The collapsin response mediator protein (CRMP) family of phosphoproteins was first characterized on the basis of their requirement for Sema3A signaling. CRMPs, also known as TOAD (turned on after division), Ulip (unc-33 like protein) or DRP (dihydropyrimidinase related protein) and are highly expressed in the nervous system. CRMPs are homologues of Caenorhabditis elegans unc-33, whose mutations cause abnormal axon growth and guidance
as well as uncoordinated movements (Li et al., 1992), demonstrating a role for these molecules in normal neuronal development and function. Currently there are five known members in the vertebrate CRMP family (CRMP1-4, and CRAM/CRMP5) (Fukada et al., 2000), of which CRMP2 is the most widely studied. Although CRMPs were initially reported to be expressed exclusively in the developing nervous system, subsequent analyses have revealed that they are also expressed in non-neuronal tissues, albeit at much lower levels (Shih et al., 2001).
2.3.1 CRMP2 switches RhoA and Rac1 morphology
Axon guidance is essential for the complexity of brain circuitry. Growth cones are thought to be a sensor for guidance molecules during development. They are localized at the tips of axons and dynamically change their morphology in response to attractive and repulsive guidance cues, thus determining the direction of growth (Dent and Gertler, 2003). The formation and directional guidance of neurites involves a dynamic and coordinated regulation of Rho family GTPases. Activation of Rac1 and Cdc42 promotes neurite outgrowth, whereas Rho activation causes neurite retraction. However, recent data have indicated that not activation itself, but the balance of Rho GTPase activities is the most important factor in the regulation of neurite outgrowth (Koh, 2006). By inhibition of Rac1, as well as by expression of dominant active Rac1, neurite outgrowth was reduced, showing that just the right amount of GTPase activity is required for neurite outgrowth (Koh, 2006). This coordinated activation of Rac1 and RhoA that is required for neurite outgrowth may be provided by CRMP2.
CRMP2 is implicated in axonal outgrowth and is a component of the semaphorin 3A (Sema 3A) pathway (Goshima et al., 1995). Sema3A leads to growth cone collapse by activation of the Neurophilin-1/Plexin-A1 receptor complex that stimulates phosphorylation of cofilin, a protein that regulates actin filament assembly (see Figure 9B) (Liu and Strittmatter, 2001). Rho kinase (Rock II), a serin-threonine kinase and probably the most important effector of RhoA in growth cones, binds to and is activated by the GTP-bound active form of RhoA ((Matsui et al., 1996), (Ishizaki et al., 1996), (Amano et al., 1997), (Arimura et al., 2000)). In
the brain, CRMP2 was found to be a prominent substrate of Rock II. It is phosphorylated by Rock II at Thr-555 in response to lysophosphatidic acid (LPA) signaling or by activation of Ephrin-5A ((Shamah et al., 2001), (Knoll and Drescher, 2004) (Sahin et al., 2005)), but not after Sema3A signaling (Arimura et al., 2000). In the case of Sema3A-induced growth cone collapse, phosphorylation of CRMP2 by Cdk5 (Ser-522) and GSK-3β (Thr-514 and Ser-518) was reported ((Brown et al., 2004), (Cole et al., 2004), (Uchida et al., 2005), (Yoshimura et al., 2005)).
Arimura and colleges (Arimura et al., 2000) therefore proposed that there may be Rock II- dependent and -independent pathways leading to growth cone collapse (Figure 8A). The role of CRMP2 in Sema3A-induced axonal outgrowth was investigated by Hall and colleges in neuroblastoma cells (Hall et al., 2001). They showed that CRMP2 switches GTPase signaling when expressed in combination with either dominant active Rac1 or RhoA, respectively. Co- expression of CRMP2 with dominant active RhoA V14 induced Rac1 morphology (cell spreading and ruffling, formation of neurites) while co-expression with dominant active Rac1 V12 inhibited Rac1 morphology (cell rounding, neurite retraction). They also observed that Rock II is a pivotal regulator of CRMP2 in neuroblastoma cells. While CRMP2 phosphorylation was required for CRMP2/Rac1 V12 inhibition, it was not necessary for the induction of Rac1 morphology by CRMP2/RhoA V14. CRMP2, regulated by Rock II, thereby was shown to promote outgrowth and collapse in response to active RhoA and Rac1, respectively, reversing their usually observed morphological effects. As a reversible switch between RhoA and Rac1 signaling pathways, CRMP2 thereby provides a mechanism for dynamic modulation of growth cone guidance (Hall et al., 2001). Sema3A-induced growth cone collapse in sensory neurons is Rac1-dependent ((Jin and Strittmatter, 1997), (Vastrik et al., 1999)) and the results from Hall and colleges suggest that CRMP2 can initiate growth cone collapse downstream of Rac1 activation (Hall et al., 2001).
While growth cone collapse and outgrowth is thought to be regulated via the actin cytoskeleton, CRMP2 was also identified as a regulator of an other major cytoskeletal component, the microtubules (Fukata et al., 2002). CRMP2 is thought to function as a carrier of tubulin heterodimers to the plus end nucleating sites of growing microtubules. It was also shown to bind to tubulin dimers with higher affinity than to microtubules. Furthermore CRMP2 promotes microtubule assembly by copolymerizing together with tubulin dimers into microtubules.
Another element that is essential for axonal growth and maintenance is the transport of tubulin and microtubules in the growing axon. Although the molecular mechanism underlying
the linkage of tubulin and microtubules to motor proteins is not yet clear, it has been shown that a CRMP2/Kinesin-1 complex regulates soluble tubulin transport to the distal part of the growing axon (Kimura et al., 2005). