Role of G-protein availability in differential signaling by alpha 2-adrenoceptors1
Introduction
The α2-adrenergic receptors (α2-ARs), represented by three genetically distinct subtypes α2A, α2B, and α2C, control the function of different organs via central and peripheral mechanisms. The effects can be either inhibitory or stimulatory depending on the organ in question [1]. This is likely to be due to the ability of these receptors to activate multiple signal transduction pathways [2]. The classical cellular response observed upon activation of endogenously expressed α2-ARs is an inhibition of stimulated cAMP production [3] or inhibition of voltage-gated Ca2+ channels in neuronal cells 4, 5. Both of these responses are mediated by Gi/o-type pertussis toxin-sensitive G-proteins. However, ectopic expression of α2-ARs in different cell lines has revealed a pertussis toxin-insensitive increase rather than a decrease in cAMP production in response to α2-AR activation 6, 7, 8, 9. In many cell lines, this can be seen as a biphasic concentration-response curve with inhibition at low and stimulation at high agonist concentrations 7, 10, 11. In some systems, in particular with the α2B subtype, an exclusively stimulatory concentration-response curve is obtained 7, 8, 9, 11, suggesting a productive receptor-Gs interaction. A large number of G-protein-coupled receptors have been shown to interact with multiple G-proteins [12]. The promiscuity can occur not only within one class of G-proteins, such as Gi and Go, but also with G-proteins from different classes. Using immunoprecipitation, it has been shown that the α2A-AR interacts with both Gi- and Gs-proteins [10]. Promiscuous G-protein coupling has often been ascribed to high expression levels of receptors, but this does not always hold true [9]. It is of course possible that promiscuous G-protein coupling seen in heterologous expression systems could be an artefact due to altered processing of the receptors. The molecular basis for receptor-G-protein coupling promiscuity is, however, not well understood and a more likely explanation is that overexpression reveals secondary coupling pathways also utilized by endogenous receptors.
Data from analysis with chimeric or mutated α2-ARs receptors suggests that the Gi/Gs coupling requires largely separate receptor domains for interaction with the two G-proteins 13, 14, 15. In addition, certain agonists can selectively direct the coupling of the α2A-AR to either inhibition [16] or stimulation [17], indicating agonist-directed signal trafficking. The above-mentioned issues strongly suggest that the two responses depend upon different conformational changes in the receptor protein altering the avidity for different G-proteins. A factor that also influences the adenylyl cyclase activity is the receptor expression level [10], suggesting that the stoichiometry between receptor and G-protein may influence coupling.
To gain insight into these issues, we chose to study the regulation of cAMP production in Sf9 cells by the human α2B-AR expressed either alone or coexpressed together with G-proteins. Insect Sf9 cells have been used successfully earlier as a cellular reconstitution system for determining receptor-G-protein interactions 18, 19, 20. The human α2B-AR produces a monophasic stimulatory cAMP response when expressed in these cells [8]. Our aim was to find out how altered levels of G-proteins affect the receptor-mediated cAMP response and in this context to get insights into partial agonism in the light of G-protein accessibility.
Section snippets
Reagents and cell culture
[3H]Adenine, [14C]cAMP, and [3H]RX821002 were purchased from Amersham Corp. [35S]GTPγS was from New England Nuclear. Noradrenaline, 3-isobutyl-1-methyl-xanthine (IBMX), and forskolin were from Sigma. 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) was from RBI. Other chemicals used were of analytical grade quality.
Sf9 cells were grown in glass spinner bottles at 26° in Grace’s insect medium supplemented with lactalbumin hydrolysate and yeastolate (Life Technologies).
Functional expression of G proteins
Stimulation of Sf9 cells expressing the human α2B-AR with noradrenaline results in a significant elevation of the cellular cAMP content compared to the basal level [8]. This stimulation presumably results from receptor activation of endogenous Gs proteins. To characterize the G-protein coupling, we coexpressed the receptor with bovine Gαs alone or in combination with βγ subunits and measured the noradrenaline-stimulated increase in [35S]GTPγS binding (Fig. 1; left panel). In membranes without
Discussion
We and others have previously shown that the α2B-AR shows preferential coupling to an increase in cAMP production in certain cell types when compared to the other subtypes, α2A-AR and α2C-AR, which seem to be primarily coupled to inhibition of stimulated cAMP production 7, 8, 11. It has been shown, however, that all three subtypes can couple to stimulation if uncoupled from Gi by pertussis toxin treatment 10, 29. The results of the present study demonstrate that the availability of G-proteins
Acknowledgements
We thank Dr. T. Haga for providing the G-protein subunit cDNAs and Drs. K. Fuxe and S. Ferré for A2A receptor cDNA. This study was funded by The Medical Research Council of Sweden (MFR K98-14X-12205-02B), The Cancer Research Fund of Sweden, The Clas Groschinsky Foundation, The Åke Wiberg Foundation, and The Lars Hierta Foundation.
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Abbreviations: α2-AR, α2-adrenergic receptor; AC, adenylyl cyclase; GTPγS, guanosine 5′-O-(3-thiotriphosphate); IBMX, 3-isobutyl-1-methyl-xanthine; UK14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine.