Spontaneous release of GDP from G i proteins and inhibition of adenylyl cyclase in cardiac sarcolemmal membranes

Abstract

Several studies have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphate transfer-resistant analog, guanosine 5'- O-(2-thiodiphosphate; GDPbetaS). Here, we studied the mechanism underlying this unconventional activation. Adenylyl cyclase activity in sarcolemmal membranes from failing human ventricular myocardium, at a low Mg2+ concentration, decreased rapidly during incubation at 37 degrees C. This decrease in enzyme activity was paralleled by a rapid release of GDP from Gi proteins, amounting to 75% release of total Gi-bound GDP within 10 min at 37 degrees C. In contrast, no GDP release was observed at 4 degrees C, and adenylyl cyclase activity remained stable for up to 20 min at 4 degrees C. GDPbetaS did not alter the initial rates of cyclic AMP formation by the adenylyl cyclase, at either 37 degrees C or 4 degrees C, but almost fully prevented the decrease in enzyme activity occurring at 37 degrees C. Hence, after 10 min of incubation at 37 degrees C, adenylyl cyclase activity in the presence of GDPbetaS was increased by 150%, while no difference in activity was observed at 4 degrees C. Under conditions where adenylyl cyclase is uncoupled from regulation by the inhibitory Gi proteins, i.e., at high concentrations of Mg2+ or Mn2+, adenylyl cyclase activity remained stable even at 37 degrees C and GDPbetaS did not stimulate activity. In conclusion, Gi proteins in sarcolemmal membranes from failing human hearts rapidly release bound GDP. The data, furthermore, suggest that this process results in adenylyl cyclase inhibition by the empty but apparently active Gi proteins.

Guanine Nucleotide Exchange-Independent Activation of GsProtein by β2-Adrenoceptor

“…Early observations that G s or G i could be activated in the presence of GDP (Iyengar and Birnbaumer, 1979;Schneyer et al, 1984;Harding and Harris, 1986;Quist et al, 1992;Piacentini et al, 1996;Lutz et al, 2002) have mostly been attributed to the conversion of GDP to GTP during the experiments (Kimura and Schimada, 1983;Kikkawa et al, 1990;Kowluru et al, 1996). Such a conversion must be nearly complete and almost instantaneous to be an explanation for the present results, because GTP and GDP supported the cyclase activation with almost identical potencies in the presence or absence of agonist.…”

“…A number of experiments in the literature show that G s (or G i ) protein can indeed be activated in the presence of GDP (Iyengar and Birnbaumer, 1979;Schneyer et al, 1984;Harding and Harris, 1986;Quist et al, 1992;Piacentini et al, 1996;Seifert et al, 1998, Hatley et al, 2003Lutz et al, 2002). These reports include observations dating from late 1970s to the present day.…”

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

“…A recent report raised the question whether G proteins have to be in the GTP-bound form to be active (Ugur et al 2005;Wieland and Michel 2005), and there is increasing evidence that not all heterotrimeric G proteins dissociate into Gα and Gβγ upon activation (Bünemann et al 2003;Frank et al 2005). Taking further into account that, in in vitro systems, even nucleotide-free Gα subunits are active (Lutz et al 2002), or heterotrimeric G proteins can be activated by pyrimidine nucleotide triphosphates under conditions excluding high energy phosphate relay (Gille et al 2005), it is evident that investigations on heterotrimeric G proteins and their interaction partners, like those performed by Karl H. Jakobs since 1970 (Jakobs and Schultz 1970), will provide interesting and relevant topics for several decades of future research.…”

“…The experimental artefacts mentioned above were due to a spontaneous release of GDP from G proteins. This GDP served as a substrate for phosphotransfer by the NDPK, and the formed GTP then bound back to the G protein (Randazzo et al 1991(Randazzo et al , 1992aLutz et al 2002). Even an approach where GDP has been cross-linked to the monomeric GTPase Rad (Zhu et al 1999) is questionable by its experimental design (Otero 2000), as NDPK is able to transfer phosphate also onto denatured proteins and thus also on the covalently linked GDP (Engel et al 1995).…”

Can a GDP-Liganded G-Protein Be Active?: Fig. 1.

“…For example, Ug ur et al (2005) report that GDP or GDP␤S enhanced agonist-stimulated adenylyl cyclase activity. A stimulatory effect of GDP and GDP␤S on adenylyl cyclase activity has been reported before, but this was interpreted as an inactivation of nucleotide free G␣ i from an active conformation that inhibits adenylyl cyclase (Piacentini et al, 1996;Lutz et al, 2002). Although the findings of Ug ur et al (2005) cannot be fully explained by a GDP-induced disinhibition of G i , the data do not unequivocally rule out that possibility.…”