Adenylate cyclase: the role of magnesium and other divalent cations.

While it is now well-established that guanine nucleotide is an important regulatory agent acting on the hormone receptor-adenylate cyclase complex, only recently has sufficient evidence accumulated to indicate that free divalent cation, particularly free Mg2+, is also an important modulatory ligand. This review discusses the interactions of free divalent cation with the receptor-cyclase complex with regard to mechanism and site of action of metal ions, receptor-mediated inhibition of Mg2+ transport, and physiological function of the Mg2+ interactions. Careful kinetic analyses indicate that the receptor-cyclase complex possesses specific sites for free Mg2+. The influence of Mg2+ on these sites is demonstrated by a Mg2+-induced increase in both agonist affinity for the hormone receptor and Vmax of adenylate cyclase catalytic activity, without change in Km for the MgATP2− substrate. Upon addition of guanine nucleotide, the effect of free Mg2+ on agonist affinity for the receptor is abolished. However, the coupling influence of nucleotide or of nucleotide plus hormone does not eliminate increased catalytic activity induced by Mg2+, but appears to increase the apparent affinity of free Mg2+ for its binding site(s). Data from specific mutant cell lines of the murine S49 lymphoma and from solubilization and reconstitution studies with S49 cells and turkey and frog erythrocytes indicate that both free Mg2+ and guanine nucleotide interact with the guanine nucleotide coupling protein at the cytoplasmic membrane face. The roles of Mn2+ and Ca2+ are less well-studied. Our assessment of published data suggests that neither of these cations is a major regulatory ligand of receptor-cyclase function. Ca2+ interacts weakly if at all with free Mg2+ sites, and the CaATP2− complex, while probably a nonproductive substrate, is a poor competitive inhibitor of the enzyme. Some evidence indicates that Ca2+ plus the Ca2+-dependent regulatory protein (calmodulin) has a potential modulatory role for a subclass of nervous system receptor-cyclase complexes. The significance of this interaction is not clear. Mn2+, unlike Ca2+, forms a productive substrate complex and in addition appears to interact with the free Mg2+ site(s). However, in contrast to the activation produced by free Mg2+, Mn2+ may be an inhibitory cation at the free metal site(s). However, no physiological significance of these interactions with Mn2+ is known. Further complexity has been added to the interactions of Mg2+ with the receptor-cyclase complex by this laboratory's description of hormonal inhibition of Mg2+ transport across the plasma membrane. Using mutant cell clones of the S49 lymphoma cell we have been able to show that this inhibition of Mg2+ transport is not mediated by cyclic AMP. The transport function appears to be a property of the receptor-cyclase complex itself or of a possible Mg2+ transport protein associated with the hormone receptor. The potential physiological significance of hormone-sensitive Mg2+ transport is discussed. Finally, we suggest, as a framework for future experimentation, a sequence of interactions of Mg2+, GTP and hormone with the receptor-cyclase complex that appears to account for the effect of and interactions between these regulatory ligands. Specifically, we suggest that activation of adenylate cyclase by hormone occurs via, a form of the receptor-cyclase complex induced by and absolutely requiring the presence of bound Mg2+. Furthermore, formation of this complex requires the absence of bound GTP. Subsequent interaction of this Mg+-containing complex with GTP results in activation of the cyclase catalytic component. We further suggest that reported differences in Mg2+/GTP interactions on receptor-cyclase complexes from different cell types are a function of minor differences (e.g. in ligand affinities) in the same fundamental characteristics. The mechanism of hormonal activation of the receptor-cyclase complex appears to be identical regardless of species or cell type and in all cases appears to require interaction with both free Mg2+ and guanine nucleotide. In this regard, it should be noted that the nucleotide coupling protein is more properly described as a metal/nucleotide coupling protein.