Corticotropin-releasing factor receptors in mouse spleen: identification, autoradiographic localization, and regulation by divalent cations and guanine nucleotides.

The radioligand [125I-Tyro]ovine CRF [( 125I] oCRF) was used to identify, characterize, and localize CRF receptors in a crude homogenate of mouse spleen. The binding was linear with increasing membrane protein concentration, saturable, and of high affinity. Scatchard analysis of [125I]oCRF saturation curves revealed a high affinity component with an apparent dissociation constant of 0.26 nM and a maximum receptor concentration of 8.74 fmol/mg protein. The relative affinities of various CRF-related and unrelated peptides at the [125I]oCRF-binding site in spleen correlate well with their reported potencies in eliciting ACTH release from the pituitary and their relative affinities for CRF receptors in brain. Consistent with a coupling of splenic CRF receptors to a guanine nucleotide regulatory protein, the binding of [125I]oCRF was increased by magnesium ions and decreased by guanine nucleotides in both mouse spleen and brain. Saturation analysis of [125I]oCRF binding at 0, 1, and 10 mM MgCl2, 10 mM MgCl2 plus 200 microM GTP, and 10 mM MgCl2 plus 50 microM 5'-guanylimidodiphosphate indicated that high affinity [125I]oCRF binding in both brain and spleen requires Mg2+; however, subtle differences were observed in the degree of sensitivity and mechanisms by which Mg2+ and guanine nucleotides regulated specific binding of [125I]oCRF to brain and spleen. Autoradiographic studies indicated that [125I]oCRF-binding sites were primarily localized to the red pulp and marginal zone regions of the spleen, which are known to contain high concentrations of macrophages. The absence of [125I]oCRF-binding sites in the periarteriolar and peripheral follicular white pulp regions suggests that neither T nor B lymphocytes have specific CRF-binding sites. In summary, we have identified a high affinity, magnesium-dependent, and guanine nucleotide-sensitive binding site for CRF in mouse spleen which appears to be localized primarily in splenic macrophages.