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Dan Larhammar - One of the best experts on this subject based on the ideXlab platform.
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earlY vertebrate chromosome duplications and the evolution of the Neuropeptide Y Receptor gene regions
BMC Evolutionary Biology, 2008Co-Authors: Tomas A Larsson, Frida Olsson, Gorel Sundstrom, Larsgustav Lundin, Sydney Brenner, Byrappa Venkatesh, Dan LarhammarAbstract:Background One of the manY gene families that expanded in earlY vertebrate evolution is the Neuropeptide (NPY) Receptor familY of G-protein coupled Receptors. Earlier work bY our lab suggested that several of the NPY Receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterYgian lineage, based on their location on chromosomes sharing several gene families. In this studY we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY Receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10.
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earlY vertebrate chromosome duplications and the evolution of the Neuropeptide Y Receptor gene regions
BMC Evolutionary Biology, 2008Co-Authors: Tomas A Larsson, Frida Olsson, Gorel Sundstrom, Larsgustav Lundin, Sydney Brenner, Byrappa Venkatesh, Dan LarhammarAbstract:One of the manY gene families that expanded in earlY vertebrate evolution is the Neuropeptide (NPY) Receptor familY of G-protein coupled Receptors. Earlier work bY our lab suggested that several of the NPY Receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterYgian lineage, based on their location on chromosomes sharing several gene families. In this studY we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY Receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10. Chromosome regions with conserved sYntenY were identified and confirmed bY phYlogenetic analYses in H. sapiens, M. musculus, D. rerio, T. rubripes and T. nigroviridis. 26 gene families, including the NPY Receptor genes, (plus 3 described recentlY bY other labs) showed a tree topologY consistent with duplications in earlY vertebrate evolution and in the actinopterYgian lineage, therebY supporting expansion through block duplications. Eight gene families had complications that precluded analYsis (such as short sequence length or variable number of repeated domains) and another eight families did not support block duplications (because the paralogs in these families seem to have originated in another time window than the proposed genome duplication events). RT-PCR carried out with several tissues in T. rubripes revealed that all five NPY Receptors were expressed in the brain and subtYpes Y2, Y4 and Y8 were also expressed in peripheral organs. We conclude that the phYlogenetic analYses and chromosomal locations of these gene families support duplications of large blocks of genes or even entire chromosomes. Thus, these results are consistent with two earlY vertebrate tetraploidizations forming a paralogon comprising human chromosomes 4, 5, 8 and 10 and one teleost tetraploidization. The combination of positional and phYlogenetic data further strengthens the identification of orthologs and paralogs in the NPY Receptor familY.
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three Neuropeptide Y Receptor genes in the spinY dogfish squalus acanthias support en bloc duplications in earlY vertebrate evolution
Molecular Biology and Evolution, 2003Co-Authors: Erik Salaneck, David H Ardell, Earl T Larson, Dan LarhammarAbstract:It has been debated whether the increase in gene number during earlY vertebrate evolution was due to multiple independent gene duplications or sYnchronous duplications of manY genes. We describe here the cloning of three Neuropeptide Y (NPY) Receptor genes belonging to the Y1 subfamilY in the spinY dogfish, Squalus acanthias, a cartilaginous fish. The three genes are orthologs of the mammalian subtYpes Y1, Y4, and Y6, which are located in paralogous gene regions on different chromosomes in mammals. Thus, these genes arose bY duplications of a chromosome region before the radiation of gnathostomes (jawed vertebrates). Estimates of duplication times from linearized trees together with evidence from other gene families supports two rounds of chromosome duplications or tetraploidizations earlY in vertebrate evolution. The anatomical distribution of mRNA was determined bY reverse-transcriptase PCR and was found to differ from mammals, suggesting differential functional diversification of the new gene copies during the radiation of the vertebrate classes.
Tomas Hokfelt - One of the best experts on this subject based on the ideXlab platform.
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subtYpes Y1 and Y2 of the Neuropeptide Y Receptor are respectivelY expressed in pro opiomelanocortin and Neuropeptide Y containing neurons of the rat hYpothalamic arcuate nucleus
Neuroendocrinology, 1997Co-Authors: Christian Broberger, Marc Landry, H Wong, John N Walsh, Tomas HokfeltAbstract:The arcuate nucleus of the hYpothalamus houses a number of neurochemicallY different cell populations. Among these, a dense cluster of small Neuropeptide-Y (NPY)-expressing neurons is located in its ventromedial subdivision and a pro-opiomelanocortin (POMC)-expressing neuron population in its ventrolateral part. Furthermore, both Neuropeptide Y Y1 and Y2 Receptors (Y1-Rs and Y2-Rs) are expressed in the arcuate nucleus. Here we analYse the co-expression of NPY and POMC/adrenocorticotropic hormone with the Y1-R and Y2-R in arcuate neurons using immunohistochemistrY and in situ hYbridization. ManY, but not all, POMC neurons expressed Y1-R mRNA and protein. ConverselY, several Y1-R-positive, POMC-negative neurons were found. NPY-positive nerve terminals were found in close apposition to Y1-R-like immunoreactivitY localized close to the dendritic and somatic cell membranes. Y2-R mRNA was found in almost all NPY mRNA-expressing neurons, but also in a group of NPY mRNA-negative cells. These results show that the POMC neurons are targets for NPY, which is presumablY present in, and released from, fibres originating in the ventromedial arcuate nucleus and which maY plaY a role in NPY-induced feeding. Release of NPY, and possible coexisting messengers, maY be controlled bY presYnaptic Y2-R expressed in NPY neurons. Taken together, the findings support the division of Y1-Rs and Y2-Rs into post- and presYnaptic Receptors, respectivelY.
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subtYpes Y1 and Y2 of the Neuropeptide Y Receptor are respectivelY expressed in pro opiomelanocortin and Neuropeptide Y containing neurons of the rat hYpothalamic arcuate nucleus
Neuroendocrinology, 1997Co-Authors: Christian Broberger, Marc Landry, H Wong, John N Walsh, Tomas HokfeltAbstract:The arcuate nucleus of the hYpothalamus houses a number of neurochemicallY different cell populations. Among these, a dense cluster of small Neuropeptide-Y (NPY)-expressing neurons is located in its v
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the effects of intrathecal Neuropeptide Y on the spinal nociceptive flexor reflex in rats with intact sciatic nerves and after peripheral axotomY
Neuroscience, 1994Co-Authors: Tomas Hokfelt, Z WiesenfeldhallinAbstract:Abstract We examined the effects of intrathecallY administered Neuropeptide Y on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats with intact sciatic nerves, or 11–39 daYs after unilateral transection of the sciatic nerve. In rats with intact sciatic nerve, intrathecal Neuropeptide Y at low doses (10 and 100ng) caused a brief facilitation of the flexor reflex. At a dose of 300 ng, the effect of Neuropeptide Y on the flexor reflex was biphasic i.e. a brief facilitation followed bY slight depression. At higher doses (1 and 10 μg), the effect of Neuropeptide Y was mainlY inhibitorY, causing substantial and usuallY prolonged depression of the flexor reflex magnitude. The reflex depression caused bY intrathecal Neuropeptide Y was not reversed bY the opioid antagonist naloxone or theα 2 adrenoceptor antagonist atipamezole. Intrathecal Neuropeptide Y at doses up to 1 and 10μg had no effect on reflex facilitation caused bY conditioning stimulation of C-fibers, intrathecal substance P or neurokinin A. Topical application of Neuropeptide Y (1 μg/μl) failed to influence the monosYnaptic reflex in normal rats. Eleven to 16 daYs after peripheral axotomY, the initial excitation of the flexor reflex to intrathecal Neuropeptide Y was significantlY enhanced in axotomized compared with normal rats. However, the depressive effect of Neuropeptide Y on the flexor reflex was unchanged. Neuropeptide Y did not influence the monosYnaptic reflex in axotomized rats at this period. In experiments performed on rats in which the sciatic nerve had been transected 31–39 daYs previouslY, the facilitatorY effect of Neuropeptide Y on the flexor reflex remained enhanced compared with normal rats. Furthermore, the inhibitorY effect of Neuropeptide Y also increased as 100 ng intrathecal Neuropeptide Y was able to produce reflex depression in a similar fashion as 300 ng Neuropeptide Y normallY and the reflex depression caused bY 1 μg Neuropeptide Y was stronger and longer lasting than in normal rats. Intrathecal Neuropeptide Y (100 ng-10 μg) in rats with intact sciatic nerves caused a moderate decrease in spinal cord dorsal surface blood flow as measured with a laser Doppler flowmeter. This effect of Neuropeptide Y was unchanged in axotomized rats. The present results support previous observations that spinal application of Neuropeptide Y in normal rats caused antinociception. As the depressive effect of Neuropeptide Y is independent of spinal opioid andα 2 -adrenergic sYstems, it maY be mediated bY its own Receptors. The biphasic effect of Neuropeptide Y maY reflect its action on different subtYpes of Neuropeptide Y Receptors. Experiments with Neuropeptide Y Receptor antagonists are required to further define the role of endogenous Neuropeptide Y in nociception normallY and after peripheral axotomY.
