Activation of signal‐transducing guanine‐nucleotide‐binding regulatory proteins by guanosine 5′‐[γ‐thio]triphosphate
Authors: WIELAND Thomas , ULIBARRI Isabel , GIERSCHIK Peter , JAKOBS Karl H. 
Published: March 2005

Signal‐transducing guanine‐nucleotide‐binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide‐binding α subunit and a βγ dimer. The influence of βγ dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide‐receptor–G‐protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5′‐[γ‐thio]triphosphate (GTP[S]) and guanosine 5′‐[βγ‐imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TDβγ was separated from TDα and its function analyzed. Addition of TDβγ isolated from TD prepared with GTP[S] (TDβγGTP[S]) to HL 60 membranes abolished high‐affinity binding of fMet‐Leu‐[3H]Phe (fMet, N‐formylmethionine) to its receptor. In contrast, TDβγ isolated from TD prepared with GTP (TDβγGTP), boiled TDβγGTP[S] and TDα prepared with GTP[S] had no or only slight effects.The inhibitory effect of TDβγGTP[S] on fMet‐Leu‐[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TDβγGTP[S], but not TDβγGTP or TDβγ isolated from TD prepared with Gpp[NH]p (TDβγGpp[NH]p), prevented fMet‐Leu‐Phe‐stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TDβγGTP was incubated with GTP[S] and TD‐depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TDβγGTP, similar to TDβγGTP[S], abolished high‐affinity binding of fMet‐Leu‐[3H]Phe to its receptor, fMet‐Leu‐Phe‐stimulated binding of [35S]GTP[S], and fMet‐Leu‐Phe‐stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TDβγ was not seen in the presence of the metabolically stable GDP analog, guanosine 5′‐[β‐thio]diphosphate. In order to obtain an insight into the modification of TDβγ apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TDα and TDβγ, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD‐depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the β band of the G protein. When apparently thiophosphorylated TDβγ was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed. This formation of [3H]GTP[S] required Mg2+, and was dependent on substrate concentrations, i. e. thiophosphorylated TDβγ and [3H]GDP, and the presence of a factor(s) present in HL 60 membranes, possibly G protein α subunits. Under optimal conditions, about 1 mol [3H]GTP[S] was formed from [3H]GDP by 1 mol added TDβγ. Evidence is presented that TDβγ can undergo a stable conformational change, apparently a thiophosphorylation of the β subunit, which is only obtained with GTP[S], and that thiophosphorylated TDβγ can serve as an intermediate in formation of GTP[S] from GDP exogenously added and/or present in HL 60 membranes, with subsequent G protein activation. As well as the classical GDP/GTP (GTP[S]) exchange at G protein α subunits, information transfer by intermediately (thio) phosphorylated βγ subunits is apparently an additional mechanism of G protein activation, with the two mechanisms possibly acting in a concerted manner.