Differential activation of an insect G protein-coupled peptide receptor by insectatachykinin-like agonists

H. Torfs 1 , R. Nachman 2 , K. Åkerman 3 , J. Poels 1 , W. Van Poyer 1 , A. De Loof 1 , G. Vassart 4 , M. Parmentier 4 , F. Guerrero 5 ? J. Vanden Broeck 1

1 Zoological Institute K.U.Leuven, Naamsestraat 59, B-3000 Leuven, Belgium; 2 Food Animal Protection Research Laboratory, U.S.D.A., A.R.S., 2881 F&B Rd., College Station, TX 77845, U.S.A.; 3 Uppsala Biomedical Center, Husargatan 3, Box 570, S-75123 Uppsala, Sweden; U.S.A.; 4 IRIBHN ULB, Route de Lennik 808, B-1070 Brussels, Belgium; 5 Knipling-Bushland U.S. Livestock Insect Research Lab, 2700 Fredericksburg Rd., Kerrville, TX 78028, U.S.A.

STKR, a G protein-coupled receptor cloned from the stable fly, Stomoxys calcitrans, which shows sequence similarity to vertebrate tachykinin (or neurokinin, NK) receptors, was functionally expressed in stably transfected Drosophila melanogaster Schneider 2 (S2) cells. Dose-dependent calcium responses were visualized by fura-2 fluorescence detection methods in transfected cells that were challenged with insectatachykinin-like peptides (e.g. locustatachykinins). Vertebrate tachykinins, such as substance P, substance K, neuromedin K and physalaemin, and eledoisin were not able to produce a detectable response at concentrations up to 10 µM. The insectatachykinin-induced response was not dependent on extracellular calcium ions and was blocked by the phospholipase C (PLC) inhibitor U73122. The involvement of PLC was confirmed by the observation of a rapid agonist-induced increase in inositol 1,4,5-trisphosphate levels. The pharmacology of the agonist-induced calcium rise was further investigated in detail by studying the activity of a large variety of natural and artificial insectatachykinin peptide analogs. The effects of the most important antagonists for mammalian NK-receptors were also evaluated on STKR-expressing insect cells. In addition, STKR receptor pharmacology was compared with mammalian NK receptors and with the cockroach hindgut contractility bio-assay. Moreover, insectatachykinin agonists also exerted a stimulatory effect on cAMP accumulation. Dose-response curves were obtained with a variety of agonists, for both calcium and cAMP effects. High concentrations of the agonist Lom-TK III elicited a stimulatory effect on adenylyl cyclase (AC) activity. The EC50 values for the AC-effect were about a 100-fold higher than for the PLC-effect. Surprisingly, recent results obtained with a few naturally occurring insectatachykinin analogs, such as the endogenous stable fly peptide Stc-TK, suggest that differential receptor-effector coupling is highly dependent on the type of insectatachykinin analog that is used.

Index terms: adenylyl cyclase, calcium, cyclic AMP, neuropeptide, phospholipase C

---