investigate the properties of achetakinin binding sites on plasma membranes from Malpighian tubules of Acheta domesticus . With optimized conditions, binding was rapid, reversible, and specific, and saturation studies revealed a single class of binding sites with K d 0.55 n M and B max 39.9 fmol/mg membrane protein. The affinities of achetakinins for binding sites on tubule membranes ranked AK-V > AK III > AK-II > AK-I ≥ AK-IV, in general agreement with their potencies in functional assays. However, IC 50 values were several orders of magnitude higher than corresponding values for EC 50 , which suggests a considerable receptor reserve.