José Ángel Narváez - One of the best experts on this subject based on the ideXlab platform.
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galanin Receptor Neuropeptide Y Receptor interactions in the central nervous sYstem
Current Protein & Peptide Science, 2014Co-Authors: Zaida Diazcabiale, Concepción Parrado, Manuel Narváez, Carmelo Millón, Araceli Puigcerver, Rafael Coveñas, Kjell Fuxe, Antonio Floresburgess, José Ángel NarváezAbstract:The presence of Galanin and Neuropeptide Y and/or their Receptors in several areas of the brain involved in memorY, mood, cardiovascular control and food intake indicates that Galanin, and Neuropeptide Y could equilibrate the phYsiological actions of each other. There is evidence for the existence of interactions between Galanin Receptor and Neuropeptide Y Receptor in the nucleus of the solitarii tract (NTS), hYpothalamus and dorsal raphe nucleus probablY taking place with the formation of heteromers between Galanin Receptor and Neuropeptide Y Y1 Receptor. The galanin fragment (Gal 1-15) preferring Receptors maY instead be formed bY the GalR1-GalR2 heteromer which in the NTS maY interact with Neuropeptide Y Y2 Receptors. These Receptor heteromers maY be one keY molecular mechanism for Galanin and its N-terminal fragment (Galanin 1-15) to modulate the function of different tYpes of glia-neuronal networks in the CNS, especiallY the emotional, metabolic and cardiovascular networks.
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galanin Receptor Neuropeptide Y Receptor interactions in the dorsal raphe nucleus of the rat
Neuropharmacology, 2011Co-Authors: Zaida Diazcabiale, Concepción Parrado, Manuel Narváez, Carmelo Millón, Araceli Puigcerver, Kjell Fuxe, Luis J Santin, José Ángel NarváezAbstract:The aim of this studY was to evaluate bY quantitative Receptor autoradiographY the interactions between Neuropeptide Y Y1 (NPY Y1) and Galanin (GAL) Receptors in the dorsal raphe nucleus (DRN) where both GAL Receptors and NPY Y1 Receptors exist. The abilitY of the GAL Receptor antagonist M35 to block the GAL action was also evaluated. Double immunocYtochemical staining of 5-hYdroxYtrYptmine and c-Fos and stereologY techniques were used to studY the specific cell activation in the DRN after the intracerebroventricular coinjections of GAL and the NPY Y1/Y5 agonist [ 125 I] Leu 31 ,Pro 34 PYY. GAL (0.3 nM) decreases [ 125 I] Leu 31 ,Pro 34 PYY binding in the DRN bY 48% (p < 0.01) as shown bY quantitative Receptor autoradiographY. This effect was reversed with the GAL Receptor antagonist M35. Intracerebroventricular coinjections of NPY Y1/Y5 agonist and GAL reduced the c-Fos expression in the serotoninergic cells induced bY the NPY Y1/Y5 agonist in DRN. These results indicate the existence of antagonistic interactions between GAL Receptors and NPY Y1 Receptors in the DRN that maY be of relevance in mood disorders.
Daniel Huster - One of the best experts on this subject based on the ideXlab platform.
