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

Tetsuya Ohtaki,Satoshi Kumano,Yoshihiro Ishibashi,Kazuhiro Ogi,Hideki Matsui,Mioko Harada,Chieko Kitada,Tsutomu Kurokawa,Haruo Onda,Masahiko Fujino

doi:10.1074/jbc.274.52.37041

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]GTPgammaS 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.

Highlights

Galanin, a C-terminally amidated peptide with amino acid residues was originally isolated from porcine intestine (1) and was later found to be ubiquitous in the central and peripheral nervous systems
We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35S]GTP␥S binding to a membrane preparation of GALR2-transfected cells
The chimeric peptides antagonizing galanin in vivo were agonists for the cloned GALR1, GALR2 (10), and GALR3 (12), which implies the involvement of an unknown galanin receptor that is antagonized by the chimeric peptides

Summary

Introduction

A C-terminally amidated peptide with amino acid residues (non-amidated peptide with residues in humans) was originally isolated from porcine intestine (1) and was later found to be ubiquitous in the central and peripheral nervous systems. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35S]GTP␥S binding to a membrane preparation of GALR2-transfected cells. A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein.

Results

Conclusion