GTP-BINDING TO G(S) DOES NOT PROMOTE SUBUNIT DISSOCIATION

The stimulatory G protein (G,) mediates activation of adenylyl cyclase . G, is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue d isplaces tightly bound guanosine diphosphate (GDP) from the guanine nu cleotide-binding site of the alpha-subunit (G(s) alpha). Divalent cati ons such as magnesium are also required for G(s) activation. Subunit d issociation can accompany G(s) activation and is thought to be critica l for this process. We investigated the effects of MgCl2, and various purine nucleotides on G(s)-subunit dissociation and activation. Subuni t dissociation was assayed by measuring the amount of G protein beta-s ubunit that was co-precipitated by G(s) alpha-specific antiserum. G(s) activation was determined by its ability to reconstitute adenylyl cyc lase activity in S49 cyc(-) membranes that lack G(s) alpha. High conce ntrations of MgCl2 caused bound GDP to dissociate from G(s) and inacti vated the protein unless high concentrations of GDP or GTP were presen t in solution. MgCl2 caused: a concentration-dependent dissociation of G(s) subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to low er concentrations of MgCl2, suggesting that GTP IS promoted subunit di ssociation. On the other hand, GDP and GTP were equally effective in s hifting the curve to higher concentration of MgCl2. These results sugg est that GTP, the compound that activates G(s) in vivo, was no more ef fective at promoting G(s) subunit dissociation than was GDP.