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the g protein coupled Neuropeptide Y Receptor tYpe 2 is highlY dYnamic in lipid membranes as revealed bY solid state nmr spectroscopY
Chemistry: A European Journal, 2014Co-Authors: Peter Schmidt, Lars Thomas, Daniel Huster, Paul Muller, Holger A ScheidtAbstract:In spite of the recent success in crYstallizing several G-protein-coupled Receptors (GPCRs), a comprehensive biophYsical characterization of these molecules under phYsiological conditions also requires the studY of the molecular dYnamics of these proteins. The molecular mobilitY of the human Neuropeptide Y Receptor tYpe 2 reconstituted into dimYristoYlphosphatidYlcholine (DMPC) membranes was investigated bY means of solid-state NMR spectroscopY. Static (15) N NMR spectra show that the Receptor performs axiallY sYmmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed bY quantitative measurements of the motional (1) H,(13) C order parameter of the CH, CH2 , and CH3 groups. In directlY polarized (13) C NMR experiments, these order parameters showed astonishinglY low values of SCH =0.55, S CH 2=0.33, and S CH 3=0.17, which corresponds to segmental amplitudes of approximatelY 50° in the backbone and approximatelY 50-60° in the side chain. At phYsiological temperature, (2) H NMR spectra of the deuterated Receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a verY broad spectrum that could stem from the transmembrane α-helices. These results suggest that the crYstal structures of GPCRs onlY represent a static snapshot of these highlY mobile molecules, which undergo significant structural fluctuations with relativelY large amplitudes in a liquid-crYstalline membrane at phYsiological temperature.
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oxidative in vitro folding of a cYsteine deficient variant of the g protein coupled Neuropeptide Y Receptor tYpe 2 improves stabilitY at high concentration
Biological Chemistry, 2013Co-Authors: Kristina Witte, Anette Kaiser, Peter Schmidt, Victoria Splith, Lars Thomas, Sandra Berndt, Daniel Huster, Annette G BecksickingerAbstract:In vitro folding of G protein-coupled Receptors into a detergent environment represents a promising strategY for obtaining sufficient amounts of functional Receptor molecules for structural studies. TYpicallY, these preparations exhibit a poor long-term stabilitY especiallY at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequentlY folded Neuropeptide Y Receptor tYpe 2. We identified the free cYsteines in the Receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cYsteine residues were mutated to alanine or serine without anY significant loss of functionalitY of the Receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cYsteines was irreversiblY formed bY applYing oxidative in vitro folding. ApplYing this strategY, the stabilitY of the functionallY folded Y2 Receptor could be increased to 20 daYs at a concentration of 15 μm in a micelle environment consisting of 1,2-diheptanoYl-sn-glYcero-3-phosphocholine and n-dodecYl-s-D-maltoside.
Annette G Becksickinger - One of the best experts on this subject based on the ideXlab platform.
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mutations in arrestin 3 differentiallY affect binding to Neuropeptide Y Receptor subtYpes
Cellular Signalling, 2014Co-Authors: Luis E Gimenez, Annette G Becksickinger, Stefanie Babilon, Lizzy Wanka, Vsevolod V GurevichAbstract:Abstract Based on the identification of residues that determine Receptor selectivitY in arrestins and the phYlogenetic analYsis of the arrestin (arr) familY, we introduced fifteen mutations of Receptor-discriminator residues in arr-3, which were identified previouslY using mutagenesis, in vitro binding, and BRET-based recruitment assaY in intact cells. The effects of these mutations were tested using Neuropeptide Y Receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected bY these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other Receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantlY reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas TYr239Thr reduced it ~ 7-fold. Thus, manipulation of keY residues on the Receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentiallY affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the Receptor involves several mechanisticallY distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific Receptors and particular activation states of the same Receptor.
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automated solid phase peptide sYnthesis to obtain therapeutic peptides
Beilstein Journal of Organic Chemistry, 2014Co-Authors: Veronika Made, Sylvia Elsheindl, Annette G BecksickingerAbstract:The great versatilitY and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last Years. In order to increase the drugabilitY of these frequentlY unstable and rapidlY cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide sYnthesis (SPPS) offers a suitable technologY to produce chemicallY engineered peptides. This review concentrates on the application of SPPS bY Fmoc/t-Bu protecting-group strategY, which is most commonlY used. Critical issues and suggestions for the sYnthesis are covered. The development of automated methods from conventional to essentiallY improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGYlation are described as covalent conjugation methods, which can be applied bY a combination of automated and manual sYnthesis approaches. The sYnthesis and application of SPPS is described for Neuropeptide Y Receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated sYnthesis technologies.
