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 receptor (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 length and ≈96% homologous to human CRF‐R2 α . The second, full‐length splice variant, derived from skeletal muscle tissue, encoded a 437‐amino acid CRF receptor protein that is ≈92% homologous to human CRF‐R2 β . Semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) amplifications and RNase protection analyses, showed that tree shrew CRF‐R2 α (tCRF‐R2 α ) and tree shrew CRF‐R2 β (tCRF‐R2 β ) were coexpressed in brain tissue but not in heart and skeletal muscle tissue. Finally, human embryonic kidney 293 (HEK293) cells stably transfected with tCRF‐R2 α and tCRF‐R2 β were used to demonstrate that the CRF analogs urocortin and sauvagine bind with significantly greater affinity (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 studies, EC 50 values were substantially lower for urocortin‐and sauvagine‐stimulated than for h/rCRF‐and oCRF‐stimulated cyclic AMP accumulation in HEK293 cells stably transfected with tCRF‐R2 α or tCRF‐R2 β cDNAs. The tree shrew therefore constitutes an important animal model in which to investigate the role of CRF receptor subtypes in the stress response.