Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

Molecular genetic analysis of a human neuropeptide Y receptor. The human homolog of the murine "Y5" receptor may be a pseudogene.

Neuropeptide Y is a 36-amino-acid peptide amide with numerous biological activities. These functions are mediated through several pharmacologically distinct receptors. To date five receptor subtypes have been cloned. Here we report the isolation, by low stringency homology cloning from a hypothalamic library, of a cDNA encoding the human homolog of the murine neuropeptide Y receptor subsequently reported (). Translation of the human Y1-like receptor clone suggested that it encoded a receptor which is truncated in the third extracellular loop. Comparison of the human Y1-like sequence to that of the human Y1 receptor suggested that the truncated receptor could have resulted from a frameshift due to a single nucleotide deletion in the sixth transmembrane domain. Southern blot analysis suggested that the gene is single copy in the human genome. The gene is located on chromosome 5q. To test the hypothesis that allelic variation of nucleic acid length within the sixth transmembrane domain of the Y1-like receptor may exist to produce a functional receptor, genomic DNA from 192 individuals of various ages, ethnic backgrounds, and degrees of obesity were analyzed electrophoretically and by direct sequencing. No variation was detected in any of the subjects, indicating that this receptor subtype may be a transcribed pseudogene in humans.

Cloning, pharmacological characterization and distribution of a novel galanin receptor.

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G-protein-coupled receptors. Through expression cloning, human and rat GALR1 receptor cDNA clones have previously been isolated and characterized. In this study, we have used homology screening to isolate a rat brain cDNA clone encoding a second galanin receptor subtype, the GALR2 receptor. The isolated cDNA encodes a 372-amino-acid G-protein-coupled receptor that shares 38% overall amino-acid identity with the rat GALR1 receptor. The pharmacological profile of the rat GALR2 receptor is similar to that of the rat GALR1 receptor. The rat GALR2 receptor binds galanin, N-terminal galanin fragments, and the putative galanin receptor antagonists galantide, C7, M35 and M40 with high affinity but it does not bind C-terminal galanin fragments. Galanin increases intracellular inositol phosphate levels in HEK 293 cells expressing the rat GALR2 receptor via a pertussis toxin-insensitive G-protein. The rat GALR2 receptor mRNA is highly expressed in several brain regions, including hypothalamus and hippocampus as well as the anterior pituitary, with lower levels of expression detected in amygdala, and regions of cortex. It is also highly expressed in the GH3 pituitary cell line and in gut tissues, and to a lower extent in spleen, lung, skeletal muscle, heart, kidney, liver and testis. These results suggest that GALR2 receptor mediates galanin's regulation of pituitary hormone secretion and possibly food intake.