Mimetic insectatachykinin analogs: Interaction with an expression with an expressed stable fly tachykinin-like receptor and active conformation

R. J. Nachman 1 , J. V. Broeck 2 , H. Williams 3 & R. E. Isaac 4

1 Veterinary Entomology Research Unit, Southern Plains Agricultural Research Center, USDA, ARS, 2881 F&B Road, College Station, TX, USA 77845;2 Laboratory for Developmental Physiology & Molecular Biology, Catholic University of Leuven, Naamsestraat 59, Leuven, Belgium, B-3000; 3 Department of Chemistry, Texas A&M University, College Station, TX, USA 77840; 4 Department of Biology, University of Leeds, Leeds, UK LS2 9JT

A G-protein-coupled receptor, labeled STKR, cloned from the stable fly, Stomoxys calcitrans, shows sequence-similarity to vertebrate NK tachykinin receptors and has been functionally expressed in stably transfected Drosophila S2 cells. Natural insectatachykinin peptides, including an insectatachykinin recently isolated from the stable fly, activate the expressed receptor system whereas vertebrate tachykinins do not. Fragment analogs of an insectatachykinin nonapeptide demonstrate that receptor activation can be achieved up to and including the pentapeptide fragment FTGMRa. An Ala-replacement series of analogs indicated that receptor activity is retained except when the highly conserved C-terminal pentapeptide residues Phe (-5) and Arg (-1) are replaced by Ala. A comparison of solution conformations of active, analogous analogs of the insectatachykinin and the vertebrate tachykinin substace P, containing the hindered residue block NMeTyr-Aib, demonstrate that they share a very similar turn shape incorporating a cis peptide bond between the NMeTyr and Aib residues. The insectachykinin analog demonstrated binding to the expressed stable fly STKR receptor. This and related Aib-containing insectatachykinin analogs demonstrate enhanced resistance to the peptidases ACE and NEP. The very similar solution conformations of the two analogous restricted conformation analogs are consistent with a distant evolutionary relationship between the tachykinins and insectatachykinins, and can be considered along with evidence obtained from comparisons of physiological cross-activity, the shared antagonist response of spantide, sequence similarities and receptor homology.

Index terms: tachykinin, ?-turn, Stomoxys, homology, peptidase-resistance.

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