A Drosophila adenosine receptor activates cAMP and calcium signaling

Abstract

Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. Here we report the first characterization of an insect AdoR, encoded by the Drosophila gene CG9753. Adenosine stimulation of Chinese hamster ovary cells carrying transiently expressed CG9753 led to a dose-dependent increase of intracellular cAMP and calcium, but untransfected controls showed no such response, showing that CG9753 encodes a functional AdoR. Endogenous CG9753 transcripts were detected in the brain, imaginal discs, ring gland and salivary glands of third-instar Drosophila larvae, and CG9753 overexpression in vivo caused lethality or severe developmental anomalies. These developmental defects were reduced by adenosine depletion, consistent with the proposed function of the CG9753 product as an AdoR. Overexpression of the G protein subunit Gα_s or of the catalytic subunit of protein kinase A (PKA) partially mimicked and enhanced the defects caused by ectopic expression of AdoR. Our results suggest that AdoR is an essential part of the adenosine signaling pathway and Drosophila offers a unique opportunity to use genetic analysis to study conserved aspects of the adenosine signaling pathway.

Introduction

Adenosine is an endogenous nucleoside that modulates numerous physiological processes, including oxygen and metabolic balance in tissues (Berne, 1963; Costa and Biaggioni, 1998), immune responses (Sitkovsky and Lukashev, 2005) and signaling in the nervous system (Masino and Dulla, 2005; Fredholm et al., 2005). Most of these roles in mammals are mediated by interaction of adenosine with specific G protein-coupled receptors (GPCRs). As in other GPCRs adenosine receptors (AdoR) have seven membrane-spanning α-helices with an extracellular amino terminus and an intracellular carboxy-terminal tail (Murphree and Linden, 2004).

Four mammalian subtypes of the AdoR have been identified and their genes cloned: A1, A2A, A2B, and A3. They have been shown to modulate intracellular levels of adenosine 3′, 5′-cyclic monophosphate (cAMP) in different ways: A1 and A3 inhibit adenylate cyclase, whereas A2A and A2B stimulate this enzyme (van Calker et al., 1979; Londos et al., 1980). In some cells, such as human kidney epithelial cell line HEK293 or canine mast cells, the A2B receptors are also coupled to the calcium-mobilizing G protein subunit, Gα_q (Auchampach et al., 1997). AdoR subtypes are differentially distributed throughout the body (Murphree and Linden, 2004; Jacobson and Gao, 2006). The A1 AdoR is expressed in the brain, heart, adipose tissue, stomach, vas deferens, testis, spleen, kidney, aorta, liver, eye and bladder. The A2A receptor is highly expressed in parts of the brain (the striatum, nucleus accumbens and olfactory tubercles), in the spleen, thymus, immune cells, heart, lung and blood vessels. The A2B receptor is expressed at low levels in almost all tissues. The A3R is expressed at low levels in the thyroid gland, brain, liver, kidney, heart and intestine. The existence of four receptors with different functions but overlapping patterns of expression, together with the pervasiveness of adenosine-mediated physiological events, pose difficult questions in efforts to design pharmacological and biochemical interventions (Nyce, 1999). Moreover, the molecular dissection of AdoR signaling is difficult due to cross talk among various GPCR receptors (Fredholm et al., 2000; Werry et al., 2003). A better understanding of the adenosine signaling pathway would help in the elucidation of these mechanisms as well as in the development of strategies for the treatment of various human diseases, such as tachycardia, sleep disorders, immune and inflammatory disorders (for a review see Jacobson and Gao (2006)).

Understanding adenosine signaling could be advanced by the use of various genetic strategies in Drosophila. Accordingly, we have characterized a mutation that eliminates the major adenosine deaminase ADGF-A, and shown that this has pleiotropic effects on development associated with increased levels of adenosine and deoxyadenosine in the hemolymph (Dolezal et al., 2005). These phenotype includes larval and pupal lethality, developmental delay with block of pupariation, fat body disintegration and amplification of hemocytes. We have also generated a loss-of-function mutation in the CG9753 gene, which encodes a putative Drosophila AdoR (Brody and Cravchik 2000; Vanden Broeck 2001). The AdoR/ADGF-A double mutants show a less extreme phenotype than the ADGF-A null mutant (Dolezal et al., 2005) confirming that the two genes are functionally related. Here we show that CG9753 shares significant structural similarity with mammalian AdoRs, and that its activation leads to strong effects on intracellular cAMP and calcium signaling pathways.