Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions

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.     

GalR2/3 mediates proliferative and trophic effects of galanin on postnatal hippocampal precursors

Understanding how neural activity is functionally linked to the stem cell niche, is assuming ever increasing importance as hippocampal neurogenesis is shown to be important for modulating the behavioural responses to stress and for certain forms of learning and memory. Neuropeptides such as neuropeptide Y and vasoactive intestinal peptide have emerged as important mediators for signalling local interneuron activity to subgranular zone precursors, however, little is known regarding the effects of neuropeptides that are extrinsic modulators of hippocampal information processing. Here, we show that the galanin GalR2/3 agonist Gal2-11 is both trophic and proliferative for postnatal subgranular precursors and proliferating neuroblasts at 10 nM and is purely trophic at doses as low as 100 pM. We found no effect mediated via GalR1. As galanin is co-released from noradrenergic and serotonergic projection neurons to the dentate gyrus, these findings support a direct effect of galanin on hippocampal neurogenesis, which may partly mediate its antidepressant effect via GalR2/3 receptors.     

Preferential activation by galanin 1–15 fragment of the GalR1 protomer of a GalR1–GalR2 heteroreceptor complex

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1–15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1–GalR2 heteromers which may play a significant role in mediating galanin fragment (1–15) signaling. In HEK293T cells evidence for the existence of GalR1–GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET² assays. PLA positive blobs representing GalR1–GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1–15) was more potent than galanin (1–29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1–GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1–15) and of galanin (1–29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1–15) vs galanin (1–29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1–29). In contrast, in NFAT reporter gene assays galanin (1–29) shows a higher efficacy than galanin (1–15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1–GalR2 heteroreceptor complexes induced by galanin (1–15) may contribute to depression-like actions since GalR1 agonists produce such effects.     

A Short Estrogen-responsive N-terminal Galanin Homologue Found in Rat Brain and Gut with Antiserum Raised Against Rat Galanin(1-16)

Galanin-like peptide (GALP) is currently the only known galanin(1-29) homologue. However, three different galanin receptors, of which GalR3 exhibits comparatively low affinity for galanin(1-29), and molecular heterogeneity of immunoreactive galanin are arguments for presence of other endogenous galanin homologues. Since antibodies recognize three-dimensional structures of 3–5 amino acids in a peptide, we raised antibodies in rabbits against galanin(1-16) conjugated to bovine serum albumin, looking for the presence of endogenous N-terminal galanin homologues in rat tissues. The antiserum selected had 7,830 times higher avidity for galanin(1-16) compared to galanin(1-29). A single immunoreactive component with a Stokes radius of about 8 amino acids was found. Immunohistochemistry strongly suggested that this immunoreactivity is localised in the same neurons as galanin(1-29). Furthermore, its concentration was increased in response to estrogen treatment in the same brain regions as galanin(1-29), although not as rapidly. The present results indicate the presence of a novel endogenous N-terminal galanin homologue.Special Issue Dedicated to Miklós Palkovits.     

Regulation of galanin and galanin receptor 2 expression by capsaicin in primary cultured dorsal root ganglion neurons

Galanin is a 29-amino-acid neuropeptide expressed in dorsal root ganglion (DRG) neurons which is thought to play a role in modulation of nociception in neuropathic states. Activation of galanin receptor 2 (GalR2) plays a pronociceptive role and enhances capsaicin-induced nociception in the periphery. GalR2 and vanilloid receptor 1 (VR1) are co-expressed in DRG neurons. Capsaicin evokes acute pain via activation of VR1 expressed in primary sensory neurons. It is not known to what extent galanin and its receptor GalR2 expression is regulated by capsaicin in DRG neurons. Effects of acute (4 h) or chronic (4 d) treatment with capsaicin at different concentrations (0.01, 0.1, 1 micromol/L) on galanin and GalR2 expression in primary cultured DRG neurons were investigated in the present study. Our results showed that acute exposure of high concentration capsaicin (1 micromol/L) increased galanin expression, whereas chronic exposure of low concentration capsaicin (0.01, 0.1 micromol/L) promoted galanin expression. Only chronic exposure of 0.1 micromol/L concentration capsaicin could elevate GalR2 expression, whereas capsaicin did not have this effect at any other conditions in this experiment. These results indicated that certain concentrations or exposure time of capsaicin stimulation may be relevant to upregulation of galanin and its receptor GalR2 expression in DRG cultures suggesting a response to peripheral neuronal stimulation. And also, capsaicin-induced GalR2 expression may be also modulated by capsaicin-induced galanin expression. The possible significance of the neurotransmission of nociceptive information involved in galanin or GalR2 expression caused by capsaicin is still to be clarified.     

