Dataset Information

Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by beta gamma-subunits and somatostatin in S49 lymphoma cell membranes.

ABSTRACT: We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.

SUBMITTER: Ransnas LA

PROVIDER: S-EPMC1130546 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.

Project description:The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D- (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G1 growth arrest. Treatment of WT, but not D-, S49 cells with 8-CPT-cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D- cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D- cells, respectively. Using an approach that combined regression analysis, clustering and functional annotation to identify transcripts that showed differential expression between WT and D- cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi and lysosomes. The 2 cell lines differed in CREB phosphorylation and expression of the transcriptional inhibitor Icer and in cAMP-regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways. Experiment Overall Design: S49 D- cells were treated with 8-CPT-cAMP over the course of 24 hours. Cells were prepared for hybridization to microarrays at times 0-untreated, 2h, 6h, and 24h after treatment with 8-CPT-cAMP. Experimental replicates were as follows n=4 for time 0 and n=3 for 2, 6, and 24h post treatment.

Dataset Information

Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by beta gamma-subunits and somatostatin in S49 lymphoma cell membranes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets