Expression and characterisation of human and rat CRF2α receptors

Abstract Rat and human CRF 2α receptors were expressed in CHO-pro5 cells and stable cell lines generated. Each receptor was characterised using [ 125 I ][tyr 0 ]sauvagine and results compared to CRF 1 receptors expressed in the same parental cell line. Under identical assay conditions, [ 125 I ][tyr 0 ]sauvagine labelled both CRF 1 and CRF 2α receptors with high affinity. The level of expression varied from 103 fmol/mg membrane protein to 1842 fmol/mg membrane protein (rat CRF 1 receptors and rat CRF 2 receptors, respectively). It was possible to establish robust scintillation proximity assays (SPA) using wheat germ agglutinin (WGA) SPA beads to trap membrane protein. The success of the SPA assay format was dependent on the level of receptor expression observed. The rank order of affinities of a series of peptide CRF receptor agonists and antagonists was similar to that described in the literature 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 α-helical CRF-(9-41). This peptide exhibited 10-fold higher affinity for rat CRF 2α receptors as compared to human CRF 2α receptors. PD 173307, PD 173602 and PD 174239 exhibited high affinity and selectivity for human CRF 1 receptors, and as such represent useful tools for probing CRF receptor function.