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 BRET(2) 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 50nM 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.

Coevolution of the spexin/galanin/kisspeptin family: Spexin activates galanin receptor type II and III.

The novel neuropeptide spexin (SPX) was discovered using bioinformatics. The function of this peptide is currently under investigation. Here, we identified SPX along with a second SPX gene (SPX2) in vertebrate genomes. Syntenic analysis and relocating SPXs and their neighbor genes on reconstructed vertebrate ancestral chromosomes revealed that SPXs reside in the near vicinity of the kisspeptin (KISS) and galanin (GAL) family genes on the chromosomes. Alignment of mature peptide sequences showed some extent of sequence similarity among the 3 peptide groups. Gene structure analysis indicated that SPX is more closely related to GAL than KISS. These results suggest that the SPX, GAL, and KISS genes arose through local duplications before 2 rounds (2R) of whole-genome duplication. Receptors of KISS and GAL (GAL receptor [GALR]) are phylogenetically closest among rhodopsin-like G protein-coupled receptors, and synteny revealed the presence of 3 distinct receptor families KISS receptor, GALR1, and GALR2/3 before 2R. A ligand-receptor interaction study showed that SPXs activate human, Xenopus, and zebrafish GALR2/3 family receptors but not GALR1, suggesting that SPXs are natural ligands for GALR2/3. Particularly, SPXs exhibited much higher potency toward GALR3 than GAL. Together, these results identify the coevolution of SPX/GAL/KISS ligand genes with their receptor genes. This study demonstrates the advantage of evolutionary genomics to explore the evolutionary relationship of a peptide gene family that arose before 2R by local duplications.

Dopamine-galanin receptor heteromers modulate cholinergic neurotransmission in the rat ventral hippocampus.

Previous studies have shown that dopamine and galanin modulate cholinergic transmission in the hippocampus, but little is known about the mechanisms involved and their possible interactions. By using resonance energy transfer techniques in transfected mammalian cells, we demonstrated the existence of heteromers between the dopamine D(1)-like receptors (D(1) and D(5)) and galanin Gal(1), but not Gal(2) receptors. Within the D(1)-Gal(1) and D(5)-Gal(1) receptor heteromers, dopamine receptor activation potentiated and dopamine receptor blockade counteracted MAPK activation induced by stimulation of Gal(1) receptors, whereas Gal(1) receptor activation or blockade did not modify D(1)-like receptor-mediated MAPK activation. Ability of a D(1)-like receptor antagonist to block galanin-induced MAPK activation (cross-antagonism) was used as a "biochemical fingerprint" of D(1)-like-Gal(1) receptor heteromers, allowing their identification in the rat ventral hippocampus. The functional role of D(1)-like-Gal receptor heteromers was demonstrated in synaptosomes from rat ventral hippocampus, where galanin facilitated acetylcholine release, but only with costimulation of D(1)-like receptors. Electrophysiological experiments in rat ventral hippocampal slices showed that these receptor interactions modulate hippocampal synaptic transmission. Thus, a D(1)-like receptor agonist that was ineffective when administered alone turned an inhibitory effect of galanin into an excitatory effect, an interaction that required cholinergic neurotransmission. Altogether, our results strongly suggest that D(1)-like-Gal(1) receptor heteromers act as processors that integrate signals of two different neurotransmitters, dopamine and galanin, to modulate hippocampal cholinergic neurotransmission.

Homodimerization and internalization of galanin type 1 receptor in living CHO cells.

Galanin is a 29- to 30-aa-long neuropeptide affecting feeding, cognitive, and sexual behavior. It exerts its effects through galanin receptors 1, 2 and 3, which are all seven transmembrane domain G-protein coupled receptors (GPCRs). The GPCRs have been shown to function as monomers, homodimers, heterodimers and oligomers. In this study, we examined the extent of galanin receptor 1 (GalR1) dimerization and internalization in living CHO cells using fluorescence resonance energy transfer (FRET) and time lapse confocal imaging. Ratio imaging analysis and emission spectral analysis revealed substantial homodimerization of GalR1. In addition, internalization of GalR1 after 1h of agonist stimulation with the GalR1 agonist galanin (1-29) was observed with time lapse fluorescence imaging, whereas stimulation with the GalR2 specific agonist galanin (2-11) did not lead to internalization. Treatment of GalR1 transfected cells with the non-selective adenylyl cyclase activator forskolin influenced the rate of internalization when administered together with galanin (1-29). These results indicate that GalR1 can act as a dimer on the cell surface and that receptor desensitization and internalization was observed after stimulation with the agonist galanin (1-29). Western blots further confirm the FRET data that GalR1-XFP dimerizes and can be detected in the cell as a monomer or dimer using antibodies to XFP. Internalization and dimerization of GalR1 is shown, contributing to the regulation of galanergic signaling.

Galanin receptor 1 has anti-proliferative effects in oral squamous cell carcinoma.

In the United States, oral cancer accounts for more deaths annually than cervical cancer, leukemias, or Hodgkin's lymphoma. Studies have shown that aberrations of chromosome 18q develop with tumor progression and are associated with significantly decreased survival in head and neck cancer patients. The G-protein-coupled receptor, galanin receptor 1 (GALR1), maps to this region of chromosome 18q. Although the role of GALR1 has been well characterized in neuronal cells, little is known regarding this receptor in non-neuronal cells. In this study, the expression, mitogenic function, and signaling mechanism of GALR1 are investigated in normal and malignant oral epithelial cells. mRNA expression was determined via reverse transcriptase-PCR. Protein quantification was done via immunoblot analysis and enzyme-linked immunosorbent assay. For functional and signaling studies, an inhibitory antibody was generated to the N-terminal ligand binding domain of GALR1. GALR1 protein and mRNA expression and GAL secretion were detected at variable levels in immortalized human oral keratinocytes and human oropharyngeal squamous cell carcinoma cell lines. Upon competitive inhibition of GALR1, proliferation was up-regulated in immortalized and malignant keratinocytes. Furthermore, studies with the inhibitory antibody and U0126, the MAPK inhibitor, show that GALR1 inhibits proliferation in immortalized and malignant keratinocytes by inactivating the MAPK pathway. GALR1s inhibitory effects on proliferation in epithelial cells raises the possibility that inactivation or disregulation of this receptor can lead to uncontrolled proliferation and neoplastic transformation.