Herbert Torfs

Characterization of a Receptor for Insect Tachykinin‐Like Peptide Agonists by Functional Expression in a Stable Drosophila Schneider 2 Cell Line

Abstract: STKR is an insect G protein‐coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin‐like peptides or “insectatachykinins,” such as Locusta tachykinin (Lom‐TK) III, produced dose‐dependent calcium responses in stably transfected S2‐STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10‐5M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom‐TK III‐induced calcium response. Further analysis showed that the agonist‐induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom‐TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5‐trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin‐dependent signaling processes.

Tachykinin-like Peptides and Their Receptors: A Review

Abstract: Tachykinin‐like peptides have been identified in many vertebrate and invertebrate species. On the basis of the data reviewed in this paper, these peptides can be classified into two distinct subfamilies, which are recognized by their respective sequence characteristics. All known vertebrate tachykinins and a few invertebrate ones share a common C‐terminal sequence motif, ‐FXGLMa. The insect tachykinins, which have a common ‐GFX1GX2Ra C‐terminus, display about 30% of sequence homology with the first group.

Functional expression of a locust tyramine receptor in murine erythroleukaemia cells

The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein‐coupled receptor. Functional agonist‐dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL‐TyrLoc). Tyramine elicited a dose‐dependent increase of cytosolic Ca2+‐ions and an attenuation of forskolin‐induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein‐coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.

Substitution of conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes partial agonism at the stomoxytachykinin receptor.

A few naturally occurring insect tachykinin‐related peptides, such as stomoxytachykinin (Stc‐TK), contain an Ala‐residue instead of the highly conserved Gly‐residue that is present in most other members of this peptide family. Stc‐TK is a potent, partial agonist of the stable fly (Stomoxys calcitrans) tachykinin receptor, STKR. By means of synthetic analogues, the Gly/Ala exchange, representing the addition of a single methyl group in the active core region of these peptides, was shown to be fully responsible for the generation of this partial agonism, which was also accompanied by an increase in agonistic potency. Surprisingly, this Ala‐dependent reduction in maximal response levels was only observed for the agonist‐induced cellular calcium rise. Stomoxytachykinin, Stc‐TK, did not display partial agonism for the STKR‐mediated cyclic AMP response. A possible explanation for this differential partial agonism is that the Gly‐containing and Ala‐replaced peptides recognize and stabilize active receptor conformations that differ in their functional coupling efficacies towards these response pathways. Drosotachykinins, Drm‐TK, tachykinin‐like peptides encoded in the fruit fly genome, were shown to be STKR‐agonists. Interestingly, one of these peptides, which contains an Ala‐residue instead of the conserved Gly‐residue, also proved to be a potent, partial agonist for STKR.

Immunolocalization of a tachykinin-receptor-like protein in the central nervous system of Locusta migratoria migratorioides and neobellieria bullata

Antisera raised against two distinct peptide regions of the Drosophila neurokinin‐like receptor NKD were used to immunolocalize tachykinin‐receptor‐like proteins in the central nervous system of two insect species: the African migratory locust, Locusta migratoria, and the gray fleshfly, Neobellieria bullata. The resulting immunopositive staining patterns were identical for both antisera. Moreover, a very similar distribution of the immunoreactive material was observed in fleshflies and locusts. Immunoreactivity was found in nerve terminals of the retrocerebral complex, suggesting a presynaptic localization of the receptor in this part of the brain. Cell bodies were stained in the subesophageal ganglion: an anterior group of four larger cells and a posterior group of about 20 cells. These cells have axons projecting into the contralateral nervus corporis allati (NCA) II, bypassing the corpus allatum and projecting through the NCA I into the storage part of the corpus cardiacum. In the glandular part of the corpus cardiacum, the glandular adipokinetic hormone‐producing cells did not show any immunopositive staining. In the locust, additional immunopositive staining was observed in internolaterally located neurons of the tritocerebrum and in important integrative parts of the neuropil such as the central body and the mushroom bodies. J. Comp. Neurol. 407:415–426, 1999.

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.

Functional analysis of synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell lines.

The activity of a series of synthetic tachykinin-like peptide analogs was studied by means of microscopic calcium imaging on recombinant neurokinin receptor expressing cell lines. A C-terminal pentapeptide (FTGMRa) is sufficient for activation of the stomoxytachykinin receptor (STKR) expressed in Schneider 2 cells. Replacement of amino acid residues at the position of the conserved phenylalanine (F) or arginine (R) residues by alanine (A) results in inactive peptides (when tested at 1microM), whereas A-replacements at other positions do not abolish the biological activity of the resulting insectatachykinin-like analogs. Calcium imaging was also employed to compare the activity of C-terminally substituted tachykinin analogs on three different neurokinin receptors. The results indicate that the major pharmacological and evolutionary difference between tachykinin-related agonists for insect (STKR) and human (NK1 and NK2) receptors resides in the C-terminal amino acid residues (R versus M). A single C-terminal amino acid change can turn an STKR-agonist into an NK-agonist and vice versa.