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

Binding of Chimeric Peptides M617 and M871 to Galanin Receptor Type 3 Reveals Characteristics of Galanin Receptor–Ligand Interaction

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 (K _i 49.2 ± 9.4 nM and >10 μM, 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.     

A Galanin Receptor Subtype 1 Specific Agonist

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.     

Stress Upregulates TPH1 but not TPH2 mRNA in the Rat Dorsal Raphe Nucleus: Identification of Two TPH2 mRNA Splice Variants

Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3′-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3′-untranslated regions (UTRs). TPH2b (with a short 3′-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3′-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.     

In vivo interaction between serotonin and galanin receptors types 1 and 2 in the dorsal raphe: implication for limbic seizures

The neuropeptide galanin suppresses seizure activity in the hippocampus by inhibiting glutamatergic neurotransmission. Galanin may also modulate limbic seizures through interaction with other neurotransmitters in neuronal populations that project to the hippocampus. We examined the role of galanin receptors types 1 and 2 in the dorsal raphe (DR) in the regulation of serotonergic transmission and limbic seizures. Infusion of a mixed agonist of galanin receptors types 1 and 2 [galanin (1-29)] into the DR augmented the severity of limbic seizures in both rats and wild-type mice and concurrently reduced serotonin concentration in the DR and hippocampus as measured by immunofluorescence or HPLC. In contrast, injection of the galanin receptor type 2 agonist galanin (2-11) mitigated the severity of seizures in both species and increased serotonin concentration in both areas. Injection of both galanin fragments into the DR of galanin receptor type 1 knockout mice exerted anticonvulsant effects. Both the proconvulsant activity of galanin (1-29) and seizure suppression by galanin (2-11) were abolished in serotonin-depleted animals. Our data indicate that, in the DR, galanin receptors types 1 and 2 modulate serotonergic transmission in a negative and a positive fashion, respectively, and that these effects translate into either facilitation or inhibition of limbic seizures.     

Effects of exogenous galanin on neuropathic pain state and change of galanin and its receptors in DRG and SDH after sciatic nerve-pinch injury in rat

A large number of neuroanatomical, neurophysiologic, and neurochemical mechanisms are thought to contribute to the development and maintenance of neuropathic pain. However, mechanisms responsible for neuropathic pain have not been completely delineated. It has been demonstrated that neuropeptide galanin (Gal) is upregulated after injury in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) where it plays a predominantly antinociceptive role. In the present study, sciatic nerve-pinch injury rat model was used to determine the effects of exogenous Gal on the expression of the Gal and its receptors (GalR1, GalR2) in DRG and SDH, the alterations of pain behavior, nerve conduction velocity (NCV) and morphology of sciatic nerve. The results showed that exogenous Gal had antinociceptive effects in this nerve-pinch injury induced neuropathic pain animal model. It is very interesting that Gal, GalR1 and GalR2 change their expression greatly in DRG and SDH after nerve injury and intrathecal injection of exougenous Gal. Morphological investigation displays a serious damage after nerve-pinch injury and an amendatory regeneration after exogenous Gal treatment. These findings imply that Gal, via activation of GalR1 and/or GalR2, may have neuroprotective effects in reducing neuropathic pain behaviors and improving nerve regeneration after nerve injury.     

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.     

Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity

The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)_o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]_o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HT_o is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]_o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein–gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HT_o sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca²⁺ influx, vesicular monoamine transporter 2–mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HT_o in the DRN and suggest that 5-HT_o may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.     

甘丙肽及其受体在嗅成鞘细胞中的表达及对细胞增殖的影响

本实验从新生大鼠嗅球中分离出嗅成鞘细胞,进行体外培养。运用RT—PCR方法检测甘丙肽及其受体在体外培养的嗅成鞘细胞中的表达;运用MTT法检测甘丙肽及其受体激动剂、拮抗剂对嗅成鞘细胞增殖的影响。结果显示：嗅成鞘细胞表达甘丙肽(GAL)及其受体GalR2,而不表达其他两种受体GalRl和GalR3;甘丙肽及两种受体激动剂GALl-11和GAL2-11能够明显地抑制体外培养的嗅成鞘细胞的增殖,这一效应可被非特异性甘丙肽受体拮抗剂M35所阻断。     

甘丙肽及其受体在嗅成鞘细胞中的表达及对细胞增殖的影响

本实验从新生大鼠嗅球中分离出嗅成鞘细胞,进行体外培养。运用RT-PCR方法检测甘丙肽及其受体在体外培养的嗅成鞘细胞中的表达;运用MTT法检测甘丙肽及其受体激动剂、拮抗剂对嗅成鞘细胞增殖的影响。结果显示:嗅成鞘细胞表达甘丙肽(GAL)及其受体GalR2,而不表达其他两种受体GalR1和GalR3;甘西肽及两种受体激动剂GAL1-11和GAL2-11能够明显地抑制体外培养的嗅成鞘细胞的增殖,这一效应可被非特异性甘丙肽受体拮抗剂M35所阻断。     

甘丙肽对PC12细胞和B104细胞增殖的不同影响

本实验运用PCI2细胞和B104细胞对甘丙肽（GAL）在神经增殖上的作用进行了研究。运用RT-PCR方法检测GAL及其受体在PCI2细胞和B104细胞中的表达：运用MTT法检测GAL及其受体激动剂、拮抗剂对两种细胞增殖的影响。结果显示：PCI2细胞表达所有三种GAL受体（GalRs）．而不表达GAL;B104细胞表达GAL及两种受体GaIR2和GalR3,而不表达GalRl;GAL及其受体激动剂GAL1-11和GAL2-11能够明显地抑制PC12细胞增殖、却会明显促进B104细胞的增殖。这些效应皆可被非特异性GAL受体拮抗剂M35所阻断。结果说明,GAL可以通过其受体影响细胞的增殖．并且不同受体可能介导不同的作用。     

Novel systemically active galanin receptor 2 ligands in depression‐like behavior

Neuropeptide galanin and its three G‐protein coupled receptors, galanin receptor type 1–galanin receptor type 3 (GalR1–GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression‐like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide‐based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant‐like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant‐like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.

     

Neuronal background of activation of estivated snails,with special attention to the monoaminergic system: a biochemical,physiological, and neuroanatomical study

Osmotic stimulation activates both estivated and inactivated specimens of Helix pomatia and increases their central arousal. High-pressure liquid chromatography has shown that, during activation, the level of both serotonin and dopamine decreases in the central nervous system (CNS) but increases in the foot and heart, organs that are involved in the eversion of the body. In isolated CNS from activated animals, the firing frequency of the heart-modulator serotonergic (RPas) neurons is significantly higher than that in the CNS of estivated or inactivated animals. These neurons innervate both the heart and the anterior aorta. In semi-intact preparations, distilled water (an osmotic stimulus) applied to the mantle collar increases their firing frequency, whereas tactile stimulation evokes their inhibition. Extracellularly applied monoamines mimic the effect of peripheral stimuli: serotonin (0.1–10 μM) increases the activity of the RPas neurons, whereas dopamine (0.1–10 μM) inhibits their activity. Tyrosine-hydroxylase immunocytochemistry and retrograde neurobiotin tracing have revealed similar bipolar receptor cells in the mantle collar and tail, organs that are exposed to environmental stimuli in estivated animals. Serotonin immunocytochemistry carried out on the same tissues does not visualize receptor cells but labels a dense network of fibers that appear to innervate neurobiotin-labeled receptor cells. The combination of neurobiotin-labeling of RPas neurons and immunolabeling suggests that RPas neurons receive direct dopaminergic inputs from receptor cells and serotonergic inputs from central serotonergic neurons, indicating that central serotonergic neurons are interconnected. Thus, the RPas neurons may belong to neuronal elements of the arousal system. This work was supported by Hungarian OTKA grants T037389, T046580, T037505, and K63451.     

Ligand binding properties of human galanin receptors

Abstract

The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.