Isoform-specific regulation of adenylyl cyclase by G alpha-s.
Item
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Title
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Isoform-specific regulation of adenylyl cyclase by G alpha-s.
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Identifier
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AAI9807938
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identifier
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9807938
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Creator
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Harry, Anya Carmen.
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Contributor
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Adviser: Ravi Iyengar
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Health Sciences, Pharmacology | Biology, Molecular
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Abstract
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Heterotrimeric guanine nucleotide binding proteins (G proteins) function as signal transducers for receptors for many hormones, neurotransmitters, autocrine and paracrine factors. The G protein links the receptor that receives the extracellular signal to a distinct intracellular effector. Effectors in G protein signaling pathways are often enzymes regulating second messenger production. The molecular multiplicity of receptors, G proteins and adenylyl cyclases have now been established. Nine distinct adenylyl cyclase (AC) isoforms have been cloned and grouped into distinct families. In this study, we determined if adenylyl cyclases from different families exhibited differential responses to {dollar}\rm G\alpha\sb{lcub}s{rcub}.{dollar} For this, we used AC1, a neuronal isoform stimulated by Ca{dollar}\sp{lcub}2+{rcub}{dollar}/CaM; AC2, largely expressed in the brain and stimulated by protein kinase C and G{dollar}\beta\gamma{dollar} subunits, and the ubiquitously expressed AC6.;Stimulation of AC2, 2 and 6 by varying concentrations of mutant (Q227L) activated {dollar}\rm G\alpha\sb{lcub}s{rcub}{dollar} (G{dollar}\rm\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}){dollar} in the presence and absence of forskolin was studied. {dollar}\rm G\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}{dollar} stimulation of AC1 exhibited simple kinetics. The apparent K{dollar}\rm\sb{lcub}act{rcub}{dollar} for G{dollar}\rm\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}{dollar} was 0.9 nM, in the presence of forskolin, it measured 0.2 nM. {dollar}\rm G\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}{dollar} stimulation of AC2 was more complex, the {dollar}\rm G\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}{dollar} concentration-effect curve best fit a two site model. The "high affinity" site was 0.7-0.9 nM and the "low affinity" site was 7-15 nM. In the presence of forskolin, {dollar}\rm G\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}{dollar} stimulation was monophasic with an apparent K{dollar}\rm\sb{lcub}act{rcub}{dollar} of 0.4 nM. Regulation of AC2 by G{dollar}\beta\gamma{dollar} subunits appeared to require occupancy of primarily the "high affinity" G{dollar}\rm\alpha\sb{lcub}s{rcub}{dollar} site. Maximal fold stimulation by G{dollar}\beta\gamma{dollar} is achieved at 2 nM G{dollar}\rm\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}.{dollar} AC6 exhibited the most complex kinetics. {dollar}\rm G\alpha\sb{lcub}s{rcub}\sp{lcub}\*{rcub}{dollar} stimulation of AC6 appeared to involve two interacting sites with apparent K{dollar}\rm\sb{lcub}act{rcub}{dollar}'s of 0.2-0.8 nM and 8-19 nM. In the presence of forskolin, stimulation was monophasic with an apparent K{dollar}\rm\sb{lcub}act{rcub}{dollar} of 0.2 nM. Thus, different adenylyl cyclase isoforms have different patterns of responses to G{dollar}\rm\alpha\sb{lcub}s{rcub}.{dollar} These data indicate that the amplitude and sensitivity of the intracellular cAMP response to hormones and neurotransmitters will be determined by which adenylyl cyclase isoforms are present in individual cells and tissues.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
