Synthesis of Gonadotropin-Releasing Hormone Receptors by Gonadotrope Cell Cultures: Both Preexisting Receptors and Those Unmasked by Protein Kinase-C Activators Show a Similar Synthetic Rate*

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Abstract
Responsiveness of gonadotropes to GnRH depends, in part, on the number of plasma membrane GnRH receptors. Since the steady state level of these plasma membrane receptors is a function of the rates of both receptor generation (synthesis, unmasking, and recycling) and loss (internalization, degradation, and inactivation) we have sought to quantify the rate of synthesis of GnTH receptors in pituitary cell cultures. Further, since the protein kinase-C activator phorbol 12-myristate 13-acetate (PMA) has been shown to unmask a class of GnRH receptors that appear to be uncoupled from phosphoinositide turnover, we have measured the rate of synthesis of this second receptor population. The present studies use the density shift technique; incorporation of densely labeled amino acids confers a higher density to newly synthesized proteins and allows their separation by physical means. Cultures of pituitary cells were prepared from female weanling rats. After cells had attached to the culture dishes, medium was replaced at 12-h intervals with medium containing either densely labeled or normal amino acids. After the incubation, GnRH receptors were covalently linked to a photoaffinity receptor agonist ([125I]Tyr5-[azido-benzoyl-D-Lys6-GnRH)], then solubilized with 1% sodium dodecyl sulfate. In some cultures PMA (50 nM) was included during the photoaffinity agonist-binding step. Newly synthesized (dense) receptors were separated from previously synthesized receptors by velocity sedimentation (0-20% sucrose in 1% sodium dodecyl sulfate-10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 .times. g for 24 h). Gradients were fractionated, and the radioactivity contained in each fraction was quantified. Newly synthesized GnRH receptors exhibited a higher density, as evidenced by further migration into the gradient, than did normal GnRH receptors. There was a delay of approximately 6 h between exposure to dense amino acids and the appearance of densely labeled GnRH receptors at the plasma membrane. Equilibrium for incorporation of dense amino acids into GnRH receptors was 48 h of exposure to dense amino acids. The time required for synthesis of half the entire population of GnRH receptors was 28 .+-. 2 h (mean .+-. SEM; n = 4). Scatchard analysis and the pattern of GnRH-stimulated LH release from densely labeled cells indicated that they bound the photoaffinity label (Kd = 0.4 nM; .apprx.1 fmol receptor/.mu.g DNA) and secreted gonadotropin normally. Additionally, treatment with PMA caused a significant increase (181 .+-. 24%) in photoaffinity agonist binding, consistent with previous observations. Although PMA treatment increased the number of binding sites for the photoaffinity agonist, no significant change in the rate of synthesis of GnTH receptors was observed (25 .+-. 1 h; n = 4) for synthesis of half of the GnRH receptor population. In summary, the rates of synthesis of both preexisting GnRH receptors and those unmasked by protein kinase-C activation are similar, requiring 25-28 h in cell culture for synthesis of half of the entire population of GnRH receptors.