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Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange
Authors:Mark M Rasenick  Steven R Childers
Institution:Department of Physiology, University of Illinois College of Medicine, Chicago 60680-6998.
Abstract:Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog 32P]P3 (4-azidoanilido)-P1-5'-GTP (32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of 32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of 32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.
Keywords:Adenylate cyclase  GTP-binding proteins  Photoaffinity labeling  Guanine nucleotides
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