Isolation and cDNA Cloning of a Novel Galanin-like Peptide (GALP) from Porcine Hypothalamus

Abstract

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35 S]GTP␥S binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9 -21) was completely identical to that of galanin-(1-13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1-60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1-24 and 41-53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1-60) had a high affinity for the GALR2 receptor (IC 50 ‫؍‬ 0.24 nM) and a lower affinity for the GALR1 receptor (IC 50 ‫؍‬ 4.3 nM). In contrast, galanin showed high affinity for the GALR1 (IC 50 ‫؍‬ 0.097 nM) and GALR2 receptors (IC 50 ‫؍‬ 0.48 nM). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.Galanin, a C-terminally amidated peptide with 29 amino acid residues (non-amidated peptide with 30 residues in humans) was originally isolated from porcine intestine (1) and was later found to be ubiquitous in the central and peripheral nervous systems. It exerts diverse regulatory functions including central modulation of cognition, nociception, and feeding behavior, endocrine control of pituitary and pancreatic hormones, and regulation of gastrointestinal smooth muscle contractions (for review, see Refs. 2, 3).Structurally, galanin is unrelated to any known family of neuropeptides or regulatory peptides, suggesting the presence of unknown members of a galanin peptide family. Indeed, the existence of a galanin-like peptide(s) in mammalian tissues has been proposed for several reasons. First, molecular heterogeneity of galanin-like immunoreactivity has been reported by several groups. Rökaeus et al. (4) first reported that rat brain and ileum contained galanin-like peptides that cross-reacted with galanin antiserum but differed from galanin in chromatographic characterization. Nevertheless, some of these immunoreactivities may be the result of galanin precursors or galaninderived peptides, as described in the subsequent studies (5). Recently, Wang et al. (6) re-evaluated the existence of a novel galanin-like peptide in rat islets by showing the presence of galanin-like immunoreactivity that cross-reacted with antibody against porcine galanin but did not with antibody against rat galanin. Second, the physiological function of the three galanin receptor subtypes, GALR1 1 (7, 8), GALR2 (9, 10), and GALR3 (11,12), is unlikely to be solely the distribution of different signals to target cells. The low affinity of human galanin for human GALR3 (12) suggests the possibility that these receptor s...

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“…Galanin-like peptide (GALP) was originally isolated from porcine hypothalamus and was shown to possess a capacity to stimulate the galanin type 2 G protein-coupled receptor (Ohtaki et al, 1999). GALP is specifically expressed in the arcuate nucleus of the hypothalamus.…”

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“…GAL binds GalR1 with similar affinity to GalR2 (Ohtaki et al, 1999). Galanin-like peptide (GALP) is a 60-amino-acid neuropeptide, expressed in the hypothalamus and discrete areas of brain, which preferentially binds to GalR2 (Langel and Bartfai, 1998).…”

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“…GALP shares a partial amino acid sequence identity with galanin and binds to several galanin receptor subtypes; however, GALP is derived from its own unique gene, distinct from galanin (Ohtaki et al, 1999). GALP is expressed in the brain, primarily in the hypothalamic arcuate nucleus (ARC), where it is regulated by various metabolic signals, including leptin, insulin, and metabolic fuels (Fraley et al, 2003a(Fraley et al, , 2004Juréus et al, 2000Juréus et al, , 2001Kumano et al, 2003).…”