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Delineating Ligand-Receptor Interactions and the Design of Subtype Selective Galanin Receptor Ligands
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

We now celebrate that it is 30 years since galanin was first isolated. During these three decades galanin has been identified in numerous tissues and physiological processes, and in an abundant number of species. In the nervous system galanin primarily displays a modulatory role. The galaninergic system consists of a number of bioactive peptides with a highlyplastic expression pattern and three different receptors, GalR1-GalR3. The lack of receptor subtype selective ligands and antibodies have severely hampered the characterization of this system. Therefore, most of the knowledgehas been drawn from experiments with transgenic animals, which has givensome major conclusions, despite the risk of inducing compensatory effects inthese animal studies. Therefore, the production of subtype selective ligandsis of great importance to delineate the galanin system and slowly experimental data from receptor subtype selective ligand trials is emerging. This thesis aims at studying galanin receptor-ligand interactions and to increase and improve the utilized tools in the galanin research field, especially the development of novel galanin receptor subtype selective ligands. Paper I demonstrates the potential to N-terminally extend galanin analogues and the successful development of a GalR2 selective ligand. In addition, a cell line stably expressing GalR3 was developed to improve and simplify future evaluations of receptor subtype selective galanin ligands. Paper II extends the number of GalR2 selective ligands and shows that i.c.v. administration of galanin receptor ligands stimulates food intake through GalR1. Paper III demonstrates the successful development of a mixed GalR1/GalR2 agonist without any detectable interaction with GalR3. Subsequently, this peptide was used to delineate which receptor subtype mediatesthe neuroprotective effects of galanin in the CA3 region of hippocampus. Furthermore, a robust protocol for detection of receptor activation was developed to ease the detection of the relative potency of novel ligands at the three galanin receptor subtypes. Paper IV describes the finding of several essential amino acids for ligand interaction in GalR3 through the performance of an L-alanine mutagenesis study. A constructed in silico homology model of GalR3 confirmed and extended these findings. In conclusion, this thesis provides a novel design strategy for galanin receptor ligands and increases the understanding of ligand interactions with the GalR3. Furthermore, published ligands together with new galanin analogues have proven to be highly receptor specific, thus implicating that a future delineation of the galaninergic system as a therapeutic target is possible.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2012. , 96 p.
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-75503ISBN: 978-91-7447-503-6 (print)OAI: oai:DiVA.org:su-75503DiVA: diva2:516922
Public defence
2012-06-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.Available from: 2012-05-10 Created: 2012-04-20 Last updated: 2015-04-21Bibliographically approved
List of papers
1. A novel GalR2-specific peptide agonist
Open this publication in new window or tab >>A novel GalR2-specific peptide agonist
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2009 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 43, no 3, 187-192 p.Article in journal (Refereed) Published
Abstract [en]

The galanin peptide family and its three receptors have with compelling evidence been implicated in several high-order physiological disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family. In the current study we present data on receptor binding and functional response for a novel galanin receptor type 2 (GalR2) selective chimeric peptide, M1145 [(RG)(2)-N-galanin(2-13)-VL-(P)(3)-(AL)(2)-A-amide]. The M1145 peptide shows more than 90-fold higher affinity for GalR2 over GalR1 and a 76-fold higher affinity over GalR3. Furthermore, the peptide yields an agonistic effect in vitro, seen as an increase in inositol phosphate (IP) accumulation, both in the absence or the presence of galanin. The peptide design with a N-terminal extension of galanin(2-13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family and opens new possibilities to apply the galanin system as a putative drug target.

Keyword
Galanin, GALP, GPCR, Agonist, GalR, Chimeric peptide, Inositol phosphate production, Receptor specificity
National Category
Neurosciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-36269 (URN)10.1016/j.npep.2009.04.004 (DOI)000267403000002 ()19467704 (PubMedID)
Available from: 2011-01-13 Created: 2010-01-22 Last updated: 2017-12-11Bibliographically approved
2. Novel galanin receptor subtype specific ligands in feeding regulation
Open this publication in new window or tab >>Novel galanin receptor subtype specific ligands in feeding regulation
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2011 (English)In: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 58, no 6, 714-720 p.Article in journal (Refereed) Published
Abstract [en]

Galanin a 29/30-residue neuropeptide has been implicated in several functions in the central nervous system, including the regulation of food consumption. Galanin and its analogues administered intraventricularly or into the hypothalamic region of brain have been shown to reliably and robustly stimulate the consumption of food in sated rodents. Three galanin receptor subtypes have been isolated, all present in the hypothalamus, but little is known about their specific role in mediating this acute feeding response. Presently, we introduce several novel GalR2 selective agonists and then compare the most selective of these novel GalR2 subtype selective agonists to known GalR1 selective agonist M617 for their ability to stimulate acute consumption of several foods shown to be stimulated by central administration of galanin. GalR1 selective agonist M617 markedly stimulated acute consumption of high-fat milk, but neither GalR2 selective agonist affected either high-fat milk or cookie mash intake. The present results are consistent with the involvement of GalR1 in mediating the acute feeding consumption by galanin and suggest an approach applicable to exploring galanin receptor specificity in normal and abnormal behavior and physiology.