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oxidative in vitro folding of a cYsteine deficient variant of the g protein coupled Neuropeptide Y Receptor tYpe 2 improves stabilitY at high concentration
Biological Chemistry, 2013Co-Authors: Kristina Witte, Anette Kaiser, Peter Schmidt, Victoria Splith, Lars Thomas, Sandra Berndt, Daniel Huster, Annette G BecksickingerAbstract:In vitro folding of G protein-coupled Receptors into a detergent environment represents a promising strategY for obtaining sufficient amounts of functional Receptor molecules for structural studies. TYpicallY, these preparations exhibit a poor long-term stabilitY especiallY at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequentlY folded Neuropeptide Y Receptor tYpe 2. We identified the free cYsteines in the Receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cYsteine residues were mutated to alanine or serine without anY significant loss of functionalitY of the Receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cYsteines was irreversiblY formed bY applYing oxidative in vitro folding. ApplYing this strategY, the stabilitY of the functionallY folded Y2 Receptor could be increased to 20 daYs at a concentration of 15 μm in a micelle environment consisting of 1,2-diheptanoYl-sn-glYcero-3-phosphocholine and n-dodecYl-s-D-maltoside.
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incorporation of ortho carbaboranYl ne modified l lYsine into Neuropeptide Y Receptor Y1 and Y2 selective analogues
Journal of Medicinal Chemistry, 2011Co-Authors: Verena M Ahrens, Annette G Becksickinger, Rene Frank, Sven Stadlbauer, Evamarie HeyhawkinsAbstract:Nontoxic ortho-carbaborane is one of the most promising structure for boron neutron capture therapY (BNCT). For directed uptake of ortho-carbaborane bY tumor cells, Receptor-subtYpe selective Neuropeptide Y (NPY) and its derivatives were modified with ortho-carbaborane. The derivative [F7, P34]-NPY has been shown to be a breast cancer selective ligand that binds to the Y1-Receptor subtYpe, whereas [Ahx(5−24)]-NPY selectivelY addresses Y2-Receptor subtYpes that are found in neuroblastoma cells. ortho-CarbaboranYl propionic acid was sYnthesized and linked to the e-amino group of Nα-Fmoc protected l-lYsine. The characterization of the compounds was performed bY NMR, IR, and MS studies. The carbaborane-modified amino acid was incorporated into NPY, [F7, P34]-NPY, and [Ahx(5−24)]-NPY bY an optimized solid phase peptide sYnthesis using Fmoc protection. Binding studies and IP accumulation assaYs confirmed nanomolar affinitY and activitY of the modified analogues despite of the large carbaborane cluster. Internal...
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agonist induced Receptor internalization of Neuropeptide Y Receptor subtYpes depends on third intracellular loop and c terminus
Cellular Signalling, 2008Co-Authors: Ilka Bohme, Jan Stichel, Cornelia Walther, Karin Morl, Annette G BecksickingerAbstract:Agonist stimulation of G-protein coupled Receptors (GPCRs) results in the redistribution of the Receptor from the cell surface into intracellular compartments through the process of endocYtosis. Monitoring ligand-mediated internalization of GPCRs in living cells has become experimentallY accessible bY applYing fluorescent reagents and fluorescence microscopY. BY using cell lines that transientlY, stablY or endogenouslY express the human Y Receptor (hYR) subtYpes hY(1)R, hY(2)R, hY(4)R and hY(5)R and differentlY fluorescentlY tagged Receptor proteins we were able to unravel further details concerning the internalization behavior of this multi-Receptor/multi-ligand sYstem. For the first time we could show that also the hY(2)R is internalized with a rate which is comparable to the hY(1)R and the hY(4)R. In contrast, the hY(5)R was internalized much slower and the rate remained unaffected bY co-expression with other hYR subtYpes. Furthermore Receptor subtYpe co-expressing cells and selectivelY binding peptides revealed a Receptor subtYpe selective internalization. BY using novel hY(5)/hY(2) Receptor chimera the Receptor subtYpe dependent differences in hY Receptor internalization could be identified on a molecular level.