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 approximately 96% homologous to human CRF-R2alpha. The second, full-length splice variant, derived from skeletal muscle tissue, encoded a 437-amino acid CRF receptor protein that is approximately 92% homologous to human CRF-R2beta. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) amplifications and RNase protection analyses, showed that tree shrew CRF-R2alpha (tCRF-R2alpha) and tree shrew CRF-R2beta (tCRF-R2beta) were coexpressed in brain tissue but not in heart and skeletal muscle tissue. Finally, human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R2alpha and tCRF-R2beta 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, EC50 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-R2alpha or tCRF-R2beta cDNAs. The tree shrew therefore constitutes an important animal model in which to investigate the role of CRF receptor subtypes in the stress response.
Palchaudhuri
M R
MR
German Primate Centre, Division of Neurobiology, Göttingen, Germany.
Hauger
R L
RL
Wille
S
S
Fuchs
E
E
Dautzenberg
F M
FM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011869:Radioligand Assay; D018019:Receptors, Corticotropin-Releasing Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D014417:Tupaia / Q000378:metabolism*
C101531:CRF receptor type 2; D018076:DNA, Complementary; D007202:Indicators and Reagents; D018019:Receptors, Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D012260:Ribonucleases