Molecular cloning and initial characterization of african green monkey (Cercopithecus aethiops) corticotropin releasing factor receptor type 1 (CRF1) from COS-7 cells.

摘要

We report the expression of endogenous CRF1 in COS-7 cells (African green monkey origin). Cloning of the coding region of CRF1 gene identified three alternatively spliced isoforms with nucleotide and predicted amino acid sequences corresponding to the membrane bound α and c and soluble e isoforms. DNA sequencing of the main isoform CRF1α showed homologies of 99%, 97% and 91% with the rhesus monkey, human and rodent genes, respectively; the deduced protein sequence differed in only one amino acid with Rhesus monkey and human. Western blot analysis with antibodies against human CRF1 demonstrated immunoreactive proteins with MW of 37, 52, 70 and 80–85 in crude membrane or cytoplasm preparation; two additional species of 40 and 60 kDa were detected only in the cytoplasmic fraction. On immunocytochemistry CRF1 was localized to both the cell surface and intracellularly. The receptor was functional, e.g., addition of CRF to COS-7 cells inhibited cell proliferation and stimulated release of arachidonic acid; nevertheless, it was poorly coupled to cAMP production (its stimulation was minimal in native cells). In conclusion, COS cells that are routinely used for the study of transfected CRF receptors do express endogenous CRF1 mRNA with splicing behavior similar to that reported in human and rodent cells, and translated into functional CRF1 receptors.