E. Vreugdenhil

Early evolutionary origin of the neurotrophin receptor family

Neurotrophins and their Trk receptors play a crucial role in the development and maintenance of the vertebrate nervous system, but to date no component of this signalling system has been found in invertebrates. We describe a molluscan Trk receptor, designated Ltrk, from the snail Lymnaea stagnalis. The full‐length sequence of Ltrk reveals most of the characteristics typical of Trk receptors, including highly conserved transmembrane and intracellular tyrosine kinase domains, and a typical extracellular domain of leucine‐rich motifs flanked by cysteine clusters. In addition, Ltrk has a unique N‐terminal extension and lacks immunoglobulin‐like domains. Ltrk is expressed during development in a stage‐specific manner, and also in the adult, where its expression is confined to the central nervous system and its associated endocrine tissues. Ltrk has the highest sequence identity with the TrkC mammalian receptor and, when exogenously expressed in fibroblasts or COS cells, binds human NT‐3, but not NGF or BDNF, with an affinity of 2.5 nM. These findings support an early evolutionary origin of the Trk family as neuronal receptor tyrosine kinases and suggest that Trk signalling mechanisms may be highly conserved between vertebrates and invertebrates.

A NOVEL G PROTEIN-COUPLED RECEPTOR MEDIATING BOTH VASOPRESSIN- AND OXYTOCIN-LIKE FUNCTIONS OF LYS-CONOPRESSIN IN LYMNAEA STAGNALIS

We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.

Characterization of a putative molluscan insulin-related peptide receptor.

In the pond snail Lymnaea stagnalis (Ls), growth and associated processes are likely to be controlled by a family of molluscan insulin-related peptides (MIP). Here we report on the cloning of a cDNA encoding a putative receptor for these MIP. This cDNA was isolated from Ls via PCR with degenerate oligodeoxynucleotides corresponding to conserved parts of the tyrosine kinase domain of the human insulin receptor and its Drosophila homologue. Many of the typical insulin-receptor features, including a cysteine-rich domain, a single transmembrane domain and a tyrosine-kinase domain are conserved in the predicted, 1607-amino acid (aa) protein. Comparison of the aa sequence of the molluscan receptor to other insulin-receptor sequences revealed strong variations in the percentage of sequence identity for the different domains, ranging from 70% sequence identity in the tyrosine-kinase domain to virtually no sequence identity in the C-terminal sequence. Striking differences are the absence of a clear tetrabasic cleavage site, and the extremely long C-terminus of 308 aa that contains seven Tyr residues. Southern blot analyses at varying stringencies, extensive screening of cDNA- and genomic libraries, and PCR experiments indicate the presence of a single putative MIP receptor. This suggests that the four different MIP may exert their functional role in Ls by binding to the same receptor.

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization

SummaryThe caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material.A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as “ectopic” CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral gangion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed. The nerves emanating from this ganglion contained many more immunoreactive axons than those from the left pedal ganglion. A polyclonal antibody raised against the synthetic fragment of CDCH stained, in addition to the neurons and axons revealed with the monoclonal antibody and the cDNA probe, three other major groups of neurons. Two are located in the cerebral ganglion, the other in the left pedal ganglion.The present findings suggest the presence of a system of neurons that contain CDCH or CDCH-like peptides. The role this system may play in the control of egg-laying and egg-laying behaviour is discussed.

Molecular cloning of the rDNA of Saccharomyces rosei and comparison of its transcription initiation region with that of Saccharomyces carlsbergensis

We have cloned one complete repeating unit of rDNA from Saccharomyces rosei and determined its physical and genetic organization. Heteroduplex analysis of the rDNA units from S. rosei and S. carlsbergensis shows that the nontranscribed spacers are largely nonhomologous in sequence, whereas the transcribed regions are essentially homologous. We also determined the transcription initiation site for the 37S precursor RNA on S. rosei rDNA. Sequence comparison of the region surrounding the site of transcription initiation for the 37S RNA with the corresponding region of S. carlsbergensis revealed extensive homology from position -9 downstream into the external transcribed spacer. Very little homology was observed between position -9 and -55, but some homologous tracts are present upstream from position -55.

Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2·10−9 M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0·10−7 M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl− current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Functional characterisation of a 5-HT2 receptor cDNA cloned from Lymnaea stagnalis.

A G-protein-coupled receptor (5-HT2Lym) resembling members of the 5-HT2 receptor subfamily was cloned from the mollusc Lymnaea stagnalis. Serotonin induces a concentration-dependent increase in intracellular inositol phospates in HEK293 cells expressing this receptor (EC50 = 114 nM). 5-HT2Lym differs from mammalian 5-HT2 receptors by the presence of a large amino-terminal region. This large domain appears to preclude an adequate level of expression of 5-HT2Lym in HEK293. Therefore, we constructed a cDNA encoding an amino-terminally truncated receptor (delta N-5-HT2Lym) that appeared to be much better expressed in HEK293 cells. delta N-5-HT2Lym-expressing cells exhibit a serotonin-induced stimulation of phosphatidylinositol bisphosphate hydrolysis (EC50 = 11.4 nM) and a high-affinity binding of the 5-HT2-selective antagonist [3H]mesulergine (Kd = 4 nM). Inhibition of this binding by several 5-HT2 antagonists and agonists revealed a pharmacological profile most closely resembling those of 5HT2Dro, 5-HT2B and 5-HT2C.

Neuropeptide Gene Families that Control Reproductive Behaviour and Growth in Molluscs

Peptidergic neuroendocrine cells play an important role in the control of complex and interrelated life processes, such as growth, reproduction and behaviour. These neurons function as transducer cells; they integrate (neural) signals carrying information on the internal and external environment and convert these signals into peptide messages, which, in a co-ordinated fashion, activate the appropriate target systems of the body to produce a specific response. For a complete understanding of the basic mechanisms underlying the functioning of peptidergic cells, information on many aspects of the cell is needed. To study these different processes (input, integrative capacities, branching patterns, ultrastructural characteristics, biosynthesis and release activities, etc.), a multidisciplinary approach is needed. Unfortunately, the peptidergic systems of most animal groups are not optimally suited for this approach. The identification of the cells in vivo is often impossible; the cells are often too small for specific techniques, such as the intracellular recording of membrane potentials; and furthermore, the physiological and behavioural systems of many animals, especially vertebrates, are very complicated, which seriously hampers studies on the role of peptidergic neurons in the control of physiological processes and behaviour.

Expression of the egg-laying hormone genes in peripheral neurons and exocrine cells in the reproductive tract of the mollusc Lymnaea stagnalis

The neuroendocrine caudodorsal cells play an important role in the control of reproduction in Lymnaea stagnalis. These neurons produce at least nine neuropeptides which are encoded by caudodorsal cell hormone-I and -II genes. The role of some of these peptides in the control of reproduction has been established. The present study demonstrates that the transcription and translation of the caudodorsal cell hormone genes also proceeds abundantly in the reproductive tract of this hermaphroditic animal. In the female part of the reproductive tract neurons were found to express gene I. These neurons are most likely involved in the control of transport of the eggs and egg-masses and in the regulation of secretory activity from the female accessory sex glands. In the male part of the reproductive tract exocrine secretory cells express gene I or gene II. The gene products are secreted into the male duct and transferred to the female copulant during copulation. Furthermore, putative sensory neurons in the skin were found to express gene I. The results indicate that in L. stagnalis the complex process of reproduction is regulated--at least in part--by a set of neuropeptides which are encoded by a small multigene family, viz. the caudodorsal cell gene family.

Molecular cloning of G protein α subunits from the central nervous system of the mollusc Lymnaea stagnalis

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein α subunits that are expressed in the Lymnaea CNS, i.e. Gαo, Gα, and Gαi. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation or G protein α subunits suggests that functional insights into G protein‐mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.