GTP binding to G s does not promote subunit dissociation

Abstract

The stimulatory G protein (G s) mediates activation of adenylyl cyclase. G s is a heterotrimeric protein ( αβγ) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the α-subunit (G s α). Divalent cations such as magnesium are also required for G s activation. Subunit dissociation can accompany G s activation and is thought to be critical for this process. We investigated the effects of MgCl 2 and various purine nucleotides on G s-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein β-subunit that was co-precipitated by G s α-specific antiserum. G s activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc − membranes that lack G s α. High concentrations of MgCl 2 caused bound GDP to dissociate from G s and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl 2 caused a concentration-dependent dissociation of G s subunits. GTP γS (a non-hydrolyzable GTP analogue) shifted the MgCl 2 concentration-response curve for subunit dissociation to lower concentrations of MgCl 2, suggesting that GTP γS promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl 2. These results suggest that GTP, the compound that activates G s in vivo, was no more effective at promoting G s subunit dissociation that was GDP.