␤-arrestins bind to G protein-coupled receptor kinase (GRK)-phosphorylated seven transmembrane receptors, desensitizing their activation of G proteins, while concurrently mediating receptor endocytosis, and some aspects of receptor signaling. We have used RNA interference to assess the roles of the four widely expressed isoforms of GRKs (GRK 2, 3, 5, and 6) in regulating ␤-arrestinmediated signaling to the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1͞2 by the angiotensin II type 1A receptor. Angiotensin II-stimulated receptor phosphorylation, ␤-arrestin recruitment, and receptor endocytosis are all mediated primarily by GRK2͞3. In contrast, inhibiting GRK 5 or 6 expression abolishes ␤-arrestin-mediated ERK activation, whereas lowering GRK 2 or 3 leads to an increase in this signaling. Consistent with these findings, ␤-arrestin-mediated ERK activation is enhanced by overexpression of GRK 5 and 6, and reciprocally diminished by GRK 2 and 3. These findings indicate distinct functional capabilities of ␤-arrestins bound to receptors phosphorylated by different classes of GRKs.angiotensin receptor ͉ extracellular signal-regulated kinase ͉ phosphorylation ͉ small interfering RNA

Netrins are a family of secreted molecules that are important for axonal outgrowth and guidance in the developing nervous system. However, the signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. Here we report that the netrin receptor DCC (deleted in colorectal cancer) interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK was expressed in developing brains and was localized with DCC in cultured neurons. Netrin-1 induced FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolished netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation.

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). We report here that the intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increased srGAP1-Robo1 interaction and inactivated Cdc42. A dominant negative srGAP1 blocked Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocked the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. We propose a signal transduction pathway from the extracellular guidance cue to intracellular actin polymerization.

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase C␥ (PLC␥) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.Proper wiring in developing brains requires neurite outgrowth and growth cone navigation. Netrins, a family of secreted factors, promote axon outgrowth (1-4). In addition, netrins are able to guide neuronal growth cones and regulate neuronal branching in the developing nervous system (1-4). Netrins act through two classes of receptors: DCC and Unc5. The DCC family includes DCC and neogenin in vertebrates (5), Unc40 in Caenorhabditis elegans (6), and Frazzled in Drosophila (7,8). DCC is required for the attractive response (9). Unc5 in C. elegans and Unc5A, -5B, and -5C in vertebrates belong to the Unc5 family, which appears to mediate the repulsive response (10 -14).DCC and Unc5 proteins are transmembrane proteins without any obvious catalytic activity, and thus it remains unknown exactly how they initiate downstream signaling to mediate or regulate axonal outgrowth and guidance. Nevertheless, perturbation of Rho family GTPases inhibits netrin-induced neurite outgrowth (15). Pharmacological inhibition of extracellular signal-regulated kinase (ERK) attenuates netrin-1-induced neurite outgrowth and growth cone turning (16,17). Inhibition of focal adhesion kinase (FAK), 3 a major tyrosine kinase localized in focal adhesions and implicated in cell spreading and migration, blocks netrin-1-induced neurite elongation and growth cone guidance (18 -20). Treatment of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, attenuates netrin-1-induced growth cone turning in Xenopus spinal neurons (27).Phosphoinositides are quantitatively minor phospholipids of cell membranes, but their metabolism is highly active and tightly regulated. They (e.g. PIP 2 ) function either as precursors of second messengers such as inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol or by directly interacting with both actin-binding and pleckstrin homology domaincontaining proteins to regulate their spatiotemporal distribution and/or activity. In addition, PIP 2 functions as a cofactor for small GTP-binding proteins (e.g. Arf) and phospholipase D (21, 22). In the nervous system, PIP 2 plays an important role in membrane trafficking at the synapse. Synaptic vesicle exocytosis and endocytosis require phosphatidylinositol 4,5-bisphosphate (23-26). Usin...