Keyword
Galanin, Galanin receptor type 2, Feeding, Agonist, GPCR, Neuropeptide
National Category
Neurosciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-68532 (URN)10.1016/j.neuint.2011.02.012 (DOI)000290139900014 ()
Note
6Available from: 2012-01-04 Created: 2012-01-04 Last updated: 2017-12-08Bibliographically approved
3. Pharmacological stimulation of galanin receptor 1 but not galanin receptor 2 attenuates kainic acid-induced neuronal cell death in the rat hippocampus
Open this publication in new window or tab >>Pharmacological stimulation of galanin receptor 1 but not galanin receptor 2 attenuates kainic acid-induced neuronal cell death in the rat hippocampus
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The neuropeptide galanin is widely distributed in the central and peripheral nervous systems. Galanin is part of a bigger family of bioactive peptides and its biological activity is mediated through three G-protein coupled receptor subtypes, GalR1-3. The last 20 years have provided data supporting a neuroprotective effect of galanin that probably is mediated through the activation of GalR1 and GalR2.

In this study, an additional galanin receptor ligand, M1154, was developed to fully address the pharmaceutical potential of galanin receptor subtype selective ligands to inhibit excitotoxic cell death. We thereafter adapted a single protocol for signaling assay for all three galanin receptors, by utilizing a non-labeled real time instrument, to ease classification of novel ligands according to their ability to activate the different galanin receptor subtypes. M1154 is a GalR1/2 selective ligand in our binding and signaling assays with no detectable interaction with GalR3.

The novel M1154 peptide was compared to earlier published selective ligands, namely M617 and M1145, in its ability to reduce the excitotoxic effects of kainic acid (KA). While no significant difference in the time course or severity of induced seizure activity was observed, administration of either M617 or M1154 before KA administration, significantly attenuated the neuronal cell death in the hippocampus. Our results clearly show the potential therapeutic value of agonists selective for GalR1 as neuroprotective agents for the prevention of excitotoxic neuronal cell death.

Keyword
GPCR, galanin, neuropeptide, galanin receptor, GalR1, label free real time technology, excitotoxicity, M1154
National Category
Neurosciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-75087 (URN)
Available from: 2012-04-05 Created: 2012-04-05 Last updated: 2016-01-29Bibliographically approved
4. Determining receptor–ligand interaction of human galanin receptor type 3
Open this publication in new window or tab >>Determining receptor–ligand interaction of human galanin receptor type 3
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2010 (English)In: Neurochemistry International, ISSN 0197-0186, E-ISSN 1872-9754, Vol. 57, no 7, 804-811 p.Article in journal (Refereed) Published
Abstract [en]

Galanin is a neuropeptide found throughout the central and peripheral nervous systems of a wide range of species, ranging from human and mouse to frog and tuna. Galanin mediates its physiological roles through three receptors (GalR1–3), all members of the G-protein coupled receptor family. In mapping these roles, receptor subtype selective ligands are crucial tools. To facilitate the ligand design, data on receptor structure and interaction points are of great importance. The current study investigates the mechanism by which galanin interacts with GalR3. Mutated receptors were tested with competitive binding analysis in vitro. Our studies identify six mutagenic constructs that lost receptor affinity completely, despite being expressed at the cell surface. Mutations of the Tyr1033.33 in transmembrane helix (TM) III, His2516.51 in TM VI, Arg2737.35 or His2777.39 in TM VII, Phe2636.63 or Tyr2707.32 in the extracellular loop III all result in complete reduction of ligand binding. In addition, docking studies of an in silico model of GalR3 propose that four of the identified residues interact with pharmacophores situated within the galanin(2–6) sequence. This study provides novel insights into the interaction between ligands and GalR3 and highlights the requirement for correct design of targeting ligands.

Keyword
Galanin, Galanin receptor type 3, hGalR3, GPCR, Docking
National Category
Neurosciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-37226 (URN)10.1016/j.neuint.2010.08.018 (DOI)000284437500013 ()
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 201924Knut and Alice Wallenberg Foundation
Available from: 2010-02-17 Created: 2010-02-17 Last updated: 2015-04-21Bibliographically approved

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