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Determining receptor–ligand interaction of human galanin receptor type 3
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2010. Vol. 57, no 7, 804-811 p.
Keyword [en]
Galanin, Galanin receptor type 3, hGalR3, GPCR, Docking
National Category
Neurosciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-37226DOI: 10.1016/j.neuint.2010.08.018ISI: 000284437500013OAI: oai:DiVA.org:su-37226DiVA: diva2:295543
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 201924Knut and Alice Wallenberg Foundation
Available from: 2010-02-17 Created: 2010-02-17 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Characterization of galanin receptors using chimeric peptides and site-directed mutagenesis
Open this publication in new window or tab >>Characterization of galanin receptors using chimeric peptides and site-directed mutagenesis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Galanin, a 29 (30) amino acid neuropeptide, is found throughout both the central and peripheral nervous systems. It signals via three receptors, GalR1-3, all belonging to the rhodopsin-like G-protein coupled receptors. Galanin and its receptors have been implicated in a vast variety of biological processes. To facilitate further characterization of the physiological/pathological roles of galanin, subtype selective ligands targeting the three receptors individually would be of great aid.

In this thesis the main objective was to provide more information about galanin receptor-ligand interactions, primarily concerning GalR2 and 3.  By using information gained from previously developed chimeric peptides, we designed and synthesized a novel peptide selective towards GalR2 (Paper I). This peptide, M871, binds GalR2 in an inhibitory manner, likely due to its truncated N-terminus and bulky character. In Paper II and IV we performed L-alanine mutagenesis assays of GalR2 and 3 respectively. By point substituting amino acid residues in the receptor sequence, we identified crucial pharmacophores for ligand binding, primarily in transmembrane regions 6 and 7. The targeted residues were selected based on knowledge concerning GalR1 and on conservation between the three receptors. For GalR3 we also conducted a computational docking assay. A homology model was first constructed using three crystallized structures of other receptors also belonging to the Rhodopsin family. Ligands galanin(2-6) and SNAP398299 were then docked to GalR3 in flexible mode. The docking resulted in characterization of GalR3-ligand interactions and conclude that this receptor display a relatively deep and narrow binding pocket. As a result of this, it was hypothesized that the C-terminus of ligands is of importance for GalR3 affinity. An L-alanine scan of ligand was performed (paper III), which confirmed this theory.

In conclusion, our results give insights into galanin receptor-ligand interactions, information that is relevant for ligand design and drug development.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2010. 70 p.
Keyword
galanin, GPCR, mutagenesis, neuropeptide
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-37259 (URN)978-91-7447-018-5 (ISBN)
Public defence
2010-04-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.Available from: 2010-03-29 Created: 2010-02-18 Last updated: 2015-04-21Bibliographically approved
2. Delineating Ligand-Receptor Interactions and the Design of Subtype Selective Galanin Receptor Ligands
Open this publication in new window or tab >>Delineating Ligand-Receptor Interactions and the Design of Subtype Selective Galanin Receptor Ligands
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:nbn:se:su:diva-75503 (URN)978-91-7447-503-6 (ISBN)
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

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Runesson, JohanSollenberg, Ulla E.Jurkowski, WiktorElofsson, ArneLangel, Ülo
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