Phosphorylation of adenylyl cyclase VI upon chronic δ-opioid receptor stimulation

doi:10.1016/S0014-2999(98)00847-4

European Journal of Pharmacology

Volume 364, Issues 2–3, 8 January 1999, Pages R1-R3

https://doi.org/10.1016/S0014-2999(98)00847-4 Get rights and content

Abstract

An immunoprecipitation method was used to measure [ $^{32} P$ ]phosphate incorporation into the adenylyl cyclase VI protein in Chinese Hamster Ovary (CHO) cells stably expressing the human δ-opioid receptor. Chronic SNC 80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide) (1 μM, 24 h) treatment increased the incorporation of [ $^{32} P$ ] into a 200 kDa protein band 2.5-fold after gel electrophoresis. The increase in phosphorylation of adenylyl cyclase VI was antagonized by naltrindole (1 μM) and the immunoprecipitation was prevented by the saturation of the antibody with the blocking peptide.

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Acknowledgements

This work was supported in part from a grant from the Arizona Disease Control Research Commission.

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These changes contribute to the development of tolerance and physical dependence [40–42], which limit the clinical use of opioids as therapeutic agents. Those adaptive changes are initiated by signaling cascades that involve regulation of membrane-delimited adenylyl cyclase (AC) enzymes [39,43,44] and act simultaneously with β-arrestin proteins signaling, whose implication to the development of analgesic tolerance is detailed below. Together, these studies indicate that G protein signaling does not only mediate the desired but also at least some unwanted effects of opioids.
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One protein that might be involved in phosphorylation of adenylyl cyclases during sensitization is Raf-1. In CHO cells, heterologous sensitization via the δ-opioid receptor leads to phosphorylation of AC6, and inhibition of Raf-1 attenuates heterologous sensitization (Varga et al., 2002, 1999; Yue et al., 2006). Moreover, prolonged morphine treatments in primary rat dorsal root ganglion neurons lead to enhanced cAMP production, which is prevented by inhibition of Raf-1 (Yue et al., 2008).
Heterologous sensitization of adenylyl cyclase (also referred to as superactivation, sensitization, or supersensitization of adenylyl cyclase) is a cellular adaptive response first described 40 years ago in the laboratory of Dr. Marshall Nirenberg. This apparently paradoxical cellular response occurs following persistent activation of Gα_i/o-coupled receptors and causes marked enhancement in the activity of adenylyl cyclases, thereby increasing cAMP production. Since our last review in 2005, significant progress in the field has led to a better understanding of the relevance of, and the cellular biochemical processes that occur during the development and expression of heterologous sensitization. In this review we will discuss the recent advancements in the field and the mechanistic hypotheses on heterologous sensitization.
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Previously we have investigated the molecular mechanisms of AC superactivation in recombinant Chinese Hamster Ovary (CHO) cell lines expressing human δ- or μ-opioid receptors. We found that sustained opioid agonist treatment leads to phosphorylation of an adenylyl cyclase isoenzyme (AC VI) in CHO cells expressing human δ-opioid receptor (Varga et al., 1999). Subsequently, we have demonstrated that multiple parallel signal transduction pathways converge at Raf-1 to mediate AC superactivation in the same CHO cell line (Varga et al., 2003a,b).
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Adenylate cyclases (AC) type 5 and 6 comprise the calcium-inhibited family of adenylate cyclase isoforms. Here we review recent discoveries in the regulation of AC5 and AC6 with a focus on posttranslational modifications including glycosylation, nitrosylation, and phosphorylation by the cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), and Raf1. We also describe novel signaling interactions such as Gα_q-mediated potentiation of AC6 activation. Novel regulators of AC5 and AC6, including small molecules and proteins that physically interact with AC5 and AC6 such as snapin, regulator of G protein signaling 2 (RGS2), protein associated with myc (PAM), and caveolin peptides are discussed. We also describe several recent studies that demonstrate the usefulness of transgenic or adenoviral overexpression of AC5 and AC6 in models for disease states such as cardiovascular hypertrophy. The discovery of novel regulatory mechanisms for AC5 and AC6 and their potential role in crucial physiological processes provide new avenues for research into therapeutic interventions targeting the cyclic AMP pathway.
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Activation of receptors coupled to inhibitory G proteins (Gα_i/o) has opposing consequences for cyclic AMP accumulation and the activity of cyclic AMP-dependent protein kinase, depending on the duration of stimulation. Acute activation inhibits the activity of adenylate cyclase, thereby attenuating cyclic AMP accumulation; in contrast, persistent activation of Gα_i/o-coupled receptors produces a paradoxical enhancement of adenylate cyclase activity, thus increasing cyclic AMP accumulation when the action of the inhibitory receptor is terminated. This heterologous sensitization of cyclic AMP signaling, also called superactivation or supersensitization, likely represents a cellular adaptive response, a mechanism by which the cell compensates for chronic inhibitory input. Recent advances in our knowledge of G protein-mediated signaling, regulation of adenylate cyclase, and other cellular signaling mechanisms have extensively increased our insight into the mechanisms and significance of this phenomenon. In particular, recent evidence points to the Gα_s–adenylate cyclase interface as a locus for the expression of the sensitized adenylate cyclase response, and to isoform-specific phosphorylation of adenylate cyclase as one mechanism that can produce sensitization. Gα_i/o-coupled receptor-induced heterologous sensitization may contribute to enhanced Gα_s-coupled receptor signaling following neurotransmitter elevations induced by the administration of drugs of abuse and during other types of neuronal function or dysfunction. This review will focus on recent advances in our understanding of signaling pathways that are involved in sensitization and describe the potential role of sensitization in neuronal function.
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Despite acutely inhibiting adenylate cyclase, prolonged activation of Gα_i/o-coupled receptors leads to a subsequent heterologous sensitization of adenylate cyclase responsiveness. Recently, protein kinase signaling and phosphorylation have been implicated in the sensitization of adenylate cyclase type 6 (AC6). To examine the sensitization specifically of AC6, we constructed human embryonic kidney cells (HEK293) cells stably expressing AC6 and the Gα_i/o-coupled D_2L dopamine receptor. In contrast to observations in δ-opioid-expressing Chinese hamster ovary (CHO) cells that express endogenous AC6 and AC7, neither protein kinase C (PKC) nor tyrosine kinase inhibitors attenuated D_2L receptor-mediated sensitization of AC6. Inhibition of Raf1 modestly inhibited the magnitude of D_2L receptor-induced sensitization of AC6; however, activation of PKC robustly enhanced D_2L receptor-mediated AC6 sensitization in a Raf1-dependent manner. These data indicate that, although PKC and Raf1 are not required for sensitization, activation of the PKC-Raf1 pathway robustly potentiated D_2L receptor-mediated sensitization of AC6.

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Abstract

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Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem.

Trends Neurosci.

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Phosphorylation of adenylyl cyclase VI upon chronic δ-opioid receptor stimulation