Abstract

We describe the binding of [¹²⁵I]tyr^osauvagine to membranes of corticotropin-releasing hormone (CRH₂) receptor expressing HEK293/EBNA (293E_CRH2α) cells. The binding of [¹²⁵I]tyr^osauvagine to CRH₂receptors was temperature, time and tissue dependent. Equilibrium was reached in 2 hr at 23°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^osauvagine binding sites were eliminated with 200 μM Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [¹²⁵I]tyr^osauvagine binding to CRH_2α receptors was: urotensin > sauvagine = urocortin > α-helical CRH_9–41 > rh-CRH≫ o-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [¹²⁵I]tyr^ooCRH binding to CRH₁ receptors: urotensin > urocortin > r/h-CRH> o-CRH = sauvagine > α-helical CRH_9–41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH₁receptor, have little or no affinity for CRH_2α receptors as labeled by [¹²⁵I]tyr^osauvagine. 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^osauvagine in combination with a specific CRH₁ antagonist to examine the anatomic distribution of CRH₂ receptors using receptor autoradiography. With a few notable exceptions the CRH₂receptors 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^osauvagine to study cloned CRH₂ receptors expressed in cell lines. In addition, [¹²⁵I]tyr^osauvagine 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.