In the present study we examined the localization and signaling properties of the RPTK, Tyro3. This receptor is detected throughout the mature cortex and is also prominently expressed in the CA1 field of hippocampus. One of the most intriguing observations to emerge from this study was that the Tyro3 protein appeared as punctae distributed throughout the dendritic compartment of cortical and hippocampal neurons. The punctate pattern suggested that the receptors were clustered and further prompted the question of whether these Tyro3 clusters may be localized to the synapse. The punctae were observed in both early (1 DIV) and later stage (8–20 DIV) cultures, implying that receptor clusters were present throughout neuronal development and at times prior to the formation of synapses. A number of dendritic proteins have been identified that exhibit a punctate pattern of expression. These include proteins enriched in the postsynaptic density such as PSD95, Shank, and the NMDA receptor subunit NR1 (Sheng and Kim, 2002
). Tyro3 exhibited limited co-localization with PSD-95, suggesting that this receptor is primarily located outside of the postsynaptic density. In addition, Tyro3 did not appear to co-localize with GABA-A receptors (A.L.P. unpublished observations), which have been described as forming large clusters at synaptic sites and smaller clusters at extrasynaptic sites (Craig et al., 1994
, Christie et al., 2002
). Tyro3 also showed limited colocalization with the GABAergic presynaptic marker GAD-65 (), which is preferentially localized to presynaptic GABAergic boutons (Benson et al., 1994
). We have been unable to determine unequivocally whether Tyro3 is localized to the mature presynaptic terminal because the optimal staining conditions required for detecting this receptor are incompatible with those required to detect the presynaptic markers synaptophysin and synapsin. An ultrastructural analysis will be required to determine whether Tyro3 is present at presynaptic and postsynaptic sites. However, based on the limited co-localization with PSD-95 at the postsynaptic compartment and GAD-65 at presynaptic terminals, Tyro3 does not appear to be primarily localized to the synapse.
Several molecules have been identified that, like Tyro3, exhibit a punctate but largely non-synaptic distribution in dendrites in vitro
. It is important to note that despite being located outside of the synapse, these molecules are capable of affecting signaling events initiated by neuronal synaptic activity. These include the signaling molecule p70S6K (Cammalleri et al., 2003
) as well as components of the protein translation machinery important for activity-dependent protein synthesis (Sutton and Schuman, 2006
) including the proteins staufen (Kiebler et al., 1999
) and pumilio 2 (Vessey et al., 2006
). In addition, clusters of the RPTK trkB have also been detected in the dendrites and axons of cortical neurons in vitro
both before and after synapse formation. However, the localization of trkB differs from that of Tyro3 in that it becomes increasingly enriched at synapses as development proceeds. In non-synaptic sites trkB was detected in both stationary and mobile clusters, the latter being consistent with possible transport in synaptic vesicles (Gomes et al., 2006
). These examples highlight the important issue that molecules localized or mobilized to the dendrites but located outside of the synapse have the ability to influence activity-dependent signaling.
Tyro3 was present not only in dendrites but also in growth cones as well as in the axonal compartment, suggesting a possible role for Tyro3 in axonal pathfinding. A role in cell adhesion and cell migration has been previously suggested for Axl (Bellosta et al., 1995
). In addition, Gas6 signaling through Axl has been shown to promote the migration of vascular muscle endothelial cells (Fridell et al., 1998
). At the structural level, TAM family members contain Ig-like domains and FN-type III repeats, motifs that are also present in molecules known to mediate cell-cell interactions such as the neural cell adhesion molecule N-CAM (Prieto and Crossin, 1995
, Kamiguchi and Lemmon, 2000
), members of the Robo receptor family that mediate the repulsive actions of the slit molecules on axons, and the deleted in colorectal cancer (DCC) receptor which mediates the attractive effects of netrins on axons (Chisholm and Tessier-Lavigne, 1999
). These observations and the prominent presence of Tyro3 in the growth cone raise the possibility that Tyro3 may also play a role in axonal pathfinding.
In addition to being expressed in neurons, Tyro3 is also expressed in glial cells. In conjunction with our earlier efforts (Prieto et al., 2000
), it is now evident that Tyro3 is expressed in radial glia, astrocytes and oligodendroglia. It appears that glia express more than one member of the TAM family since Axl is co-expressed with Tyro3 in Schwann cells where Gas6 induces cell proliferation (Li et al., 1996
). Axl was also detected in oligodendroglia (Prieto et al., 2000
, Shankar et al., 2003
). Axl has also been identified in microglia (Funakoshi et al., 2002
) and in human malignant glioma cells (Vajkoczy et al., 2006
). Mer has been detected in developing oligodendrocytes (Prieto et al., 2000
) and in microglia (our unpublished observations). It will be important to determine the relative expression of each of the TAM receptors in these cell types as it has been reported that the levels of Tyro3 and Mer protein are co-regulated in retinal epithelial cells (Prasad et al., 2006
Our previous studies have indicated that Gas6 mRNA and protein are expressed throughout the rat cortex and in most pyramidal cells in the hippocampus, as well as in a number of other regions in the CNS. In contrast, the expression of protein S mRNA was highly restricted with low levels of expression observed in the locus coeruleus and high levels in the choroid plexus (Prieto et al., 1999
). In the rabbit brain, expression of protein S mRNA and protein has been reported in the cortex, hippocampal pyramidal neurons and granule neurons of the dentate gyrus (He et al., 1995
). Protein S has also been detected in cells of the glial lineage in vitro
, including several glioblastomas and one neuroblastoma cell line (Phillips et al., 1993
). Given the increased interest in the role of protein S as a TAM family ligand, it would be important to address these apparent discrepancies by evaluating the expression profiles of protein S and Gas6 in additional species, and by further exploring how the postranslational modifications of these gene products may influence their signaling abilities.
