Human CRF2 alpha and beta splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay.

Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF1 and CRF2). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF2 subtype receptors, CRF2alpha and CRF2beta, have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF2, receptor. We have used radioligand binding with [125I]-tyr0-sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [125I]-tyr0-sauvagine binding to the hCRF2beta receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF2alpha receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF2alpha receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [125I]-tyr0-sauvagine to both hCRF2 receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF2 isoforms (urocortin > sauvagine > urotensin 1 > r/hCRF > alpha-helical CRF(9-41) > oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist alpha-helical CRF(9-41) exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF2 receptor isoforms. Taken together, these results indicate that the pharmacological profiles of the CRF2 splice variants are identical. This indicates that the region of the N-terminus that varies between the receptors is probably not important in the binding of peptide CRF receptor ligands or functional activation of the receptor.