| Abstract: |
The effects of Mg2+or ethylenediaminetetraacetic acid (EDTA) on125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5’-triphosphate (GTP), maximal binding of125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+(EC50=0.3 mM) while EDTA or higher concentrations of Mg2+diminish binding. Response to exogenous Mg2+or EDTA depends on the concentration of Mg2+in the membranes and may vary with the method used for membrane isolation. Solubilized125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+or 5 mM EDTA diminishes receptor affinity for hormone (from KD= 1.2 ± 0.1 nM to KD= 2.6 ± 0.3 nM) and the fraction of125I-glucagon in high molecular weight receptor-Nscomplexes without affecting site number (Bmax= 1.8 ± 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Nscomplexes, Mg2+or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD= 9.0 ± 3.0 nM), low molecular weight receptors uncoupled from Ns. Binding site affinity is diminished by 40 mM Mg2+or 5 mM EDTA (KD= 20 ± 5 nM) and increased by 0.5 mM Mg2+(KD= 3.0 ± 0.4 nM) without significantly changing the amount of125I-glucagon in high molecular weight receptor-Nscomplexes. The ability of Mg2+to alter binding to receptors uncoupled from Nssuggests the presence of a cation binding site in the glucagon receptor. GTP enhances hormone dissociation from control membranes or membranes incubated with 0.5 mM Mg2+(EC50= 270 nM). EDTA (5 mM) diminishes (EC50= 500 nM GTP) while Mg2+(40 mM) enhances (EC50= 30 nM GTP) the sensitivity of glucagon-receptor complexes to GTP-induced dissociation. Thus, divalent cations also affect the sensitivity of hormone-receptor-regulatory protein complexes to GTP-induced disaggregation. © 1988, American Chemical Society. All rights reserved. |
