Isolation and Pharmacological Characterization of Two Functional Splice Variants of Corticotropin‐Releasing Factor Type 2 Receptor from Tupaia belangeri

Abstract

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.

International Union of Pharmacology. XXXVI. Current Status of the Nomenclature for Receptors for Corticotropin-Releasing Factor and Their Ligands

“…Complementary DNAs for the CRF 2 receptor have been isolated from man (Liaw et al, 1996;Valdenaire et al, 1997;Kostich et al, 1998), tree shrew (Palchaudhuri et al, 1999), rat (Lovenberg et al, 1995b), mouse (Kishimoto et al, 1995;Perrin et al, 1995;Stenzel et al, 1995), frog (Dautzenberg et al, 1997), and fish (Arai et al, 2001;Pohl et al, 2001). Three functional splice variants (Lovenberg et al, 1995b;Kostich et al, 1998) and a truncation variant (Miyata et al, 1999) have been identified for the mammalian CRF 2 receptor.…”

Site-specific overexpression of corticotropin-releasing hormone (CRH) in the mouse brain – modelling central CRH system hyperactivity

“…The splice variants have a common 377 amino acid C-terminal part, whereas the extreme N-termini, which are encoded by one or two exons (Dautzenberg and Hauger, 2002), differ substantially from one another. The CRH 2α receptor, a 411-413 amino acid protein, has been isolated from mammalian and amphibian species (Dautzenberg et al, 1997) (Palchaudhuri et al, 1999). In contrast, CRH 2β receptors, which are 430-438 amino acids in size, have only been reported in mammals (Kishimoto et al, 1995) (Palchaudhuri et al, 1999) (Valdenaire et al, 1997).…”

“…The CRH 2α receptor, a 411-413 amino acid protein, has been isolated from mammalian and amphibian species (Dautzenberg et al, 1997) (Palchaudhuri et al, 1999). In contrast, CRH 2β receptors, which are 430-438 amino acids in size, have only been reported in mammals (Kishimoto et al, 1995) (Palchaudhuri et al, 1999) (Valdenaire et al, 1997). Finally, the 397 amino acid CRH 2γ receptor is exclusively found in humans (Kostich et al, 1998) (Palchaudhuri et al, 1999).…”

“…In contrast, CRH 2β receptors, which are 430-438 amino acids in size, have only been reported in mammals (Kishimoto et al, 1995) (Palchaudhuri et al, 1999) (Valdenaire et al, 1997). Finally, the 397 amino acid CRH 2γ receptor is exclusively found in humans (Kostich et al, 1998) (Palchaudhuri et al, 1999). In addition to the functional splice variants, a truncated CRH 2α mutant (CRH 2avar) has been identified in the rat (Miyata et al, 1999).…”

“…The physiological role of this receptor variant needs to be determined. Because of its existence in mammalian and amphibian species and its higher degree of sequence conservation between the species (Palchaudhuri et al, 1999), it is suggestive that the CRH 2α receptor arose very early during evolution. The CRH 2β receptor, which is conserved to a lower extent, has developed in mammalian species only and thus probably arose later in evolution than the CRH 2α receptor.…”