Adenosine receptor signaling and the activation of mitogen-activated protein kinases

Abstract: The nucleoside adenosine is present in all cells and body fluids of all living organisms; its production, both intracellularly and extracellularly, is tightly coupled to energy consumption resulting in increasing extracellular adenosine levels with increased energy consumption. Adenosine is metabolized by adenosine deaminase and adenosine kinase. Deamination leads to the production of inosine, which appears in concentrations similar to those of adenosine. Adenosine exerts modulatory effects on many body functions and is known to regulate, for example, the cardiovascular and central nervous system, pain modulation, mast cells and immune function. These physiological effects are mediated by four pharmacologically and biochemically distinct adenosine receptors, which belong to the family of G protein coupled receptors activating heterotrimeric G proteins. The adenosine A, and A3 receptors generally couple to Gj proteins, whereas the adenosine A2A and A2B receptors activate Gs proteins. In order to describe adenosine receptor pharmacology and signaling the recombinant human adenosine receptors were stably transfected into Chinese hamster ovary (CHO) cells, which normally do not express adenosine receptors. This is of advantage for the description of single adenosine receptor subtypes since sufficiently specific pharmacological tools are not available and since most cells express more than one adenosine receptor subtype. Furthermore, this recombinant cellular system allows the comparison of the human adenosine receptors in front of a similar cellular background. Human adenosine receptors are, indeed, activated by adenosine, whereas its metabolite inosine is only active at the adenosine A, and A3 receptor, and even there inosine has low potency and efficacy. Thus, adenosine is the main ligand for adenosine receptors. Moreover, adenosine A, and A2A receptors may be constitutively activated at basal adenosine levels (30 - 300 nM), whereas the A2B and A3 receptor are only activated when adenosine levels are increased. Inosine - at high concentrations - may have effects at the adenosine A3 receptor, at least where it is highly expressed. Adenosine affects cell growth positively and negatively. An important role in mitogenesis is played by the family of m itogen -activated protein kinases (MAPK) and we show that all adenosine receptors expressed in CHO cells mediate signaling to the MAPK extracellular signal-regulated kinase 1/2 (ERK1/2). ERK1/2 phosphorylation is time- and dose-dependently increased upon stimulation with the unspecific adenosine receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) and with the endogenous ligand adenosine. Adenosine receptors showed different efficacy in mediating ERK1/2 phosphorylation: A3>A1>A2A>A2B. The EC50 value for the adenosine A,, A2A, and A3 receptor mirrored that for effects on e.g. adenylyl cyclase, but NECA-induced ERK1/2 phosphorylation in CHO A2B cells was half-maximal already at 20 nM, compared to a half-maximal cAMP production at 1.4 muM. Due to the high efficacy and the high potency of agonists to induce ERK1/2 activation via the adenosine A3 and A2B receptor, respectively, we specifically examined these intracellular signaling pathways, using both pharmacological and molecular biological tools. Pharmacological tools such as kinase and adenylyl cyclase inhibitors, however, caused unexpected problems. Due to structural similarity, inhibitors binding to the ATP-binding site of such enzymes may also bind to and block the ligand-binding site of adenosine receptors, which could be shown by competitive radioligand binding experiments. The adenosine A3 receptor signals to ERK1/2 by the release of beta-gamma subunits from pertussis toxin sensitive G proteins, activation of phosphatidyl i nositol-3- kinase (P13K), the small GTP-binding protein Ras and the MAPKK MEK. The Gs-coupled adenosine A2B receptor, on the other hand, accomplishes ERK1/2 activation in a cAMP-dependent but cAMP-dependent protein kinase (PKA)-independent manner. Furthermore, this pathway uses P13K and transactivation of the epidermal growth factor receptor, whereas activation of the small GTP-binding protein Rap1 is not necessary for ERK1/2 activation. Signaling to the cAMP response element binding protein (CREB) and the stress-activated protein kinase p38 involves cAMP and PKA - independent of P13K. The molecular aspects of adenosine receptor signaling in vivo are still obscure, but the knowledge of intracellular signaling pathways activated by single adenosine receptor subtypes in recombinant cell systems may present some support for mechanisms in the in vivo situation.