CHARACTERIZATION OF [I-125] SAUVAGINE BINDING TO CRH2 RECEPTORS - MEMBRANE HOMOGENATE AND AUTORADIOGRAPHIC STUDIES

We describe the binding of [I-125]tyr degrees sauvagine to membranes o f corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EB NA (293E(CRH2 alpha)) cells. The binding of [I-125]tyr degrees sauvagi ne to CRH2 receptors was temperature, time and tissue dependent. Equil ibrium 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 a nd low affinity states, respectively. The high affinity [I-125]tyr deg rees sauvagine binding sites were eliminated with 200 mu M Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [I-125]tyr degrees sauvagine binding to CRH2 alpha receptors was: urotensin > sauvagine = urocortin > alpha-helica l CRH9-41 > rh-CRH much greater than o-CRH. This was in contrast to th e rank order potency of the peptides at inhibiting [I-125]tyr degrees oCRH binding to CRH1 receptors: urotensin > urocortin > r/h-CRH> o-CRH = sauvagine > alpha-helical CRH9-41. We show that two recently identi fied small molecule CRH antagonists with nanomolar potency at the CRH1 receptor, have little or no affinity for CRH2 alpha receptors as labe led by [I-125]tyr degrees sauvagine. Two selective CRH1 receptor antag onists enabled us to examine comparative densities of CRH1 and CRH2 re ceptors in several brain areas. We also used [I-125]tyr degrees sauvag ine in combination with a specific CRH1 antagonist to examine the anat omic distribution of CRH2 receptors using receptor autoradiography. Wi th a few notable exceptions the CRH2 receptors demonstrated autoradiog raphically in this study match the data obtained by in situ hybridizat ion 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 usi ng [I-125]tyr degrees sauvagine to study cloned CRH2 receptors express ed in cell lines. In addition, [I-125]tyr degrees sauvagine used in co njunction with saturating concentrations of a specific CRH1 receptor a ntagonist allows the study of CRH2 receptors in brain tissues using bo th in vitro homogenate binding and receptor autoradiography techniques .