The presence of Tyro3 in dendrites, a compartment critical for integrating synaptic inputs, suggests that it may initiate signaling events capable of modulating activity-dependent neuronal plasticity. In support of this hypothesis, it has been reported that young adult Tyro3 knockout mice exhibit a decrease in LTP (Lemke and Lu, 2003
). Our findings have revealed that Gas6 stimulation of Tyro3 is capable of activating several components of the MAPK and PI(3)K signaling pathways, both of which play key roles in the molecular events underlying neuronal plasticity (Thomas and Huganir, 2004
). Therefore, the other major finding of this study is that Tyro3 may serve as a heretofore unrecognized modulator of MAPK and PI(3)K signaling in neuronal dendrites.
We have demonstrated that Gas6 can activate the MAPK pathway through the phosphorylation of two MAP kinases, ERK 1 and 2 (see ). Gas6 has been previously shown to induce ERK1/2 phosphorylation in mature mouse osteoclasts (Katagiri et al., 2001
). One possible mechanism of MAPK/Erk1/2 activation is by the direct or indirect recruitment of Grb2 by RPTKs (Kouhara et al., 1997
). Our data have indicated that Grb2 can directly interact with Tyro3 (), providing one avenue for the activation of the MAPK pathway. In neurons, activation of the MAPK signaling pathways through Grb2 has been reported for several members of the Eph (Pasquale, 2005
) and trk RPTK families (Reichardt, 2006
What are the possible targets of Gas6-induced ERK1/2 activation? Two candidates have been identified in this study, RSK and CREB. RSK has been previously shown to regulate CREB phosphorylation at ser133 in response to growth factors such as EGF and the neurotrophins (Ginty et al., 1994
), and we have observed that Gas6 activation also leads to phosphorylation on this residue. CREB and its coactivators have been shown to function as molecular regulators of gene expression leading to the transcriptional regulation of a large set of genes (West et al., 2001
) implicated in the alteration of complex processes such as the consolidation of memories, addictive behaviors, and circadian rhythms (Lonze and Ginty, 2002
). Additional studies will be required to determine the functional consequences of ser133CREB phosphorylation in response to Gas6.
Our studies have also demonstrated that Gas6 activates multiple components the PI(3)K pathway in neurons. Tyro3 bears a single consensus binding site for the p85 regulatory subunit of PI(3)K. Although we have been able to detect a direct association between Tyro3 and p85 in a fibroblastic cell line overexpressing p85 (not shown), we have been unable to demonstrate a direct association in neurons. Thus the precise mechanism of the Gas6-mediated PI(3)K activation in neurons is not understood and it is conceivable that the PI(3)K signaling pathway is being activated indirectly, possibly through ras (Cantley, 2002
We have also detected Gas6/Tyro3 mediated activation of three other members of the PI(3)K signaling pathway, AKT, which along with PDK1 serves as one of the main targets of PI(3)K (Cantley, 2002
), mTOR and p70S6K which can act downstream of AKT. What are the potential consequences in cortical neurons of Tyro3 activation of these molecules? One possibility, although speculative, is that Tyro3 activation can regulate protein translation, since activated mTOR can phosphorylate and activate p70S6K, which in turn phosphorylates the S6 ribosomal protein (Shaw and Cantley, 2006
). mTOR can also phosphorylate inhibitors of translation such as 4E-BP1, resulting in enhanced translation.
In neurons, the PI(3)K pathway has also been implicated as a modulator of synaptic plasticity (Sweatt, 2001
, Opazo et al., 2003
). Activity-dependent protein translation is mediated by p70S6K in the dendritic compartment even though this kinase is primarily found in the dendritic shaft and to a lesser extent in the dendritic spines (Aakalu et al., 2001
, Cammalleri et al., 2003
). It should be noted that ERK1/2 activated by Gas6/Tyro3 could also induce mTOR activation. The relative contribution of the MAPK and AKT pathways to the Gas6-mediated activation of mTOR remains to be established. The ability of Gas6 to stimulate the phosphorylation of AKT/mTOR/p70S6kinase suggests a potential role for Tyro3 as a regulator of translation in dendrites.
In summary, our studies have revealed that Tyro3 is a receptor protein tyrosine kinase expressed in dendrites that is capable of activating signaling pathways known to modulate synaptic plasticity. These findings suggest that Gas6 activation of Tyro3 may influence synaptic transmission.