Corticotropin-releasing factor receptor type 1 from Tupaia belangeri--cloning, functional expression and tissue...

A cDNA clone encoding corticotropin-releasing factor (CRF) type 1 (CRF-R1) has been isolated from the tree shrew Tupaia belangeri with a PCR-based approach. The full-length cDNA encoded a 415-amino-acid protein with highest sequence identity (approximately 98%) to human CRF-R1 and slightly less identity to rat or mouse CRF-R1 (approximately 97%). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the N-terminus, residue 232 in transmembrane domain 4 and residue 410 in the C-terminus) differed between tree shrew CRF-R1 (tCRF-R1) and human CRF-R1 (hCRF-R1). tCRF-R1 mRNA was detected by semiquantitative RT-PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF-R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R1 showed that the CRF agonists ovine CRF (KD = 1.28 nM), human/rat CRF (KD = 1.09 nM), urocortin (KD = 0.37 nM) and sauvagine (KD = 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (KD = 12.4 nM). In agreement with the binding data half maximum effective EC50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF-R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF-R1.