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Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions
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
2007 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 103, no 5, 1774-1784 p.Article in journal (Refereed) Published
Abstract [en]

To define the specific role of the galanin receptors when mediating the effect of galanin, effective tools for distinct activation and inhibition of the different receptor subtypes are required. Several of the physiological effects modulated by galanin are implicated to be mediated via the GalR2 subtype and have been distinguished from GalR1 effects by utilizing the Gal(2–11) peptide, recognizing only GalR2 and GalR3. In this study, we have performed a mutagenesis approach on the GalR2 subtype and present, for the first time, a molecular characterization of the interactions responsible for ligand binding and receptor activation at this receptor subtype. Our results identify four residues, His252 and His253 located in transmembrane domain 6 and Phe264 and Tyr271 in the extracellular loop 3, to be of great significance. We show evidence for the N-terminal tail of GalR2 to participate in ligand binding and that selective binding of Gal(2–11) includes interaction with the Ile256 residue, located at the very top of TM 6. In conclusion, we present a mutagenesis study on GalR2 and confer interactions responsible for ligand binding and receptor activation as well as selective recognition of the Gal(2–11) peptide at this receptor subtype. The presented observations could be of major importance for the design and development of new and improved peptide and non-peptide ligands, selectively activating the GalR2 subtype.

Place, publisher, year, edition, pages
2007. Vol. 103, no 5, 1774-1784 p.
National Category
Biochemistry and Molecular Biology Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-20667DOI: 10.1111/j.1471-4159.2007.04959.xISI: 000250985200008PubMedID: 17953676OAI: oai:DiVA.org:su-20667DiVA: diva2:187193
Available from: 2007-11-28 Created: 2007-11-28 Last updated: 2015-04-21Bibliographically approved
In thesis
1. Subtype selective activation and molecular characterization of galanin receptors
Open this publication in new window or tab >>Subtype selective activation and molecular characterization of galanin receptors
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Showing an extensive distribution in the nervous system, and often in co-localization with the classical neurotransmitters, neuropeptides are functioning as important modulators of neuronal signaling. Subsequently, compelling evidence has implicated a modulatory role for the neuropeptide galanin in several physiological functions. The effect of galanin is trancduced intracellularly by three different receptors, and defining the explicit effect from these receptor subtypes is of outmost interest, and likely to result in future therapeutic utilization of the galanin system.

The main aim of this thesis was to improve the development of subtype selective ligands utilized to differentiate between the galanin receptor subtypes. To achieve this, we have designed and developed novel galanin receptor ligands and characterized the molecular interactions necessary for ligand bindig at the GalR2 subtype.

The major findings include the introduction and characterization of two galanin receptor ligands, selectively activating GalR1 or inhibiting GalR2. Although having moderate selectivity, the two ligands have been utilized in a number of studies, pursuing their initial presentation, in order to differentiate between the galanin receptors and to establish their specific function. Further optimization is likely to improve the selectivity and utilization of these ligands. By identifying the major pharmacophores in the Gal(2-11) ligand and the residues in the GalR2 subtype participating in ligand binding, we have been able to characterize the binding site in this receptor subtype and interactions that are of significance for recognition of subtype specific ligands. Together, these findings on GalR2 and Gal(2-11) are of importance for future design of ligands acting on this receptor.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2007. 87 p.
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-1344 (URN)91-7155-325-8 (ISBN)
Public defence
2007-02-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2007-01-03 Created: 2007-01-03 Last updated: 2010-02-24Bibliographically approved
2. 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

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