No abstract

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G‐protein‐coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N‐terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C‐terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

Galanin is a neuropeptide that activates specific receptors to modulate several physiological functions including food intake, nociception, and learning and memory. The molecular nature of the interaction between galanin and its receptors and the fate of the galanin/receptor complex after the binding event are not understood. A fluorescein-N-galanin (F-Gal) was generated to measure the interaction between galanin and rat GalR1 galanin receptor (rGalR1) and rGalR1-mediated ligand internalization using flow cytometry in transfected Chinese hamster ovary (CHO) cells. Like galanin, F-Gal bound rGalR1 with high affinity and stimulated intracellular signaling events. Fluorescence quenching by soluble KI of rGalR1-bound F-Gal revealed a highly protected environment around the fluorescein, suggesting that the N-terminal portion of galanin, which constitutes the binding site of galanin for the receptor, binds to a protected hydrophobic binding pocket within the receptor. Exposure to F-Gal stimulated rapid (t1/2 approximately 10 min) and extensive (78%) internalization of surface F-Gal into rGalR1/CHO cells at 37 degreesC but not at 0 degreesC. In addition, the internalization did not occur in parental CHO cells at either 0 or 37 degreesC and was inhibited by addition of 0.25 M sucrose in the medium, indicating a GalR1-mediated energy-requiring endocytic process. These results revealed a hydrophobic interaction between galanin and the GalR1 receptor, which is in contrast to those of other G protein-coupled receptors that mainly require hydrophilic interaction with their peptide ligands near or outside the plasma membrane surface, and illustrated that the initial binding interaction is followed by rapid cellular internalization of the agonist/GalR1 complex.

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The rat GalR1 galanin receptor was used as a prototypic G protein-coupled receptor to test the feasibility of heterologous expression in a retrovirus-based system. The system utilizes an independent retroviral vector pMX, a virus-packaging cell line BOSC23 and a pre-B cell line BA/F3 as the host for expression. A polyclonal cell population that expresses high ligand affinity (KD = 0.18 nM) and high level (7 pmol/mg) of GalR1 was generated within days with no drug sensitivity-based selection. The expression represented a 20-fold increase over the expression level of GalR1 achieved in CHO cells. The affinity of galanin for the expressed receptor was decreased by 19-fold in the presence of GTP-gamma-S, suggesting that the expression system can produce active galanin receptor functionally coupled to G proteins. The fast and efficient method to generate stable cell lines and to prepared large quantities of receptors may provide a general application for expression of other G protein-coupled receptors.