Receptor‐stimulated guanine‐nucleotide‐triphosphate binding to guanine‐nucleotide‐binding regulatory proteins

Abstract

In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5'-[y-thioltriphosphate, GTP [S], and guanosine 5'-[j?,y-imino]triphosphate, p [NH]ppG, and the regulation of binding by the fonnyl-peptide-receptor agonist, Net-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For met-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of Met-Leu-Phe-stimulated binding of GTP [S] Guanine-nucleotide-binding regulatory proteins (G proteins) participate in many transmembrane signalling pathways, coupling a large variety of membrane receptors to second-messenger-generating enzymes and ion channels. These G proteins are heterotrimers composed of a, j?, and y subunits, the latter two forming a j?y dimer under non-denaturating conditions [l-31. The activation of G proteins is generally assumed to be caused by receptor-induced exchange of GDP bound to G protein a subunits by GTP. We have recently reported that / 3 subunits of the retinal G protein

Nucleoside diphosphate kinase activity in soluble transducin preparations

“…Both ATP and GTP can support phosphorylation of G‐protein β subunits [17–23]. In agreement with data obtained in other systems [19,22,23], [γ‐ 32 P]GTP induced, in a time‐dependent manner, phosphorylation of the 36‐kDa protein (and autophosphorylation of rhodopsin kinase) in soluble transducin preprations (Fig.…”

“…In contrast with NTP[S]s, p[NH]ppNs cannot serve as donors in trans(thio)phosphorylation reactions [20,32]. Thus, through competition with NTP[S]s, p[NH]ppNs might be expected to act as inhibitors of trans(thio)phosphorylation reactions.…”

In Vitro Autoradiographic Visualization of Guanosine‐5’‐O‐(3‐[35S]Thio)triphosphate Binding Stimulated by Sphingosine 1‐Phosphate and Lysophosphatidic Acid

“…The GTP analogues GTPγS and GppNHp have been shown to discriminate between different G proteins (Vitale et al., 1994) or GTP‐dependent processes (Ahnert‐Hilger et al, 1992 ; Parsons and Hartzell, 1993 ; Kaldenberg‐Stasch et al, 1994). We have shown that 5‐HT 1A , 5‐HT 1B , and CB 1 receptor agonists stimulate [ 3 H]‐GppNHp binding with a spatial distribution identical to that observed with [ 35 S]GTPγS (authors’ unpublished data).…”

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…The identity of the proposed membrane-bound histidine kinase to catalyse this reaction remained, however, elusive (Nürnberg et al 1996, Kowluru 2002. Also, in this phosphotransfer reaction, the phosphorylation of GDP bound to Gα was a matter of debate (Wieland et al 1993;Kaldenberg-Stasch et al 1994;Hohenegger et al 1996;Kowluru et al 1996;Niroomand et al 1997). The same experimental constraints, which cannot exclude the spontaneous release of the Gα-bound GDP and re-binding of formed GTP, apply here, and some experimental data are clearly arguing against a direct transfer on Gα-bound GDP (Hohenegger et al 1996).…”

“…These G proteins still can be activated by the classical pathway of GPCR-induced GDP/GTP exchange. Nevertheless, a variety of data accumulated by different laboratories in the early days of investigations on NDPK and G protein activation (reviewed in Otero 1990;Piacentini and Niroomand 1996) as well as some data on G protein activation by phosphorylated Gβ subunits (Kaldenberg-Stasch et al 1994) argued for a role of phosphate transfer in GPCR-induced G protein activation. These speculations culminated in a hypothesis in which the GPCR/G protein complex functions as GTP synthase and a phosphorelay would be the essential mechanism of G protein activation (Nederkoorn et al 1998).…”