Abstract

Many different types of receptors couple to the inhibitory guanine nucleotide-binding protein (G protein) Gi. In NG108-15 neuroblastoma-glioma cells, alpha 2b-adrenergic, m4 muscarinic, and delta-opiate receptors all use Gi as a transducer. According to the ternary complex model of receptor-G protein interactions, agonists bind to these receptors with high affinity only in their G protein-associated form. Conversely, G protein affinity for the receptor is increased by agonist binding. We have developed an extended ternary complex model in which multiple receptors couple to a single G protein and we have examined two consequences of the model theoretically and experimentally. First, the simple ternary complex model can account for the observed high and low affinity agonist binding only when G protein is limiting; however, measurements show a significant excess of G protein over receptor. Could this paradox be explained by other receptors competing for the same G protein and limiting the amount of free G protein so that high and low affinity agonist binding would be seen? Our theoretical simulations show that this does not occur unless the receptors and G protein are present in a precise stoichiometric ratio and have an extremely high affinity, such as when agonists for both receptors are present. The second prediction of this model is that binding of an agonist at one receptor should produce competition for G protein used by another receptor. If the G protein pool were limiting and freely mobile, this would result in an unlabeled agonist at one receptor decreasing binding of a radiolabeled agonist to another receptor. Experimentally, the G protein was made limiting by a partial pertussis toxin treatment. Radioligand binding to alpha 2b-adrenergic and m4 muscarinic receptors in these pertussis toxin-treated NG108-15 membranes showed no cross-talk with the delta-opiate or muscarinic receptors, which are present in excess. This could occur because the different receptors interact with structurally different G proteins (e.g., distinct beta or gamma subunits). More likely it is because of limitations of the mobility of G proteins in the membrane due to 1) attachment to structural elements, such as the cytoskeleton, 2) sequestration in lipid pools, or 3) organization into slowly exchanging supramolecular complexes. These results show that we must reexamine the assumptions of the collision coupling and ternary complex models.