GTP binding to Gs does not promote subunit dissociation.
The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) 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 alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.
Basi
N S
NS
Membrane Biochemistry Section, National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Okuya
S
S
Rebois
R V
RV
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology*; D011685:Purine Nucleotides / Q000378:metabolism
D011685:Purine Nucleotides; D015636:Magnesium Chloride; D006153:Guanosine Diphosphate; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins