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D Larhammar

Publications and source records attributed to D Larhammar.

At least 37 records · Page 2Linked to original sources
PubMed2000

Evolution of the neuropeptide Y receptor family: gene and chromosome duplications deduced from the cloning and mapping of the five receptor subtype genes in pig.

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.

Animals↗
PubMed2000

Neuropeptide Y family of peptides: structure, anatomical expression, function, and molecular evolution.

Evolutionary relationships between neuroendocrine peptides are often difficult to resolve across divergent phyla due to independent duplication events in different lineages. Thanks to peptide purification and molecular cloning in many different species, the situation is beginning to clear for the neuropeptide Y (NPY) family, which also includes peptide YY (PYY), the tetrapod pancreatic polypeptide (PP) and the fish pancreatic peptide Y (PY). It has long been assumed that the first duplication to occur in vertebrate evolution generated NPY and PYY, as both of these are found in all gnathostomes as well as lamprey. Evidence from other gene families show that this duplication was probably a chromosome duplication event. The origin of a second PYY peptide found in lamprey remains to be explained. Our recent cloning of NPY, PYY and PY in the sea bass proves that fish PY is a separate gene product. We favour the hypothesis that PY is a duplicate of the PYY gene and that it may have occurred late in fish evolution, as PY has so far only been found in acanthomorph fishes. Thus, this duplication seems to be independent of the one that generate PP from PYY in tetrapods, although both tetrapod PP and fish PY are expressed in the pancreas. Studies in the sea bass and other fish show that PY, in contrast to PP, is expressed in the nervous system. We review the literature on the distribution and functional aspects of the various NPY-family peptides in vertebrates.

Amino Acid Sequence↗
PubMed1999

Identification of an amino acid residue important for binding of methiothepin and sumatriptan to the human 5-HT(1B) receptor.

Site-directed mutagenesis of the human 5-HT1B receptor was performed to investigate the role of the amino acid residues cysteine 326 and tryptophan 327 in transmembrane region VI and aspartic acid 352 in transmembrane region VII in ligand binding. Binding studies were performed with the antagonist radioligand [3H]GR125743 on mutant and wild-type receptors stably expressed in Chinese hamster ovary cells (CHO)-K1 cells. Substitution of tryptophan 327 by alanine resulted in decreased affinities of all ligands tested. The most prominent changes in affinity were observed for the antagonist methiothepin and the antimigraine drug sumatriptan, which were reduced approximately 300- and 60-fold, respectively. Nevertheless, the affinity of 5-HT remained the same. Replacement of the aspartic acid 352 by alanine reduced high-affinity binding of 5-HT. Substitution of cysteine 326 by alanine had minor effects on ligand binding. Some of these results agree with the results from mutagenesis studies of the corresponding amino acids in other receptors. However, some notable differences also emerge showing that functional roles of individual amino acid residues must be tested experimentally in each receptor subtype.

Amino Acid Sequence↗
PubMed1999

Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor.

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides for which five different G-protein-coupled receptor subtypes have been cloned in mammals. To identify additional subtypes we have performed PCR with degenerate primers in different species. We describe here the cloning and pharmacological profile of a unique NPY receptor subtype in the zebrafish that has tentatively been called the zYa receptor. It has 46-50% amino acid identity to the mammalian Y1, Y4 and y6 receptors and the previously cloned zebrafish receptors zYb and zYc, and only about 27% to Y2 and Y5. The zYa receptor binds NPY and PYY from mammals as well as zebrafish with high affinities and has a K(d) of 28 pM for porcine (125)I-PYY. It has a unique binding profile displaying some features in common with each of the mammalian Y1, Y2 and Y5 receptors. In a microphysiometer assay the receptor responds with extracellular acidification. Chromosomal mapping in the zebrafish genome of zYa, zYb and zYc receptor genes indicates a possible orthologous relationship between zYc and mammalian y6, but identifies no obvious mammalian ortholog for zYa (zYb is a recent copy of zYc in the fish lineage). These results imply that previous studies of NPY in fishes, which have striven to interpret the effects within the framework of mammalian Y1, Y2, and Y5 receptors, need to be reevaluated. Thus, the sequence comparisons, pharmacological properties, and chromosomal localization suggest that the zYa receptor is a novel NPY receptor subtype which is likely to be present also in mammals.

Amino Acid Sequence↗
PubMed1999

Characterization of the cloned atlantic cod neuropeptide Y-Yb receptor: peptide-binding requirements distinct from known mammalian Y receptors.

Five members of the neuropeptide Y (NPY) receptor family have been cloned in mammals. The recently cloned NPY receptor in the Atlantic cod seems to be distinct from the mammalian subtypes as it has only 50% identity to Y1, Y4, and y6 and only 30% to Y2 and Y5. In most of the other families of G-protein-coupled receptors, species homologues have 65-90% identity between fishes and mammals. The functional expression and detailed pharmacological characterization of this cod NPY receptor, designated Yb, is reported. Membranes of cells transiently transfected with cod Yb showed saturable [(125)I]PYY binding with a K(d) of 45 pM. The pharmacological profile is similar to those of both the zebrafish Yb and Yc receptors and distinct from those of the mammalian NPY receptors. In competition experiments the cod Yb receptor had the following rank order of potencies: porcine PYY = porcine NPY = p[Leu(31), Pro(34)]NPY > zebrafish PYY > zebrafish NPY >> NPY2-36 = NPY3-36 > NPY18-36 > bovine PP = [D-Trp(32)]NPY > BIBP3226. This is in sharp contrast to the high selectivity of BIBP3226 for the Y1 receptor from all mammalian species. Together with the low amino acid identity of cod Yb with the mammalian Y1, Y4, and y6 receptors, this is further support for the notion that fish Yb constitutes a distinct NPY receptor subtype.

Animals↗
PubMed1999

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function.

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.

Animals↗
PubMed1999

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.

Amino Acid Sequence↗
PubMed1999

The highly conserved synapse protein SNAP-25 displays sequence variability in the cockroach Leucophaea maderae.

SNAP-25 (synaptosome-associated protein of 25 kD) is attached to the intracellular side of presynaptic membranes where it serves as a target receptor for the vesicle docking machinery prior to release of neurotransmitter. SNAP-25 displays a high degree of sequence conservation between vertebrates and Drosophila melanogaster. To obtain more information about conserved regions of SNAP-25, we have isolated cDNA clones from the cockroach Leucophaea maderae. One clone (Lm1) encoded a full-length SNAP-25 protein and its deduced amino acid sequence is 77% identical to Drosophila SNAP-25. Surprisingly, the cockroach protein is 17 amino acids shorter than Drosophila SNAP-25 at the carboxy terminus. Four other cDNA clones encode parts of SNAP-25 and each clone has distinct characteristics, including amino acid replacements and unique carboxy termini. Thus, the highly conserved protein SNAP-25 displays unexpected sequence variability in the cockroach that may indicate specialized SNAP-25 isoforms.

Amino Acid Sequence↗
PubMed1999

Cloning the neuropeptide Y exon 2 from sea bass (Dicentrarchus labrax).

A partial genomic library of sea bass DNA was constructed and screened with a goldfish NPY cDNA probe. Two identical clones were isolated and sequenced. The clones contain a segment with high identity to exon 2 of the NPY gene in tetrapods. This segment encodes a 62-amino acid peptide consisting of a signal peptide of 28 amino acids and the main portion of the mature NPY (34 amino acids). In the latter extension, sea bass NPY shows high identity with the human and deduced ancestral gnathostome sequences (88 and 91%, respectively). The open reading frame is followed by a consensus splice donor site. Northern blot hybridization to examine tissue distribution detected a 1-kb RNA transcript restricted to brain tissue. These data show that the NPY gene of this teleost fish has the same intron positions as tetrapods for at least two of the gene's three introns. In addition, the high evolutionary conservation of NPY is corroborated since sea bass NPY exhibits the same identity to both goldfish and human NPY.

Amino Acid Sequence↗
PubMed1999

Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP-25 with the Semliki Forest virus vector.

