Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [ 35S]GTPγS to Dictyostelium discoideum membranes

Abstract

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTPγS>GTP>8-N 3-GTP; the binding of GTPγS and GTP, but not of 8-N 3-GTP, was stimulated by receptor agonists. [ 3H]GTP binding to D. discoideum membranes has been described previously by a single binding type ( K d = 2.6 μM, B max = 85 nM). More detailed studies with [ 35S]GTPγS showed heterogeneous binding composed of two forms of binding sites with respectively high ( K d = 0.2 μM) and low ( K d = 6.3 μM) affinity. cAMP derivatives enhanced GTPγS binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTPγS binding showed aclose correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTPγS binding, but eliminated the cAMP stimulation of GTP and GTPγS binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.