BACKGROUND: Numerous studies suggest that cAMP signaling pathways play important roles in the development of and predisposition to alcoholism. Our previous study showed that cAMP generation by various isoforms of adenylyl cyclase (AC) exhibits a broad spectrum of responses to ethanol in the human embryonic kidney (HEK) 293 cell system overexpressing individual AC isoforms. These findings suggest that the target of ethanol's action in the cAMP-generating system is AC. However, it is unknown if the action of ethanol is direct or indirect. METHODS: The effect of a series of n-alkanols (ethanol to decanol) on dopamine (DA)-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected HEK293 cells by cAMP accumulation assay. RESULTS: n-Alkanols increased DA-stimulated cAMP production in an AC isoform-specific manner, and displayed the alcohol cutoff phenomenon (defined as the carbon chain length beyond which there is no further increase in the potency of an ascending series of n-alkanols). The n-alkanol cutoffs for AC6, AC7, and AC9 are butanol (C4), pentanol (C5), and equal to or greater than decanol (C10), respectively. CONCLUSION: The results clearly indicate that, in the HEK293 expression system, the alcohol cutoff effect for n-alkanol potentiation of DA-stimulated AC activity is AC isoform specific. These results strongly suggest that alcohols interact directly with AC molecules.
Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2. The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit alpha (Galpha(s)). In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD protein can inhibit the Galpha(s)- and/or forskolin-activated activity of both soluble and membrane-bound AC7. Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli slyD-deficient strain. This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6. This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an ethanol stimulus.
BACKGROUND: Numerous studies suggest that cAMP signaling pathways play important roles in the development of and predisposition to alcoholism. Our previous study showed that cAMP generation by various isoforms of adenylyl cyclase (AC) exhibits a broad spectrum of responses to ethanol in the human embryonic kidney (HEK) 293 cell system overexpressing individual AC isoforms. These findings suggest that the target of ethanol's action in the cAMP-generating system is AC. However, it is unknown if the action of ethanol is direct or indirect. METHODS: The effect of a series of n-alkanols (ethanol to decanol) on dopamine (DA)-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected HEK293 cells by cAMP accumulation assay. RESULTS: n-Alkanols increased DA-stimulated cAMP production in an AC isoform-specific manner, and displayed the alcohol cutoff phenomenon (defined as the carbon chain length beyond which there is no further increase in the potency of an ascending series of n-alkanols). The n-alkanol cutoffs for AC6, AC7, and AC9 are butanol (C4), pentanol (C5), and equal to or greater than decanol (C10), respectively. CONCLUSION: The results clearly indicate that, in the HEK293 expression system, the alcohol cutoff effect for n-alkanol potentiation of DA-stimulated AC activity is AC isoform specific. These results strongly suggest that alcohols interact directly with AC molecules.
The process in which a signal is passed on to downstream components within the cell, which become activated themselves to further propagate the signal and finally trigger a change in the function or state of the cell.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of the nucleotide cAMP (cyclic AMP, adenosine 3',5'-cyclophosphate).
Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2. The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit alpha (Galpha(s)). In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD protein can inhibit the Galpha(s)- and/or forskolin-activated activity of both soluble and membrane-bound AC7. Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli slyD-deficient strain. This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6. This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.
BACKGROUND: Numerous studies suggest that cAMP signaling pathways play important roles in the development of and predisposition to alcoholism. Our previous study showed that cAMP generation by various isoforms of adenylyl cyclase (AC) exhibits a broad spectrum of responses to ethanol in the human embryonic kidney (HEK) 293 cell system overexpressing individual AC isoforms. These findings suggest that the target of ethanol's action in the cAMP-generating system is AC. However, it is unknown if the action of ethanol is direct or indirect. METHODS: The effect of a series of n-alkanols (ethanol to decanol) on dopamine (DA)-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected HEK293 cells by cAMP accumulation assay. RESULTS: n-Alkanols increased DA-stimulated cAMP production in an AC isoform-specific manner, and displayed the alcohol cutoff phenomenon (defined as the carbon chain length beyond which there is no further increase in the potency of an ascending series of n-alkanols). The n-alkanol cutoffs for AC6, AC7, and AC9 are butanol (C4), pentanol (C5), and equal to or greater than decanol (C10), respectively. CONCLUSION: The results clearly indicate that, in the HEK293 expression system, the alcohol cutoff effect for n-alkanol potentiation of DA-stimulated AC activity is AC isoform specific. These results strongly suggest that alcohols interact directly with AC molecules.
Enzyme that catalyzes the cleavage of C-C, C-O, C-S, C-N or other bonds by other means than by hydrolysis or oxidation, with two substrates in one reaction direction, and one in the other. In the latter direction, a molecule (of carbon dioxide, water, etc) is eliminated, thus creating a new double bond or a new ring.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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