Molecular Cloning and Characterization of Two Novel Retinoic Acid-Inducible Orphan G-Protein-Coupled Receptors (GPRC5B and GPRC5C)

• Published in: Genomics (San Diego, Calif.) Calif.), 2000-07, Vol.67 (1), p.8-18
• Main Authors: Robbins, M.J., Michalovich, D., Hill, J., Calver, A.R., Medhurst, A.D., Gloger, I., Sims, M., Middlemiss, D.N., Pangalos, M.N.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Brain - physiology; Chromosomes, Human, Pair 16; Chromosomes, Human, Pair 17; Cloning, Molecular; DNA Primers - chemistry; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genes. Genome; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Humans; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Multigene Family; Phylogeny; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - analysis; Sequence Homology, Amino Acid; Signal Transduction; Tissue Distribution; Transfection; Tretinoin - pharmacology; Tumor Cells, Cultured - cytology; Tumor Cells, Cultured - drug effects
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1006/geno.2000.6226

Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABAB receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative TaqMan PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405–D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352–D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.