Stimulation of the Mitogen-Activated Protein Kinase by the A2A-Adenosine Receptor: Identification of the Signaling Pathway in Human Endothelial Cells and Stable Cell Lines

Under physiological conditions, de novo formation of blood vessels from preexisting capillaries (=angiogenesis), is intimitately involved in tissue repair (e.g. posttraumatic or in localized inflammatory reactions). Angiogenesis is, however, also a prerequisite for the growth of solid tumours and their metastases and is also involved in diabetie retinopathy or in the chronic inflammatory response that leads to cartilage destmetion in rheumatoid arthritis. During angiogenesis, quiescent endothelial cells are recruited into the cell cycle by growth factors. In previous work, we have shown that adenosine stimulates endothelial proliferation; this mitogenic effect is elicited by an A2A-adenosine receptor-mediated stimulation of MAP kinase (mitogen-activated protein kinase). The A 2A- adenosine receptor is a prototypical Gs -coupled receptor and, thus, activates adenylyl cyclase and - via the resulting cAMP elevation protein kinase A (PKA). It is unclear, whether this signalling cascade is upstrearn of MAP kinase stimulation. PKA can activate (via the GTP-binding protein rapl and the rafB-kinase) and suppress MAP kinase (via inhibition of the interaction between p21ras and the raf1-kinase). Both effects are observed in transfected cell lines. In endothelial cells, MAP kinase is not activated after transient, direct stimulation of PKA by membrane-permeable cAMP-analogues and persistent actiavtion of PKA results in growth inhibition. These findings and related observations argue for an additional signalling pathway that links the endothelial A2A- adenosine receptor to cell proliferation. The current grant application aims at identifying the components of this additional signalling cascade; we intend to test three models, which are supported by evidence from preliminary experiments, in endothelial cells and in stably transfected cell lines: (i) The A2A-adenosin receptor activates MAP kinase via a signalling pathway that is entirely independent of G s - cAMP-PKA; this requires the interaction with a G protein other than G s . (ii) The mechanism of MAP kinase activation is analogous to that recently described for the b 2 -adrenergic receptor, i. e. via PKA-mediated phosphorylation of the receptor; this modification switches the G protein specificity of the receptor, such that it interacts with G i (or another G protein) und thereby stimulates thecascade upstream of MAP kinase. (iii) The stimulation of MAPK is achieved via the signal pathway composed of PKA - rap1-rafB. This is in contrast to the observation that membrane-permeable cAMP-analogues fail to activate MAP kinase. This discrepancy may arise from intracellular compartimentalisation of cAMP generated by receptor activation. These models can be differentiated by introducing plasmids that encode mutated, dominant negativ versions of signalling proteins (e.g. G proteins, sos, p21ras etc.). Thus, the aims of the current grant application are to define the signalling cascade(s) that link(s) the A 2A-adenosine receptor to MAP kinase activation and to understand the process of signal integration between the cAMP- and the tyrosine kinase-dependent regulation of MAP kinase. These pathways play a pivotal role in the control of cell proliferation; hence, the results can be expected to be of general interest from the point of view of basic research. Given that endothelial cell proliferation is a prerequisite for angiogenesis, we believe that insights into the mechanism by which the A2A-adenosine receptor stimulates endothelial cell proliferation will be, in the long-term, of relevance to human pharmacotherapy.

G proteins play a central role as molecular switches. More than 1000 individual human cell- surface receptors signal via G proteins (the hormone or neurotransmitter is on the outside - a biological response has to generated within the cell based on computational integration of the available information). The A2A-adenosine receptor is a prototypical G protein-coupled receptor. Adenosine is a locally produced tissue hormone (autacoid) and affects the function of almost all tissues and organs (via interaction with 4 subtypes of receptors). Adenosine is released in large amounts by hypoxic tissues and is considered a candidate mediator of angiogenesis (=de novo formation of blood vessels from preexisting capillary endothelium). Preceding projects documented the mitogenic action of adenosine on endothelial cells (= increase in proliferation). The current grant application was based on these earlier findings and aimed at identifying the signalling pathways that mediate the mitogenic effect. The experiments demonstrated that at least two signalling pathways were required, namely the stimulation of p70S6 kinase and of mitogen-activated protein kinase (MAP kinase); the observations also showed that the A 2A- receptor impinges on MAP kinase via two distinct pathways. These depend on the cell type in which the receptor is expressed and include a cAMP- und rap1-dependent cascade as well as a pathway that relies on p21ras but that is independent of Gs and cAMP. In fact, at the current stage, it is most reasonable to conclude that this second pathway does not require any heterotrimeric G protein. Recently, numerous additional proteins (other than G proteins) have been identified that bind directly to G protein-coupled receptors. These accessory proteins also participate in signalling by receptors. In collaboration with Dr. Betz (Max-Planck Institut Frankfurt) we showed that the Ca2+-binding protein calmodulin is such an accessory protein: in group III metabotropic glutamate receptors (in particular mGluR-7), the interaction between receptor and calmodulin supports coincidence detection; the presence of agonist and the binding of calmodulin are both required for signalling. In contrast, binding of calmodulin to the D2 -dopamine receptor inhibits agonist-dependent signalling. Finally, we also observed the recruitment of multiple signalling pathways by other P-receptor family members (P 1 = adenosine receptors; P2 = nucleotide receptors). Accordingly, the current project achieved the goals originally defined in the grant application in a rather satisfactory way; we obtained new and - in part - surprising insights into the mechanisms of signal transduction. The resulting publications have already been met with some interest in the field (cf. Science Citation Index). The collaboration with Dr. Betz will be continued in a EU-sponsor concerted action. From the perspective of technology and methodology, we consider our establishing FRET-microscopy as the most innovative aspect of this funding period. FRET-microscopy allows to follow protein-protein interactions in living cells. This addition to our methodological repertoire provides for a competitive edge that will be instrumental in keeping the traditional strength of basic research in pharmacology in Austria.