Characterization of the gene for the calcitonin gene-related peptide (CGRP) receptor component protein (RCP), and generation of RCP +/- embryonic stem cells.

摘要

The Dickerson lab discovered a 148 as intracellular protein named calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) that is required for signal transduction at receptors for the neuropeptide CGRP. RCP works with two other proteins to constitute a functional G-protein coupled receptor: calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP1). CRLR is a 7-transmembrane G-protein coupled receptor and requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. We demonstrated the requirement for RCP for CGRP receptor function using an antisense approach in NIH3T3 cells, where RCP expression was diminished and correlated with reduction in CGRP-mediated signal transduction. RCP was detected in a complex with RAMP1 and CRLR by co-immunoprecipitation from lysates prepared from NIH3T3 cells, mouse embryonic stem cells and guinea pig cerebellum.; I have recently extended our studies on RCP to generate a knockout mouse for the RCP gene. To genetically engineer RCP-deficient mice by homologous recombination, I have constructed a targeting vector that replaced 55% of the coding region of the RCP gene in mouse embryonic stem cells (ES) with a cassette containing the enhanced green fluorescent protein (EGFP) and the positive selectable marker neomycin phosphotransferase. RCP +/− ES cells have been identified by PCR and confirmed by Southern blot analysis, and demonstrated 50% or more reduction in RCP protein expression when compared to wild type.; Identification of the RCP promoter is a key element in understanding the regulation of RCP gene expression, which is commonly initiated by identification of the transcription start site(s). I identified the transcription start sites in a three step process, which involved a preliminary approach using a series of reverse transcriptase reactions, followed by Ribonuclease Protection Assay (RPA) using probes that started at the translation start site (ATG) and moved upstream. Finally, I precisely determined the transcription start sites by Primer Extension Analysis. RCP mRNA appears to be transcribed from a TATA-less GC-rich promoter, and transcription is initiated from at least four start sites that fall within a 50 bp-region.