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 (PKCalpha) 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 PKCalpha 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 PKCalpha-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 PKCalpha-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.

Protein interacting with C-kinase 1/protein kinase Calpha-mediated endocytosis converts netrin-1-mediated repulsion to attraction.

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

Independent roles of SOCS-3 and SHP-2 in the regulation of neuronal gene expression by leukemia inhibitory factor.

The neurokine leukemia inhibitory factor (LIF) initiates signaling through heterodimerization of the low affinity LIF receptor (LIFR) and gp130. Tyrosine 759 of gp130 is required for the negative regulation of LIF-mediated signaling by both the protein tyrosine phosphatase SHP-2 and the suppressor of cytokine signaling-3 (SOCS-3). We find that SOCS-3 is expressed in the neuronal cell lines SN56 and IMR32 and negatively regulates LIF-stimulated neuronal gene expression. Studies using antisense oligonucleotides targeted to SHP-2 or SOCS-3 indicate that either protein can negatively regulate LIF-stimulated neuronal gene expression independently of the other. Mutagenesis of the cytoplasmic domain of gp130 demonstrates that the four signal transducer and activators of transcription (STAT) binding sites within gp130 are necessary for the induction of vasoactive intestinal peptide (VIP) and choline acetyltransferase (ChAT) reporter genes, with the sites surrounding tyrosines 905 and 915 (Y905 and Y915) being most important in gp130-mediated reporter gene expression. While there are four STAT binding sites within gp130, only those surrounding Y905 and Y915 can mediate STAT1 activation; these results indicate that STAT1 may be essential for normal gp130-stimulated VIP and ChAT expression. Additionally, the negative regulation of signaling mediated by Y759 of gp130 is dependent upon intact STAT sites within the receptor. This indicates that STAT signaling is necessary for LIF- and CNTF-stimulated VIP and ChAT expression and Y759 of gp130 mediates the activities of SHP-2 and SOCS-3, which act to negatively regulate STAT activity.