Human Type 2 Neuropeptide Y Receptor Gene: Isolation and Characterization

Our interest in G-protein signaling events involved in the specialized functions of the retinal pigment epithelium led us to investigate the pattern of G-protein-coupled receptor expression in this cell type. We used reverse transcriptase-coupled polymerase chain reaction (PCR) to amplify cDNAs derived from bovine tissue using degenerate oligonucleotide primers corresponding to conserved sequences in receptor transmembrane helices two and seven (1 ). One of the sequences identified encoded the partial sequence of a novel protein most closely related to the type 1 neuropeptide Y (NPY) receptor, the only member of the NPY receptor family characterized by molecular cloning at that time (2 ,3 ). To identify DNA sequences encoding the corresponding full-length protein necessary to verify NPY receptor activity, we chose to clone the human genomic DNA (4 ). This approach offers a number of advantages over cDNA library screening, especially for identification of clones corresponding to low-abundance transcripts. In a genomic DNA library, each gene is represented equally, whereas representation in a cDNA library is proportional to expression level. In addition, the large insert size favors the possibility that one clone will encompass the entire gene and allows the gene structure to be determined. Furthermore, the large genomic DNA fragments obtained are suitable for use in determining chromosomal localization by hybridization analysis of somatic cell hybrid panels and in situ hybridization of metaphase chromosomes.