We have examined whether the Semliki Forest virus (SFV) expression vector can be used to manipulate the exocytotic machinery in cultured hippocampal neurons. Autaptic responses were recorded in individually identified neurons which overexpressed either a non-synaptic protein, the transferrin receptor, or the synaptic SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDA). In neurons overexpressing the transferrin receptor, autaptic responses occurred in a similar proportion and had similar amplitudes (12-18 h postinfection) as in uninfected control neurons. With increasing time after the infection, an increasing proportion of the transferrin receptor-overexpressing neurons showed changes in the shape of the cell body, but the autaptic responses appeared normal as long as recordings could be performed (up to 30 h postinfection). In contrast, in SNAP-25-overexpressing neurons, the proportion of responding cells was reduced 12-18 h after the infection, and the amplitude of the autaptic current in responding neurons was also reduced. The sensitivity to exogenously applied glutamate was, however, unchanged. Biochemical analysis showed that 50% of the overexpressed SNAP-25 was palmitoylated. The levels of two other SNAREs, syntaxin and synaptobrevin (also called vesicle-associated membrane protein), were not affected. Our results indicate that the SFV vector can provide an effective tool to study the function of proteins participating in neurotransmitter release.

Animals↗
PubMed1998

Cloning of two loci for synapse protein Snap25 in zebrafish: comparison of paralogous linkage groups suggests loss of one locus in the mammalian lineage.

Synaptosome-associated protein of 25 kDa (Snap25) is an intracellular protein that is defined as a target receptor for synapse vesicles prior to neurotransmitter release. Snap25 is highly conserved, with 61% identity between human and Drosophila melanogaster. Whereas mammals and chicken have a single locus for Snap25, the tetraploid goldfish has at least three loci. We report that the zebrafish has two loci with 91% amino acid identity to each other. The alternative splicing of exon 5 arose before the gene duplication. The expression patterns of the two loci are virtually identical in adult zebrafish. The two zebrafish snap25 loci are located in paralogous linkage groups that seem to correspond to human chromosome 20, which harbors the SNAP locus, and human chromosome 14. Because no additional Snap25 homologue has been reported for any mammal or chicken, snap25.2 may have been lost in the amniote or even tetrapod lineage.

Amino Acid Sequence↗
PubMed1998

Preprocholecystokinin mRNA-expressing neurons in the rat parabrachial nucleus: subnuclear localization, efferent projection, and expression of nociceptive-related intracellular signaling substances.

The pontine parabrachial nucleus (PB) is a major target for ascending fibers from nociresponsive dorsal horn neurons. Several different neuropeptides have been identified in the PB. By using double-labeling methods that combine in situ hybridization histochemistry with retrograde tract tracing and immunohistochemistry, we have examined the subnuclear localization of preprocholecystokinin mRNA (ppCCK)-containing neurons, investigated their efferent projection, and analyzed their expression of intracellular signaling substances that may be of importance for nociceptive processing. The results show that neurons containing ppCCK are preferentially localized to the superior lateral subnucleus (PBsl), whereas other subnuclei, such as the dorsal lateral, external lateral, central lateral, and ventral lateral subnuclei, and the Kölliker-Fuse nucleus, contain only moderate to small numbers of such neurons. Injections of the retrograde tracer cholera toxin subunit b into the ventromedial hypothalamus demonstrated that ppCCK-containing neurons in PBsl were projection neurons. Following nociceptive stimulation, the ppCCK-containing neurons expressed FOS protein as well as phosphorylated cyclic AMP-responsive element-binding protein (CREB). In addition, Ca2+/calmodulin-dependent kinase II (CaMKII) was heavily and rather selectively expressed in PBsl and was co-localized to ppCCK-containing neurons. These observations show that nociceptive stimuli activate a cholecystokinin pathway from the parabrachial nucleus to the ventromedial hypothalamus that may be important for homeostatic responses to tissue damage, and point to a putative intracellular route for Ca2+-mediated FOS transcription via CaMKII and CREB for the regulation of ppCCK transcription.

Animals↗
PubMed1998

Cloning and functional expression of the guinea pig neuropeptide Y Y2 receptor.

