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Characterization of galanin receptors using chimeric peptides and site-directed mutagenesis
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
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 [en]
galanin, GPCR, mutagenesis, neuropeptide
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
URN: urn:nbn:se:su:diva-37259ISBN: 978-91-7447-018-5 (print)OAI: oai:DiVA.org:su-37259DiVA: diva2:297887
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
List of papers
1. M871 – a novel peptide antagonist selectively recognizing the galanin receptor type 2
Open this publication in new window or tab >>M871 – a novel peptide antagonist selectively recognizing the galanin receptor type 2
2006 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 12, no 2, 115-119 p.Article in journal (Refereed) Published
Abstract [en]

Galanin and its three receptors have been linked to a wide variety of physiological processes and are distributed in both the central and peripheral nervous systems. Further knowledge of the properties of galanin-activated signaling systems can best be obtained by the availability of peptide and non-peptide ligands that are selective for the different receptor subtypes. The current study describes binding and signaling data for the chimeric peptide, galanin-(2–13)-Glu-His-(Pro)3-(Ala-Leu)2-Ala-amide (M871). This compound binds to the galanin receptor type 2 with more than 30-fold higher affinity than to the galanin receptor type 1 and exhibits antagonist actions at galanin receptor type 2, blocking increased release of inositol phosphate produced by galanin in CHO cells. This peptide opens new possibilities for the study of galanin receptor physiology.

National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-23019 (URN)10.1007/s10989-005-9008-x (DOI)
Available from: 2007-01-03 Created: 2007-01-03 Last updated: 2015-04-21Bibliographically approved
2. Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions
Open this publication in new window or tab >>Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions
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.

National Category
Biochemistry and Molecular Biology Neurosciences
Identifiers
urn:nbn:se:su:diva-20667 (URN)10.1111/j.1471-4159.2007.04959.x (DOI)000250985200008 ()17953676 (PubMedID)
Available from: 2007-11-28 Created: 2007-11-28 Last updated: 2017-12-13Bibliographically approved
3. Binding of Chimeric Peptides M617 and M871 to Galanin Receptor Type 3 Reveals Characteristics of Galanin Receptor–Ligand Interaction
Open this publication in new window or tab >>Binding of Chimeric Peptides M617 and M871 to Galanin Receptor Type 3 Reveals Characteristics of Galanin Receptor–Ligand Interaction
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.

National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-37229 (URN)10.1007/s10989-009-9197-9 (DOI)000275791700003 ()
Available from: 2010-02-17 Created: 2010-02-17 Last updated: 2015-04-22Bibliographically 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
Show others...
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: 2017-12-12Bibliographically approved

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