Role of G-protein availability in differential signaling by alpha 2-adrenoceptors

doi:10.1016/S0006-2952(01)00730-4

Biochemical Pharmacology

Volume 62, Issue 7, 1 October 2001, Pages 913-922

https://doi.org/10.1016/S0006-2952(01)00730-4 Get rights and content

Abstract

The impact of G-protein expression on the coupling specificity of the human α_2B-adrenergic receptor (α_2B-AR) was studied in Sf9 cells. The α_2B-AR was shown to activate both coexpressed G_s- and G_i-proteins in a [³⁵S]GTPγS binding assay. Noradrenaline and the synthetic agonist UK14,304 were equally potent and efficacious in stimulating G_i activation. At the effector level (adenylyl cyclase), both ligands stimulated cAMP production. In the presence of forskolin, the effects of the agonists were more complex. Noradrenaline stimulated cAMP production, while UK14,304 showed a biphasic concentration-response curve with inhibition of stimulated cAMP production at low agonist concentrations and further stimulation at high agonist concentrations. G_s coexpression caused a monophasic stimulatory response with both ligands. Coexpression with G_i resulted in a biphasic concentration-response curve for noradrenaline and a monophasic inhibition with UK14,304. Experiments with a panel of agonists demonstrated that the more efficacious an agonist is in stimulating cAMP production, the weaker is its ability to couple to inhibition of cAMP accumulation via exogenous G_i. To be able to explain the mechanistic consequences of dual G-protein coupling described above, we developed a mathematical model based on the hypothesis that an agonist induces different conformations of the receptor having different affinity for different G-proteins. The model reproduced the profiles seen in the concentration-response curves with G_s and G_i coexpression. The model predicts that the affinity of the receptor conformation for G-proteins as well as the availability of G-proteins will determine the ultimate response of the receptor.

Introduction

The α₂-adrenergic receptors (α₂-ARs), represented by three genetically distinct subtypes α_2A, α_2B, and α_2C, 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 G_i/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 α_2B subtype, an exclusively stimulatory concentration-response curve is obtained 7, 8, 9, 11, suggesting a productive receptor-G_s 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 G_i and G_o, but also with G-proteins from different classes. Using immunoprecipitation, it has been shown that the α_2A-AR interacts with both G_i- and G_s-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 G_i/G_s 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 α_2A-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 α_2B-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 α_2B-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 α_2B-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 G_s proteins. To characterize the G-protein coupling, we coexpressed the receptor with bovine Gα_s 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 α_2B-AR shows preferential coupling to an increase in cAMP production in certain cell types when compared to the other subtypes, α_2A-AR and α_2C-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 G_i 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 A_2A 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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¹: 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.

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Role of G-protein availability in differential signaling by alpha 2-adrenoceptors1

Abstract

Introduction

Section snippets

Reagents and cell culture

Functional expression of G proteins

Discussion

Acknowledgements

Trends Pharmacol Sci

Biochim Biophys Acta

J Biol Chem

Eur J Pharmacol

Eur J Pharmacol

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

FEBS Lett

FEBS Lett

Anal Biochem

Biochem Pharmacol

J Biol Chem

Cell Signal

J Biol Chem

Eur J Pharmacol

Trends Pharmacol Sci

J Biol Chem

J Biol Chem

Role of G-protein availability in differential signaling by alpha 2-adrenoceptors¹