Five neuropeptide Y (NPY) receptor subtypes have been cloned in mammals. The degree of sequence conservation differs considerably between subtypes as well as between evolutionary lineages. To shed further light on this, we have cloned the five NPY receptors in the guinea pig. Here, we report the cloning of the guinea pig Y2 receptor. The Y2 receptor is generally highly conserved, with 90-95% identity between different orders of mammals, including the guinea pig. The guinea pig receptor has a divergent cytoplasmic tail, indicating possible differences in regulation of signalling and/or down regulation. COS-7 cells transiently transfected with the gpY2 receptor show saturable 125I-PYY binding with a Kd = 6 pM. In displacement experiments, the gpY2 receptor was similar to the human and rat receptors with the following rank order of potencies: pNPY > pPYY > pNPY13-36 = pNPY22-36 >> [Leu31Pro34]NPY > BIBP3226. Thus, the guinea pig Y2 receptor is well conserved in comparison with human and rat with regard to both amino acid sequence and pharmacological profile.

Amino Acid Sequence↗
PubMed1998

The cloned guinea pig pancreatic polypeptide receptor Y4 resembles more the human Y4 than does the rat Y4.

Pancreatic polypeptide (PP) is involved in gastrointestinal functions and forms, together with neuropeptide Y (NPY) and peptide YY (PYY), the PP-fold family of peptides. The PP-binding receptor subtype Y4 has so far been cloned in human, rat, and mouse, and displays extensive species differences regarding sequence, pharmacology, and distribution. To explore this variability further, we have cloned the Y4 receptor in the guinea pig, which is evolutionarily equally distantly related to both humans and rodents. The guinea pig Y4 receptor is 84% identical to the human Y4 receptor, but only 74-75% identical to the rat and mouse receptors. The two latter are 75-76% identical to human Y4. The guinea pig Y4 receptor bound 125I-hPP with a dissociation constant (Kd) of 29+/-3 pM. The pharmacological profile of guinea pig Y4 has the following rank order of potencies: PP > NPY approximately = PYY approximately = LP-NPY approximately = LP-PYY > NPY2-36 >> [D-Trp32]NPY. Thus, the guinea pig receptor is more similar to the human Y4 than to the rat Y4 both in sequence and pharmacology. This agrees with the greater identity between guinea pig and human PP compared to rat PP. These comparisons suggest that the rodent PPs and Y4 receptors have an accelerated replacement rate.

Amino Acid Sequence↗
PubMed1998

Cloning of a neuropeptide Y/peptide YY receptor from the Atlantic cod: the Yb receptor.

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides that bind to G protein-coupled receptors. Five different receptor subtypes have recently been cloned in mammals and we have found another three receptor genes in the zebrafish, called zYa, zYb, and zYc, that appear to be distinct subtypes as deduced from their widely different sequences. To elucidate the evolutionary relationships between the mammalian and zebrafish receptors, we have used the zebrafish probes to isolate genomic clones from another teleost fish, the Atlantic cod, Gadus morhua. We present here the sequence of the cod Yb gene, whose deduced protein sequence is equally identical to the zebrafish Yb (69%) and Yc proteins (66%). The two zebrafish receptors are 76% identical to each other, suggesting that they arose by gene duplication in the zebrafish lineage after divergence from the cod lineage. The five cloned mammalian NPY-family receptors and the three cloned zebrafish NPY receptors indicate that this is the largest receptor family among all peptide receptors that belong to the superfamily of G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1998

Site-directed mutagenesis of the human 5-HT1B receptor.

Site-directed mutagenesis was used to investigate the molecular interactions involved in ligand binding to the human 5-HT1B receptor. Six mutants were constructed at four positions and expressed in Chinese hamster ovary cells. Substitution of the amino acid F185 in transmembrane region IV by an alanine increased the affinities of sumatriptan, methysergide and 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) 3-4-fold and substitution by a methionine increased the affinities of methysergide and methiothepin 2- and 3-fold, respectively. Substitution of amino acid S334 in transmembrane region VI by an alanine increased the affinity of 8-OH-DPAT 5-fold. In accordance with this, the EC50 value of 8-OH-DPAT was decreased 7-fold. This suggests that the serine at position 334 causes steric hindrance for 8-OH-DPAT binding that is lost in the S334A mutant. Mutation of F354 in transmembrane region VII, which differs between receptor subtypes, increased the affinity of methiothepin 2-3-fold but the affinities of the other compounds tested were essentially unchanged.

Amino Acid Substitution↗