Dietmar Richter

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin‐like receptor 1 (SLC‐1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC‐1 and G protein‐gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N‐terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK‐mediated currents but also by the induction of Ca2+ dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the Gi‐ or Gq‐mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.

Reverse physiology in drosophila: identification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family.

By using degenerate oligonucleotide primers deduced from the conserved regions of the mammalian somatostatin receptors, a novel G‐protein‐coupled receptor from Drosophila melanogaster has been isolated exhibiting structural similarities to mammalian somatostatin/galanin/opioid receptors. To identify the bioactive ligand, a ‘reverse physiology’ strategy was used whereby orphan Drosophila receptor‐expressing frog oocytes were screened against potential ligands. Agonistic activity was electrophysiologically recorded as inward potassium currents mediated through co‐expressed G‐protein‐gated inwardly rectifying potassium channels (GIRK). Using this approach a novel peptide was purified from Drosophila head extracts. Mass spectrometry revealed an octapeptide of 925 Da with a sequence Ser‐Arg‐Pro‐Tyr‐Ser‐Phe‐Gly‐Leu‐NH2 reminiscent of insect allatostatin peptides known to control diverse functions such as juvenile hormone synthesis during metamorphosis or visceral muscle contractions. Picomolar concentrations of the synthesized octapeptide activated the cognate receptor response mediated through GIRK1, indicating that we have isolated the 394‐amino‐acid Drosophila allatostatin receptor which is coupled to the Gi/Go class of G proteins.

Molecular cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis

A cDNA clone encoding a novel putative G‐protein coupled receptor has been isolated from a rat testis cDNA library using a PCR‐amplified cDNA fragment as a hybridization probe. Northern blot analysis reveals that a corresponding 1.5 kb mRNA is exclusively expressed in testis. By in situ hybridization experiments this mRNA has been localized in spermatocytes and spermatids but not in spermatogonia, Leydig or sertoli cells. Ontogenic studies show that expression of the receptor‐encoding mRNA and sexual maturation are correlated reaching highest levels during the second and third months. Although the ligand for this receptor has not yet been identified, this receptor may play a role during reproduction.

Identification of a cis -Acting Dendritic Targeting Element in MAP2 mRNAs

In neurons, a limited number of mRNAs have been identified in dendritic processes, whereas other transcripts are restricted to the cell soma. Here we have investigated the molecular mechanisms underlying extrasomatic localization of mRNAs encoding microtubule-associated protein 2 (MAP2) in primary neuronal cultures. Vectors expressing recombinant mRNAs were introduced into hippocampal and sympathetic neurons using DNA transfection and microinjection protocols, respectively. Chimeric mRNAs containing the entire 3′ untranslated region of MAP2 transcripts fused to a nondendritic reporter mRNA are detected in dendrites. In contrast, RNAs containing MAP2 coding and 5′ untranslated regions or tubulin sequences are restricted to the cell soma. Moreover, 640 nucleotides from the MAP2 3′ untranslated region (UTR) are both sufficient and essential for extrasomatic localization of chimeric mRNAs in hippocampal and sympathetic neurons. Thus, a cis-acting dendritic targeting element that is effective in two distinct neuronal cell types is contained in the 3′ UTR of MAP2 transcripts. The observation of RNA granules in dendrites implies that extrasomatic transcripts seem to assemble into multimolecular complexes that may function as transport units.

Differential Responses to Osmotic Stress of Vasopressin-Neurophysin mRNA in Hypothalamic Nuclei

mRNA encoding the vasopressin-neurophysin precursor was quantitated in individual hypothalamic nuclei of rats by a liquid hybridization assay. Drinking of 2% saline for 14 days, a treatment that increased the plasma vasopressin concentration 9-fold, resulted in a 5- and 2-fold increase in mRNA levels in the supraoptic and paraventricular nucleus, respectively. This osmotic stimulus had no effect on vasopressin-neurophysin mRNA content of the suprachiasmatic nucleus. This dissociation in regulation of vasopressin-neurophysin mRNA in hypothalamic nuclei indicates the existence of two separate vasopressin systems that are independently activated.

Axonal transport of neuropeptide encoding mRNAs within the hypothalamo-hypophyseal tract of rats

Hypothalamic vasopressin and oxytocin transcripts have been detected in the posterior pituitary suggesting either transcription of the respective genes in pituicytes or axonal mRNA transport from the hypothalamus to the nerve terminals of the posterior pituitary. The concept of axonal mRNA transport is supported firstly, by Northern blot and in situ hybridization analysis indicating that vasopressin and oxytocin mRNAs are also present in the neural stalk; secondly, by intron analysis and transcription run on experiments demonstrating the absence of primary vasopressin and oxytocin transcripts in non‐neuronal cells of the posterior pituitary; thirdly, by embryonic developmental studies showing that appearance of vasopressin transcripts in the hypothalamus and the pituitary anlage is correlated. Furthermore, during axonal transport the respective mRNAs are subject to specific modification at the poly(A) tails.

Somatostatin Receptor Interacting Protein Defines a Novel Family of Multidomain Proteins Present in Human and Rodent Brain

By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile α motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.

Fragile X mental retardation protein regulates the levels of scaffold proteins and glutamate receptors in postsynaptic densities.

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1–3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3′-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

Ovulation triggers oxytocin gene expression in the bovine ovary.

Oxytocin Neurophysin Corpus luteum Estrous cycle Gene expression

Differential expression and dendritic transcript localization of Shank family members: identification of a dendritic targeting element in the 3′ untranslated region of Shank1 mRNA

Shank proteins are scaffolding proteins in the postsynaptic density of excitatory synapses in the mammalian brain. In situ hybridization revealed that Shank1/SSTRIP and Shank2/ProSAP1 mRNAs are widely expressed early in postnatal brain development whereas Shank3/ProSAP2 expression increases during postnatal development especially in the cerebellum and thalamus. Shank1 and Shank3 (but not Shank2) mRNAs are present in the molecular layers of the hippocampus, consistent with a dendritic transcript localization. Shank1 and Shank2 transcripts are detectable in the dendritic fields of Purkinje cells, whereas Shank3 mRNA is restricted to cerebellar granule cells. The appearance of dendritic Shank mRNAs in cerebellar Purkinje cells coincides with the onset of dendrite formation. Expression of reporter transcripts in hippocampal neurons identifies a 200-nucleotide dendritic targeting element (DTE) in the Shank1 mRNA. The widespread presence of Shank mRNAs in dendrites suggests a role for local synthesis of Shanks in response to stimuli that induce alterations in synaptic morphology.