Netrin-1-independent adenosine A2b receptor activation regulates the response of axons to netrin-1 by controlling cell surface levels of UNC5A receptors

Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKC alpha) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCa and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKC alpha-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKC alpha-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.