Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) |
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Authors: | Timothy M Palmer Miles D Houslay |
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Institution: | Molecular Pharmacology Group, Institute of Biochemistry, University of Glasgow, GlasgowU.K. |
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Abstract: | Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi α-2, Gi α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), pNH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC50-60 nM) of pNH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, pNH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and pNH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by pNH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals. |
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Keywords: | G-protein ADP-ribosylation Diabetes G-protein Guanine nucleotide-binding regulatory protein the stimulatory G-protein of adenylyl cyclase family of G-proteins originally assigned the function of inhibiting adenylyl cyclase p[NH]ppG guanylyl 5′-[β γ-imido]diphosphate SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis concentration at which 50% of the maximal response was achieved Ptx pertussis toxin Ctx cholera toxin |
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