H. Van Heerikhuizen

Functional characteristics of heterologously expressed 5-HT receptors

Over the past 10 years, molecular cloning has revealed the presence of 15 serotonin (5-hydroxytryptamine; 5-HT) receptor subtypes, which can be subdivided in seven subfamilies. Except for the 5-HT3 receptors, which are ligand-gated ion channels, all 5-HT receptors belong to the superfamily of G-protein-coupled receptors. The large multiplicity of 5-HT receptor subtypes has been suggested to be a direct result of the evolutionary age of the 5-HT system. Molecular information on G-protein-coupled 5-HT receptors is currently available for several mammalian species as well as for a limited number of invertebrate species (insects, molluscs). The aim of this review is to give an overview of all cloned 5-HT receptor subtypes belonging to the superfamily of G-protein-coupled receptors with specific emphasis on the pharmacological and signaling properties of the receptors upon expression in several heterologous expression systems.

Towards Understanding the Role of Insulin in the Brain: Lessons from Insulin-related Signaling Systems in the Invertebrate Brain

Insulin is a molecule that has played a key role in several of the most important landmarks in medical and biological research. It is one of the most extensively studied protein hormones, and its structure and function have been elucidated in many vertebrate species, ranging from man to hagfish and turkey. The structure, function as well as tissue of synthesis of vertebrate insulins are strictly conserved. The structural identification of insulin-related peptides from invertebrates has disrupted the picture of an evolutionary stable peptide hormone. Insulin-related peptides in molluscs and insects turned out to be a structurally diverse group encoded by large multi-gene families that are uniquely expressed in the brain and serve functions different from vertebrate insulin. In this review, we discuss invertebrate insulins in detail. We examine how these peptides relate to the model role that vertebrate insulin has played over the years; however, more importantly, we discuss several unique principles that can be learned from them. We show how diversity of these peptides is generated at the genetic level and how the structural diversity of the peptides is linked to the exclusive presence of a single type of neuronal insulin receptor-related receptor. We also discuss the fact that the invertebrate peptides, in addition to a hormonal role, may also act in a synaptic and/or nonsynaptic fashion as transmitters/neuromodulators on neurons in the brain. It can be expected that the use of well-defined neuronal preparations in invertebrates may lead to a further understanding of these novel functions and may act as guide preparations for a possible role of insulin and its relatives in the vertebrate brain.

Chaperonin complex with a newly folded protein encapsulated in the folding chamber.

A subset of essential cellular proteins requires the assistance of chaperonins (in Escherichia coli, GroEL and GroES), double-ring complexes in which the two rings act alternately to bind, encapsulate and fold a wide range of nascent or stress-denatured proteins. This process starts by the trapping of a substrate protein on hydrophobic surfaces in the central cavity of a GroEL ring. Then, binding of ATP and co-chaperonin GroES to that ring ejects the non-native protein from its binding sites, through forced unfolding or other major conformational changes, and encloses it in a hydrophilic chamber for folding. ATP hydrolysis and subsequent ATP binding to the opposite ring trigger dissociation of the chamber and release of the substrate protein. The bacteriophage T4 requires its own version of GroES, gp31, which forms a taller folding chamber, to fold the major viral capsid protein gp23 (refs 16–20). Polypeptides are known to fold inside the chaperonin complex, but the conformation of an encapsulated protein has not previously been visualized. Here we present structures of gp23–chaperonin complexes, showing both the initial captured state and the final, close-to-native state with gp23 encapsulated in the folding chamber. Although the chamber is expanded, it is still barely large enough to contain the elongated gp23 monomer, explaining why the GroEL–GroES complex is not able to fold gp23 and showing how the chaperonin structure distorts to enclose a large, physiological substrate protein.

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.

A system for the analysis of yeast ribosomal DNA mutations.

To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.

Molecular cloning and chromosomal assignment of the human homolog of int-1, a mouse gene implicated in mammary tumorigenesis.

Viral mammary tumorigenesis in mice is frequently initiated by proviral activation of a highly conserved cellular gene called int-1. We have cloned the human homolog of this putative mammary oncogene and compared its structure to that of the mouse gene by heteroduplex analysis. The human int-1 gene was localized on chromosome 12 by use of somatic cell hybrids.

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.