Expression Cloning of a Rat Hypothalamic Galanin Receptor Coupled to Phosphoinositide Turnover

Kelli E Smith,Carlos Forray,Mary W Walker,Kenneth A Jones,Joseph A Tamm,Jonathan Bard,Theresa A Branchek,David L Linemeyer,Christophe Gerald

doi:10.1074/jbc.272.39.24612

Kelli E Smith, Carlos Forray + Show 7 more

Open Access

https://doi.org/10.1074/jbc.272.39.24612

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Abstract

The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a 125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of 125I-pGAL to transiently expressed GALR2 receptors was saturable (KD = 0.15 nM) and displaceable by galanin peptides and analogues in rank order: porcine galanin approximately M32 approximately M35 approximately M40 >/= galanin-(1-16) approximately M15 approximately [D-Trp2]galanin-(1-29) > C7 >> galanin-(3-29). This profile resembles that of the rat GALR1 receptor with the notable exception that [D-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C.

Highlights

Galanin is a neuropeptide with a broad distribution in both central and peripheral nervous systems
The new galanin receptor subtype, termed GALR2, has the same predicted amino acid sequence as the galanin receptor cDNA recently described by Howard et al [22] which was reported to have a pharmacological profile similar to the GALR1 receptor
To clone additional members of the galanin receptor family, we used an expression cloning strategy based on the hypothesis that there were multiple galanin receptors present in hypothalamus [17, 29]

Summary

Introduction

Galanin is a neuropeptide with a broad distribution in both central and peripheral nervous systems. Receptor-mediated activation of phosphatidylinositol turnover was measured in CHO cells stably expressing the rat GALR2 receptor.

Results

Conclusion