Isolation and pharmacological characterization of two functional splice variants of corticotropin-releasing factor type 2 receptor from Tupaia belangeri

From brain, heart and muscle tissue of the tree shrew (Tupaia belangeri), a higher order mammal, cDNA clones were isolated that encoded two functional splice variants of the corticotropin-releasing factor (CRF) type 2 recepto r (CRF-R2). The first, full-length splice variant, amplified from brain and heart tissue, encoded a CRF receptor protein that is 410 amino acids in le ngth and approximate to 96% homologous to human CRF-R2 alpha. The second, f ull-length splice variant, derived from skeletal muscle tissue, encoded a 4 37-amino acid CRF receptor protein that is approximate to 92% homologous to human CRF-R2 beta, Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) amplifications and RNase protection analyses, showed tha t tree shrew CRF-R2 alpha (tCRF-R2 alpha) and tree shrew CRF-R2 beta (tCRF- R2 beta) were coexpressed in brain tissue but not in heart and skeletal mus cle tissue, Finally, human embryonic kidney 293 (HEK293) cells stably trans fected with tCRF-R2 alpha and tCRF-R2 beta were used to demonstrate that th e CRF analogs urocortin and sauvagine bind with significantly greater affin ity (21- to 140-fold) to these two CRF-R2 splice variants than do human/rat and ovine CRF analogs. In keeping with these results of our CRF binding st udies, EC50 values were substantially lower for urocortin-and sauvagine-sti mulated than for h/rCRF-and oCRF-stimulated cyclic AMP accumulation in HEK2 93 cells stably transfected with tCRF-R25 alpha or tCRF-R2 beta cDNAs. The tree shrew therefore constitutes an important animal model in which to inve stigate the role of CRF receptor subtypes in the stress response.