Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits

Autor: Isabel Ulibarri, Peter Gierschik, Thomas Wieland, Karl H. Jakobs
Rok vydání: 1991
Předmět:
Zdroj: European journal of biochemistry. 196(3)
ISSN: 0014-2956
Popis: Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding M subunit and a fly dimer. The influence of fly 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’-[y-thio]triphosphate (GTP[S]) and guanosine 5’-[py-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TDjy was separated from TDM and its function analyzed. Addition of TDPy isolated from TD prepared with GTP[S] (TDPYGT~[S]) to HL 60 membranes abolished high-affinity binding of Met-Leu[3H]Phe (met, N-formylmethionine) to its receptor. In contrast, TDPy isolated from TD prepared with GTP (TDPyGTp), boiled TDjyGTPISl and TDcl prepared with GTP[S] had no or only slight effects. The inhibitory effect of TDPyGTPISl on f’Met-Le~-[~H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TDPYGTP[S], but not TDjyGTp or TDPy isolated from TD prepared with Gpp[NH]p (TDPyGpp[NHlp), prevented met-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TDPyGTp was incubated with GTP[S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TDPyGTp, similar to TDPyGTPISl, abolished high-affinity binding of fMet-Le~-[~H]Phe to its receptor, Met-Leu-Phe-stimulated binding of [35S]GTP[S], and Met-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TDPy was not seen in the presence of the metabolically stable GDP analog, guanosine 5’-[P-thio]diphosphate. In order to obtain an insight into the modification of TDPy apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TDM and TDPy, all prepared in the presence of GTP, were incubated with [3’S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the band of the G protein. When apparently thiophosphorylated TDPj 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 Mgzf, and was dependent on substrate concentrations, i. e. thiophosphorylated TDPy and [3H]GDP, and the presence of a factor(s) present in HL 60 membranes, possibly G protein M subunits. Under optimal conditions, about 1 mol [3H]GTP[S] was formed from [3H]GDP by 1 mol added TDPy. Evidence is presented that TDPy can undergo a stable conformational change, apparently a thiophosphorylation of the /3 subunit, which is only obtained with GTP[S], and that thiophosphorylated TDPy 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 M subunits, information transfer by intermediately (thi0)phosphorylated By subunits is apparently an additional mechanism of G protein activation, with the two mechanisms possibly acting in a concerted manner.
Databáze: OpenAIRE
Externí odkaz: