Guanine Nucleotide and Magnesium Ion Regulation of the Interaction of Gonadotropic and β-Adrenergic Receptors with Their Hormones: A Comparative Study Using a Single Membrane System*

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Abstract
The effects of guanine nucleotides and magnesium (Mg) on the interaction of catecholamines and gonadotropins with their receptors were studied in rabbit luteal membanes. The affinity of the luteal β-receptor for the agonists isoproterenol, epinephrine, and norepinephrine, as measured by inhibition of [125I]iodohydroxybenzylpindolol ([125I]IHYP) binding, was increased by 2 mM MgCl2. The addition of 100 μM GTP or MgCl2 in combination with GTP resulted in a reduction in the affinity of the β-receptor for these agonists. These effects of Mg and GTP were agonist specific, as the affinity of the luteal β-receptor for the antagonists propranolol and IHYP were not altered by Mg or GTP. The potencies with which isoproterenol, epinephrine, and norepinephrine competed with [125I]IHYP for the luteal β-receptor and activated adenylyl cyclase activity indicated that the rabbit luteal adrenergic receptors were of the β1 type. Comparison of the Kd values for these agonists calculated from binding experiments in the absence and presence of Mg, GTP, and combinations thereof with the Kact values (the concentrations of catecholamines required to stimulate adenylyl cyclase activity half-maximally) obtained in adenylyl cyclase assays demonstrated a positive correlation. This correlation was best when the Kact values were compared to the Kd values obtained in the presence of Mg plus GTP, suggesting that low affinity binding of catecholamines in the presence of guanine nucleotide is the biologically relevant affinity. In contrast, the affinity of the luteal gonadotropin receptor for hCG, human LH (hLH), ovine LH (oLH), and bovine LH (bLH) was not altered by GTP, as measured by direct Scatchard analysis of [125I]iodo-hCG binding or inhibition of [125I]iodo-hCG binding by hCG, hLH, oLH, or bLH. Mg slightly lowered the affinity of the receptor for [125I] iodo-hCG, hCG, and hLH, while Mg increased the affinity of the receptor for oLH and bLH. However, in no instance did the addition of GTP alter the affinity of the receptor from that observed in the presence of Mg alone. A positive correlation existed between the Kd values calculated from receptor-binding experiments with Kact values obtained in adenylyl cyclase assays, suggesting that the specific gonadotropin-binding sites present in rabbit luteal membranes represent receptors which mediate the stimulatory effect of LH. Thus, it is concluded that the binding function of these two classes of hormone receptors are regulated differently.

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