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Solubilization of Receptors for Thyrotropin-Releasing Hormone from GH₄C₁ Rat Pituitary Cells: Demonstration of Guanyl Nucleotide Sensitivity

Laboratory of Toxicology, Harvard School of Public Health and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Boston, Massachusetts 02115

Address requests for reprints to: Armen H. Tashjian, Jr., Laboratory of Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115.

Abstract

TRH receptors have been solubilized from GH₄C₁ cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH ([³H]MeTRH) attained equilibrium within 2–3 h at 4 C, and it was reversible, dissociating with a t of 7 h. Analysis of [³H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (k_d) of 3.8 nM and a total binding capacity (B_max) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [³H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC₅₀ of 11µM. When [³H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [³H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [³H]MeTRH dissociation by 10µM GTPS occurred within about 45 min at 22 C. GTPS, GTP, GDPβS, and GDP were all effectors of [³H]MeTRH dissociation, exhibiting EC₅₀S in the range of 14–450 nM. The rank order of potency of the tested nucleotides was GTPS > GTP GDPβS > GDP ATPS > GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [³H]MeTRH to GH₄C₁ cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.

FOOTNOTES

This investigation was supported in part by Research Grant DK-11011 from the NIDDK.

^* Recipient of a Canadian Heart Foundation Postdoctoral Fellowship.

Received for publication January 12, 1987. Accepted for publication September 22, 1987.