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Binding of Chimeric Peptides M617 and M871 to Galanin Receptor Type 3 Reveals Characteristics of Galanin Receptor–Ligand Interaction
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.ORCID iD: 0000-0001-6107-0844
2010 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 16, no 1, 17-22 p.Article in journal (Refereed) Published
Abstract [en]

The neuropeptide galanin is ascribed to a variety of biological effects, but selective compounds to examine the specific roles of the three receptor subtypes are currently lacking. The recently introduced chimeric peptide ligands M617 and M871 target the galanin receptors GalR1 and GalR2, respectively. These peptides have been used to examine receptor function in vitro and in vivo, but their affinity to GalR3 has not been tested. Here, we report the binding affinity of these peptides at human GalR3 and demonstrate that M617 binds GalR3 and stimulates this receptor in an agonistic manner, whereas M871 shows very low affinity towards GalR3 (Ki 49.2 ± 9.4 nM and > 10 microM respectively). An L-alanine scan of M617 revealed the importance of the ligand C-terminus in GalR3 binding, which stands in contrast to the structural requirements for binding to GalR1 and GalR2. These data provide insights into galanin receptor ligand binding that should be considered when using these compounds in functional studies.

Place, publisher, year, edition, pages
2010. Vol. 16, no 1, 17-22 p.
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-37229DOI: 10.1007/s10989-009-9197-9ISI: 000275791700003OAI: oai:DiVA.org:su-37229DiVA: diva2:295981
Available from: 2010-02-17 Created: 2010-02-17 Last updated: 2015-04-22Bibliographically 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. Galanin receptor ligands
Open this publication in new window or tab >>Galanin receptor ligands
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

In the nervous system galanin primarily displays a modulatory role. The galaninergic system consists of a number of bioactive peptides with a highly plastic expression pattern and three different receptors. The lack of receptor subtype selective ligands and antibodies have severely hampered the charac-terization of this system. Therefore, most of the knowledge has been drawn from experiments with transgenic animals, which has given some major conclusions, despite the compensatory effects seen in several animal studies. Therefore, the production of subtype selective ligands is 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, espe-cially the development of novel galanin receptor subtype selective ligands. Paper I demonstrates the potential to N-terminally extend galanin ana-logues and the successful development of a galanin receptor 2 (GalR2) selec-tive ligand. In addition, a cell line stably expressing galanin receptor 3 (GalR3) was developed, to improve and simplify future evaluations of sub-type selective galanin ligands. Paper II measures the affinities of M617 and M871 to GalR3 and demon-strates that M871 preferentially binds GalR2. Furthermore, the relatively high affinity of M617 was evaluated by assessing the contribution in recep-tor interaction of individual amino acid residues in the C-terminal part of the M617.

In conclusion, this thesis has provided a novel design strategy for galanin receptor ligands and increased the understanding of ligand interactions with the GalR3. Furthermore, M1145 has together with new analogues proven to be highly GalR2 specific, holding promises to future delineation of the galaninergic system as a therapeutic target.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2009. 50 p.
Keyword
Galanin, GALP, GPCR, Agonist, GalR, Chimeric peptide, Inositol phosphate production, Receptor specificity
National Category
Natural Sciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-59743 (URN)978-91-7155-915-9 (ISBN)
Presentation
2009-09-11, Heilbronnsalen, Institutionen för neurokemi, Svante Arrhenius väg 21A, Stockholm, 12:15 (English)
Opponent
Supervisors
Available from: 2011-11-03 Created: 2011-07-11 Last updated: 2015-04-21Bibliographically approved

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