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Mouse Corticotropin-Releasing Factor Receptor Type 2 Gene: Isolation, Distribution, Pharmacological Characterization and Regulation by Stress and Glucocorticoids

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie Vale, Ph.D., Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu.

Effects of the corticotropin-releasing factor (CRF) family of peptides are mediated through activation of two receptors, CRF receptor (CRFR) 1 and CRFR2. Based on the homology between known mammalian CRFR genes, we have isolated a cDNA encoding the mouse CRFR2 (mCRFR2) ortholog from brain. The isolated cDNA encodes a 411-amino acid protein with high identity to the rat (97%) and human (93%) receptors. Central and peripheral expression of mCRFR2, determined by RT-PCR followed by Southern hybridization, revealed that mCRFR2 is restricted mainly to brain structures, with highest levels in the hypothalamus and olfactory bulb. In situ hybridization showed mCRFR2 localization in discrete brain regions, including the lateral septum and the ventromedial hypothalamus, whereas mCRFR2ß is found only in the choroid plexus. Binding and signaling of CRF-related ligands was studied using COS-M6 or HEK293T cells transiently transfected with mCRFR2. Urocortins (Ucns) show different affinities for binding to mCRFR2: Ucn 3 binds mCRFR2 with approximately 11-fold lower affinity than Ucn 2, which displays an affinity similar to Ucn 1 (1 nM). Cyclase activation, determined by intracellular cAMP accumulation and cAMP response element-luciferase activity, showed no differences between CRFR2 and CRFR2ß in response to stimulation by Ucn 1, Ucn 2, and Ucn 3. Interestingly, Ucn 3 was less efficacious than Ucn 1 or Ucn 2 in activating MAPK (ERK1/2-p44/p42) via CRFR2, but all three Ucns showed equivalent efficacy for activating MAPK through mCRFR2ß. We found a significant reduction in hypothalamic mCRFR2 mRNA levels after acute and chronic restraint stress in mice. Hypothalamic mCRFR2 gene transcription in mice was inhibited by glucocorticoid administration and elevated by adrenalectomy. In addition, we demonstrated that the mCRFR2 gene is increased in the hypothalamus of the CRFR1-null compared with wild type mice. The predicted mCRFR2 promoter region was isolated and fused to a luciferase reporter gene and found to be decreased by glucocorticoids in a dose and time-dependent manner when transfected into CATH.a cells. Computer analysis revealed the presence of 23 putative half-palindromic glucocorticoid response element sequences within 2.4 kb of the mCRFR2 5' flanking region. Elucidation of the structure and processing of the mCRFR2 gene and examination of the mCRFR2 gene regulation in various conditions will enable better understanding of the involvement of this receptor in the central response to stress in normal and transgenic mice models.

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