Insect G protein-coupled receptors and signal transduction.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins (7-TM) that transduce extracellular signals into cellular physiological responses through the activation of heterotrimeric guanine nucleotide binding proteins (alpha beta gamma subunits). Their general properties are remarkably well conserved during evolution. Despite this general resemblance, a large variety of different signals are mediated via this category of receptors. Several GPCR-(sub)families have an ancient origin that is situated before the divergence of Protostomian and Deuterostomian animals. Nevertheless, an enormous diversification has occurred since then. The availability of novel sequence information is growing very rapidly as a result of molecular cloning experiments and of metazoan genome (Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens) and EST (expressed sequence tags) sequencing projects. The Drosophila Genome Sequencing Project will certainly have an important impact on insect signal transduction and receptor research. In parallel, convenient expression systems and functional assay procedures will be needed to investigate insect receptor properties and to monitor the effects of natural and artificial ligands. The study of the evolutionary aspects of G protein-coupled receptors and of their signaling pathways will probably reveal insect-specific features. More insight into these features may result in novel methods and practical applications. Arch.

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

High affinity displacement of [(3)H]NPY binding to the crude venom of conus anemone by insect neuropeptides.

The venom from Conus anemone contains a protein, named ANPY toxin, which displayed high affinity (IC(50) in nanomolar range) to neuropeptide Y (NPY), [Leu(31), Pro(34)]NPY, peptide YY, pancreatic polypeptide, the Y(1) antagonist 1229U91, and C-terminal NPY fragments. N-terminal fragments and the free acid form of NPY did not bind to ANPY. The truncated NPY fragments displayed very low affinity to Y(1) receptors and partially inhibited [(3)H]NPY binding to anti-NPY antiserum. Several insect neuropeptides, the sequences of which related to the C-terminal fragments of NPY, were observed to bind with similar affinity or even 20 times higher (Lom-MS and Scg-NPF) affinity than NPY. In contrast, no significant binding of these insect peptides was observed for Y(1) receptors and anti-NPY antiserum. Therefore, ANPY can be viewed as an acceptor that binds with very high affinity to a broad spectrum of vertebrate and invertebrate neuropeptides that share a similar C-terminal amino acid sequence.

Immunocytochemical distribution of locustamyoinhibiting peptide (Lom-MIP) in the nervous system of Locusta migratoria.

Locustamyoinhibiting peptide (Lom-MIP) is one of the 4 identified myoinhibiting neuropeptides, isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion complexes of the locust, Locusta migratoria. An antiserum was raised against Lom-MIP for use in immunohistochemistry. Locustamyoinhibiting peptide-like immunoreactivity (Lom-MIP-LI) was visualized in the nervous system and peripheral organs of Locusta migratoria by means of the peroxidase-antiperoxidase method. A total of 12 specific immunoreactive neurons was found in the brain. Processes of these neurons innervate the protocerebral bridge the central body complex and distinct neuropil areas in the proto- and tritocerebrum but not in the deuterocerebrum nor in the optic lobes. The glandular cells of the corpora cardiaca, known to produce adipokinetic hormones, are contacted by Lom-MIP-LI fibers. The corpora allata were innervated by the nervus corporis allati I containing immunoreactive fibers. Lom-MIP-LI cell bodies were also found in the subesophageal ganglion, the metathoracic ganglion and the abdominal ganglia I-IV. In peripheral muscles, Lom-MIP-LI fibers innervate the heart, the oviduct, and the hindgut. In the salivary glands, Lom-MIP-LI was detected in the intracellular ductule of the parietal cells. Possible functions of Lom-MIP are discussed.

The pivotal role of the plasma membrane-cytoskeletal complex and of epithelium formation in differentiation in animals.

1. If a few exceptions are disregarded, the several somatic cell types of a differentiated organism all have an identical genome. They all differ in their plasma membrane-cytoskeletal complex. 2. Differences in plasma membrane properties usually result in differences in ionic concentrations/activities in the cytoplasm and nucleoplasm. A basic question therefore is whether there exists a causal relationship between the ionic environment of the nucleus and differential gene expression/protein synthesis. 3. Development is switched on by a "Ca2+ explosion", often accompanied by pH changes and plasma membrane depolarisation. The penetration of the spermatozoon in the plasma membrane acts as a trigger. 4. All animal species develop from a blastula. At this stage they organise themselves as an epithelium enclosing an inner (fluid) compartment. This suggests that epithelium formation is absolutely essential in animal development. 5. As development proceeds, more and more compartments, lined by different epithelia, are formed. Differentiated organisms largely consist of folded epithelia. Some cells leave their original epithelial environment and become free floating (e.g. blood cells) or engage in other types of organisation. 6. Epithelial cells have the ability to segregate some membrane proteins, e.g. receptors, ion pumps, ion channels etc., so as to make selective transcellular transport possible. The cytoskeleton plays an important role in this segregation and in the interconnection of epithelial cells. 7. Transembryonic electric currents which have been measured by the vibrating probe technique, are due to electrogenic ion transport by epithelia. 8. Segregation of membrane proteins is not an exclusive property of epithelial cells but it is probably a property of all animal cell types, single cells inclusive; asymmetry is the rule, symmetry--if it exists at all--the exception. 9. Differences in several plasma membrane proteins (receptors, ion transporting molecules, cell adhesion molecules and signal transducing systems) are not only causally related to differential transcellular transport but also indirectly to differential protein synthesis and hence to differentiation. There are already a few well documented examples of "electrical" control of gene expression. 10. The major "strategy" which applies in differentiation seems to be to keep the genome constant but to change over and over its ionic and macromolecular environment, both acting in a complementary way. The first one may be considered as the coarse tuning mechanism of gene expression-protein synthesis, the second as the fine one. In our opinion this might be a principle universal to differentiation processes in all animal species.

Immunocytochemical localization of human growth hormone- and prolactin-like antigenic determinants in the insects, Locusta migratoria and Sarcophaga bullata.

1. By use of the peroxidase-antiperoxidase immunocytochemical method, substances immunoreactive to antisera directed against human growth hormone (hGH) and prolactin (hPrl) were localized in the nervous system of larval and adult Locusta migratoria and of adult Sarcophaga bullata belonging to different age groups. 2. No major differences in the distribution of cerebral immunoreactive materials were observed between males and females or between juvenile and adult insects. 3. Differential immuno-labeling of alternating tissue sections demonstrated that materials resembling hGH or hPrl are present in distinct neurons in the locust, whereas neurons immunoreactive to both antisera were detected in the fleshfly (Sarcophaga).