guanylyl-imidodiphosphate and sauvagine

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.

Topics: Alternative Splicing; Amphibian Proteins; Animals; Brain; Cell Line; Cell Membrane; Genes, Reporter; Genetic Variation; Guanylyl Imidodiphosphate; Humans; Iodine Radioisotopes; Kinetics; Luciferases; Peptide Hormones; Peptides; Promoter Regions, Genetic; Protein Isoforms; Radioligand Assay; Rats; Receptors, Corticotropin-Releasing Hormone; Recombinant Fusion Proteins; Transfection; Vasoactive Intestinal Peptide

We describe the binding of [125I]tyr(o)sauvagine to membranes of corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EBNA (293ECRH2 alpha) cells. The binding of [125I]tyr(o)sauvagine to CRH2 receptors was temperature, time and tissue dependent. Equilibrium was reached in 2 hr at 23 degrees C. Saturation data best fit a two-site model with affinity constants of 44 pM and 4.1 nM for high and low affinity states, respectively. The high affinity [125I]tyr(o)sauvagine binding sites were eliminated with 200 microM Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [125I]tyr(o)sauvagine binding to CRH2 alpha receptors was: urotensin > sauvagine = urocortin > alpha-helical CRH9-41 > rh-CRH >> o-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [125I]tyr(o)oCRH binding to CRH, receptors: urotensin > urocortin > r/h-CRH > o-CRH = sauvagine > alpha-helical CRH9-41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH1 receptor, have little or no affinity for CRH2 alpha receptors as labeled by [125I]tyr(o)sauvagine. Two selective CRH1 receptor antagonists enabled us to examine comparative densities of CRH1 and CRH2 receptors in several brain areas. We also used [125I]tyr(o)sauvagine in combination with a specific CRH1 antagonist to examine the anatomic distribution of CRH2 receptors using receptor autoradiography. With a few notable exceptions the CRH2 receptors demonstrated autoradiographically in this study match the data obtained by in situ hybridization studies on the localization of CRH2 mRNA. The anatomic overlap of the autoradiographic and in situ hybridization data suggest that CRH2 receptors are postsynaptic. This study demonstrates the utility of using [125I]tyr(o)sauvagine to study cloned CRH2 receptors expressed in cell lines. In addition, [125I]tyr(o)sauvagine used in conjunction with saturating concentrations of a specific CRH1 receptor antagonist allows the study of CRH2 receptors in brain tissues using both in vitro homogenate binding and receptor autoradiography techniques.

Topics: Amphibian Proteins; Animals; Autoradiography; Brain; Cell Line; Guanylyl Imidodiphosphate; Humans; Iodine Radioisotopes; Male; Peptide Hormones; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Temperature