Expression and characterisation of human and rat CRF2 alpha receptors

Rat and human CRF2 alpha receptors were expressed in CHO-pro5 cells and sta ble cell lines generated. Each receptor was characterised using [I-125][tyr (0)]sauvagine and results compared to CRF1 receptors expressed in the same parental cell line. Under identical assay conditions, [I-125][tyr(0)]sauvag ine labelled both CRF1 and CRF2 alpha receptors with high affinity. The lev el of expression varied from 103 fmol/mg membrane protein to 1842 fmol/mg m embrane protein (rat CRF, receptors and rat CRF2 receptors, respectively). It was possible to establish robust scintillation proximity assays (SPA) us ing wheat germ agglutinin (WGA) SPA beads to trap membrane protein. The suc cess of the SPA assay format was dependent on the level of receptor express ion observed. The rank order of affinities of a series of peptide CRF recep tor agonists and antagonists was similar to that described in the literatur e for the two receptor subtypes as determined using radioligand binding and cAMP accumulation No pharmacological differences were apparent between rat and human cloned receptors with the exception of alpha-helical CRF-(9-41). This peptide exhibited 10-fold higher affinity for rat CRF2 alpha receptor s as compared to human CRF2 alpha receptors. PD 173307, PD 173603 and PD 17 4239 exhibited high affinity and selectivity for human CRF1 receptors, and as such represent useful tools for probing CRF receptor function. (C) 1999 Elsevier Science B.V. All rights reserved.