Jürgen Krieger

Two classes of olfactory receptors in xenopus laevis

Xenopus laevis possess a gene repertoire encoding two distinct classes of olfactory receptors: one class related to receptors of fish and one class similar to receptors of mammals. Sequence comparison indicates that the fish-like receptors represent closely related members of only two subfamilies, whereas mammalian-like receptors are more distantly related, most of them representing a different subfamily. The fish-like receptor genes are exclusively expressed in the lateral diverticulum of the frogs nose, specialized for detecting water-soluble odorants, whereas mammalian-like receptors are expressed in sensory neurons of the main diverticulum, responsible for the reception of volatile odors.

Binding proteins from the antennae of Bombyx mori.

From an antennal library of Bombyx mori cDNA clones encoding different binding proteins have been isolated. The deduced amino acid sequences showed only moderate homology to each other but shared several common structural features. Based on a sequence comparison with the antennal binding proteins from different moth species, one of the clones appears to encode a pheromone binding protein, whereas two others represent new members of the two general odorant binding protein families. A fourth clone encodes a protein which is related to antennal binding proteins so far found only in Drosophila melanogaster.

Selective Activation of G Protein Subtypes in the Vomeronasal Organ upon Stimulation with Urine-derived Compounds

Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones related to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate Gα-subunits, activation dependently labeled with [α-32P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of Gi as well as Go subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only Gi proteins were activated, whereas Go activation was elicited by α2u-globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with Gimay be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with Go seem to be specialized to interact with pheromonal components of proteinaceous nature.

Odorant binding proteins of Heliothis virescens

cDNA clones coding for three different binding proteins were isolated from an antennal library of Heliothis virescens. The deduced amino acid sequences showed only moderate homology to each other but shared several common structural features. Based on a comparison with the predicted primary structures of antennal binding proteins from different moth species, one of the clones (Hel-1) was found to encode a pheromone binding protein, whereas the two others (Hel-10 and -11) encode general odorant binding proteins.

Cloning of biogenic amine receptors from moths (Bombyx mori and Heliothis virescens)

Based on the similarity of genes which code for guanine-nucleotide binding protein- (G-protein-) coupled receptors, cDNA clones encoding new members of the receptor family have been isolated from Bombyx mori and Heliothis virescens. The deduced protein structures exhibit highest similarity to tyramine/octopamine and serotonin receptors of Drosophila. One of the receptor clones (K50Hel) was permanently expressed in the mammalian cell line LLC-PK1. In stimulation experiments its responded to octopamine leading to an inhibition of adenylate cyclase activity in a dose-dependent manner. Pharmacological studies revealed a higher affinity for mianserin than for yohimbine suggesting, that the K50Hel clone encoded a neuronal type 3 octopamine receptor. As revealed by in situ hybridization, this receptor type is expressed in the central nervous system and antennae of moth.

Molecular cloning of an insect pheromone-binding protein

Pheromone binding protein; cDNA cloning; Nucleotide sequence; Primary structure; RNA blot hybridization

Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity

Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. The expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed.

A novel class of binding proteins in the antennae of the silk moth Antheraea pernyi

Abstract Using recombinant DNA approaches a cDNA clone was isolated from antennal libraries of Antheraea pernyi which encodes the complete sequence of a putative pheromone or odorant binding protein. The deduced protein sequence consist of a signal peptide of 19 amino acids and a mature protein of 141 amino acid residues. It shares a number of structural features with previously identified pheromone binding proteins although the predicted primary structures show only moderate homologies; Furthermore, the identified clone is preferentially expressed in female antennae.

Primary structure of a pheromone-binding protein from Antheraea pernyi: homologies with other ligand-carrying proteins

SummaryAn antennal cDNA clone encoding the complete sequence (163 amino acids) of a pheromone-binding protein precursor from the male silk moth, Antheraea pernyi, was isolated using oligonucleotide probes. The cloned cDNA was expressed and the translation product detected by specific antibodies. The deduced protein sequence consists of a signal peptide of 21 amino acids and a mature binding protein of 142 amino acid residues. The predicted structure of this protein is homologous to binding-proteins from different insect species which have previously been identified, but shows no similarities to odorant-binding proteins from vertebrates, suggesting that soluble odorant-binding proteins in insects and vertebrates represent an evolutionary convergence.

Identification of a cyclic nucleotide- and voltage-activated ion channel from insect antennae

From an antennal cDNA library of Heliothis virescens a clone has been isolated encoding a polypeptide of 678 amino acids. Data base comparisons and primary structure analysis of the deduced protein sequence (HvCNG) indicated significant homology to cyclic nucleotide and voltage-activated ion channels including six putative membrane spanning domains, a putative cyclic nucleotide binding site, a pore region and a voltage-sensor motif. Heterologous expression of the cloned cDNA in Sf9 cells resulted in a polypeptide of the predicted molecular mass. Patch clamp analysis allowed to record the activity of the identified HvCNG channels; they were activated by cAMP but also by hyperpolarization. The channel displayed in potassium solution a conductance of 30 pS; the ion selectivity was calculated as PK/PNa approximately 3. Northern blot analysis revealed that the channel is highly expressed in the antennae; weaker signal were detected in heads and legs. In situ hybridization of tissue sections through the antennae showed a spatial distribution of reactive cells; they are located beneath sensillar hairs. Thus, a novel channel type has been identified which may play an important role in antennal cells.