RT Journal Article
SR Electronic
T1 Characterization of [<sup>125</sup>I]Sauvagine Binding to CRH<sub>2</sub> Receptors: Membrane Homogenate and Autoradiographic Studies
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 459
OP 468
VO 286
IS 1
A1 Rominger, David H.
A1 Rominger, Cynthia M.
A1 Fitzgerald, Lawrence W.
A1 Grzanna, Reinhard
A1 Largent, Brian L.
A1 Zaczek, Robert
YR 1998
UL http://jpet.aspetjournals.org/content/286/1/459.abstract
AB We describe the binding of [125I]tyrosauvagine to membranes of corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EBNA (293ECRH2α) cells. The binding of [125I]tyrosauvagine 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]tyrosauvagine 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]tyrosauvagine binding to CRH2α receptors was: urotensin &gt; sauvagine = urocortin &gt; α-helical CRH9–41 &gt; rh-CRH≫ o-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [125I]tyrooCRH binding to CRH1 receptors: urotensin &gt; urocortin &gt; r/h-CRH&gt; o-CRH = sauvagine &gt; α-helical CRH9–41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH1receptor, have little or no affinity for CRH2α receptors as labeled by [125I]tyrosauvagine. Two selective CRH1 receptor antagonists enabled us to examine comparative densities of CRH1 and CRH2 receptors in several brain areas. We also used [125I]tyrosauvagine 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]tyrosauvagine to study cloned CRH2 receptors expressed in cell lines. In addition, [125I]tyrosauvagine 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. The American Society for Pharmacology and Experimental Therapeutics