Abstract
The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the $Department of Pharmacology, University of Texas Health Science Center at Dallas, Dallas, Texas75235 and the lIDepartment of Physiological Chemistry,Faculty of Pharmaceutical Sciences, Hokkaido University,Sapporo, Japan
Inhibition caused by GTP plus somatostatin pendent hormonal inhibitionof adenylate cyclase and or thapt roduced by GTPyS is relievebdy the 8 subunit that caused by guanine nucleotide analogsseem to re- in cyc- membranes, while the a subunit of Gi relieves sult from dissociationof the subunitsof Gi
We propose that a major fraction of the inhibition caused by guanine nucleotide and somatostatin in cyc- membranes is the result of the action of the a subunit, rather than thpesubunit,of Gi.this does not appear to be the case for S49 wild type membranes, since the observable effects of the resolved subunits of Gi are totally opposite in membranes that have functional G
Summary
Guanine nucleotide analogs, such as GTPrS, inhibited the adenylate cyclase activity of both S49 cell lines with. Adenylate Cyclase and G, Assays-Plasma membranes from S49 cyc- cells were reconstituted with G., as described by Sternweis et al. The adenylate cyclase assay contained(in 100 pl) 30 pgocfyc- or wild type membranes, 50 mM sodium Hepes (pH 8), 0.25 mM ATP with -lo cpm of Ic~-~']ATP2, mM MgCl,, 1 mM sodium EDTA, 10 pg/ml of pyruvate kinase, 0.1 mg/ml of bovine serum albumin, 3 mM potasslum phosphoenolpyruvate, and 0.1 mM 4-(3-butoxy-4-methoxybenzyl)-2-. Gi in Wild Type and cyc- S49 Cell Membranes and GTPyS resulted in immediate inhibition of adenylate cyclase activity to a steady statelevel similar to thatinduced [ACyc-.
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