The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.

Galanin, a 29-30-amino acid neuropeptide, is widely distributed in central and peripheral systems and mediates a variety of physiological functions. Pharmacological studies have suggested the existence of multiple receptor subtypes but only the type I (GalR1) galanin receptor has been cloned. Now we report the cloning by a combination of sib selection and rapid amplification of cDNA ends of a cDNA encoding a new galanin receptor (GalR2) from rat hypothalamus. The receptor is 372 amino acids in length and shares only 40% homology with the rat GalR1 receptor. It contains seven putative transmembrane domains with the amino and carboxyl termini being least identical to GalR1. Northern blot analyses revealed a 2-kilobase pair mRNA species distributed in several tissues, suggesting a broader functional spectrum than GalR1. 125I-Labeled human galanin binding to rat GalR2 receptor expressed in COS-1 cells was saturable (Kd = 0.59 nM) and could be displaced by galanin, several galanin fragments, and chimeric peptides. The pharmacological profiles of GalR1 and GalR2 receptors were distinguishable by galanin fragment(2-29), which bound the cloned GalR2 receptor with markedly higher affinity than the GalR1 receptor. Activation of the cloned receptor by galanin led to inhibition of forskolin-stimulated intracellular cAMP production. The cloning of this new receptor subtype should provide further insights into the mechanisms by which galanin mediates its diverse physiological functions. The identification of galanin(2-29) as a receptor-specific ligand should enhance the understanding of specificity of galanin-receptor interactions.

Galanin, a 29 -30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G proteincoupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors.125 I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (K d ‫؍‬ 0.6 nM) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin.Galanin is a 29 -30 amino acid neuropeptide with no significant homology to any known family of biologically active peptides (1). Galanin is widely distributed in the central and peripheral nervous systems and is highly expressed in various regions of the brain. Many physiological processes are modulated by galanin, including neurotransmitter and hormone release (2), spinal reflexes, nociception (3), firing of noradrenergic neurons, and contraction of gastrointestinal smooth muscle (4, 5). Like neuropeptide Y, centrally administered galanin potently stimulates food intake in animals (6), suggesting a role for galanin in control of body weight.A large body of evidence suggests that galanin mediates the various physiological functions through interaction with distinct receptor subtypes. Pharmacological studies with several peptidic agonists and antagonists of galanin receptor suggest the existence of multiple receptor subtypes (7-9). The first of these receptors (GalR1) has been cloned from several species (10 -13). More recently, a second galanin receptor subtype (GalR2) was cloned. GalR2 is markedly dissimilar to the GalR1 receptor, sharing only 40% sequence homology (14,15). Hydropathy analysis suggests that both GalR1 and GalR2 receptors have seven hydrophobic transmembrane domains, typical of members of the G protein-coupled receptor superfamily (16). The GalR2 receptor is distinguished pharmacologically from the GalR1 receptor by its high affinity for ligand galanin-(2-29). In addition to the pharmacological differences, the GalR2 transcript is widely distributed in both central and peripheral tissues (14, 15), whereas the expression of GalR1 is more restricted to brain and spinal cord (12,13).Given that a large number of physiological actions are modulated by galanin, it is unlikely that the two cloned galanin receptors mediate all the functions of galanin. A complete understanding of the roles of galanin requir...

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The neuropeptide galanin mediates a diverse array of physiological functions through activation of specific receptors. Roles of the three recently cloned galanin receptors (GalRs) in rat intestinal contraction and food intake were examined using GalR-selective ligands and the results were compared with the pharmacological profiles of defined GalRs. The action profile of these ligands in jejunal contraction resembled only that of GalR2 and only a high level of GalR2 mRNA was detected in the tissue, supporting GalR2 as the receptor mediating jejunal contraction. The action profile for food intake in rats excluded GalR2, GalR3 and the putative pituitary galanin receptor as the`feeding receptor', suggesting that either GalR1 or an unidentified GalR is responsible for mediating this function.z 1998 Federation of European Biochemical Societies.