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.     

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.     

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.     

Cholesterol content drives distinct pharmacological behaviours of micro-opioid receptor in different microdomains of the CHO plasma membrane

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human micro-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.     

Cholesterol content drives distinct pharmacological behaviours of µ-opioid receptor in different microdomains of the CHO plasma membrane

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human µ-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.     

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.     

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.     

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.     

Retrovirus-Mediated Expression of the Galr1 Galanin Receptor: Implication for Efficient Stable Expression of Functional G Protein-Coupled Receptors

Abstract

The rat GalR1 galanin receptor was used as a prototypic G protein-coupled receptor to test the feasibility of heterologous expression in a retrovirus-based system. The system utilizes an independent retroviral vector pMX, a virus-packaging cell line BOSC23 and a pre-B cell line BA/F3 as the host for expression. A polyclonal cell population that expresses high ligand affinity (K_D = 0.18 nM) and high level (7 pmol/mg) of GalR1 was generated within days with no drug sensitivity-based selection. The expression represented a 20-fold increase over the expression level of GalR1 achieved in CHO cells. The affinity of galanin for the expressed receptor was decreased by 19-fold in the presence of GTP-γ-S, suggesting that the expression system can produce active galanin receptor functionally coupled to G proteins. The fast and efficient method to generate stable cell lines and to prepared large quantities of receptors may provide a general application for expression of other G protein-coupled 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.     

Galanin is highly expressed in bone marrow mesenchymal stem cells and facilitates migration of cells both in vitro and in vivo

Galanin peptide has recently been found to be highly abundant in early embryonic mouse mesenchyme, while galanin and its receptors are expressed in embryonic mouse stem cells. Bone marrow mesenchymal stem cells (BMMSCs) represent the primary source for adult stem cell therapy. In this study we examined the abundance of galanin and its receptors in BMMSCs and evaluated its possible function. Galanin mRNA and protein were highly expressed in BMMSCs cultures up to four passages, while among the three galanin receptor subtypes (GalR1, GalR2, and GalR3) only GalR2 and to a lesser extent GalR3 were expressed. Using chemotaxis and wound assays we found that galanin protein increased the migration of BMMSCs. Furthermore, increased serum galanin levels in a galanin transgenic model enhanced the mobilization (homing) of injected BMMSCs in vivo. These data suggest a role for galanin in BMMSC migration, probably through activation of the GalR2 receptor.     

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.     

Novel galanin receptor subtype specific ligands in feeding regulation

Galanin a 29/30-residue neuropeptide has been implicated in several functions in the central nervous system, including the regulation of food consumption. Galanin and its analogues administered intraventricularly or into the hypothalamic region of brain have been shown to reliably and robustly stimulate the consumption of food in sated rodents. Three galanin receptor subtypes have been isolated, all present in the hypothalamus, but little is known about their specific role in mediating this acute feeding response. Presently, we introduce several novel GalR2 selective agonists and then compare the most selective of these novel GalR2 subtype selective agonists to known GalR1 selective agonist M617 for their ability to stimulate acute consumption of several foods shown to be stimulated by central administration of galanin. GalR1 selective agonist M617 markedly stimulated acute consumption of high-fat milk, but neither GalR2 selective agonist affected either high-fat milk or cookie mash intake. The present results are consistent with the involvement of GalR1 in mediating the acute feeding consumption by galanin and suggest an approach applicable to exploring galanin receptor specificity in normal and abnormal behavior and physiology.     

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.     

Molecular modelling study of the role of cholesterol in the stimulation of the oxytocin receptor.

Cholesterol, an integral component of membranes in Eucaryota, is a modifier of membrane properties. In vivo studies have demonstrated that cholesterol can also modulate activities of some G protein-coupled receptors (GPCRs), which are integral membrane proteins. This can result either from an effect of cholesterol on the membrane fluidity or from specific interactions of the membrane cholesterol with the receptor, as recently demonstrated for the cholecystokinin type beta (CCKRbeta) or the oxytocin receptor (OTR). Using molecular modelling, we studied conformational preferences of cholesterol and several of its analogues. Subsequently, we simulated the distributions of their preferred conformations around the surface of OTR, CCKRbeta and a chimeric oxytocin/cholecystokinin receptor. Consequently, we suggest residues on the surface of OTR which are potentially significant in the OTR/cholesterol interaction.     

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。     

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。     

Novel Galanin Receptor Subtype Specific Ligand in Depression Like Behavior

Neuropeptide galanin and its three receptors, galanin receptor type 1–galanin receptor type 3, are known to be involved in the regulation of numerous psychological processes, including depression. Studies have suggested that stimulation of galanin receptor type 2 (GalR2) leads to attenuation of the depression-like behavior in animals. However, due to the lack of highly selective galanin subtype specific ligands the involvement of different receptors in depression-like behavior is yet not fully known. In the present study we introduce a novel GalR2 selective agonist and demonstrate its ability to produce actions consistent with theorized GalR2 functions and analogous to that of the anti-depressant, imipramine.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.