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Subtype selective activation and molecular characterization of galanin receptors
Stockholm University, Faculty of Science, Department of Neurochemistry.
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: urn:nbn:se:su:diva-1344ISBN: 91-7155-325-8 (print)OAI: oai:DiVA.org:su-1344DiVA: diva2:189922
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
List of papers
1. A galanin receptor subtype 1 specific agonist
Open this publication in new window or tab >>A galanin receptor subtype 1 specific agonist
Show others...
2005 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3904, Vol. 11, no 1, 17-27 p.Article in journal (Refereed) Published
Abstract [en]

The chimeric peptide M617, galanin(1–13)-Gln14-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (K i=0.23±.12 nM) while lower affinity was seen towards GalR2 (K i=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca2+, high Mg2+, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.

Keyword
cAMP production, chimeric peptide, feeding behavior, galanin, galanin receptor, inositol phosphate production, locus coeruleus, nociception, receptor internalization, receptor specificity
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23018 (URN)10.1007/s10989-004-1717-z (DOI)
Available from: 2007-01-03 Created: 2007-01-03 Last updated: 2015-04-21Bibliographically approved
2. 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
3. Important pharmacophores for binding to galanin receptor 2
Open this publication in new window or tab >>Important pharmacophores for binding to galanin receptor 2
2005 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 39, no 3, 169-171 p.Article in journal (Refereed) Published
Abstract [en]

Galanin(2–11) has been introduced as a receptor subtype selective ligand for the GalR2 subtype of the galanin receptors, and has gained use in pharmacological studies of galaninergic signaling in the past two years. By introducing l-Ala substitutions in the galanin(2–11) sequence, we have examined the amino acid residues which are of importance for binding to the GalR2 receptor. Our study shows that Trp2, Asn5, Gly8 and Tyr9 are of great importance for high affinity binding. When placed in an α-helical conformation, the side chains of these residues are, with the exception of Tyr9, displayed on the same “side” of the peptide. This information is useful in the rational design of non-peptide type GalR2 receptor ligands.

Keyword
Galanin, Galanin receptors, Galanin(2–11), Galanin receptor selective ligand, L-Ala substitution
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:su:diva-23020 (URN)
Available from: 2007-01-03 Created: 2007-01-03 Last updated: 2015-04-21Bibliographically approved
4. 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: 2015-04-21Bibliographically approved

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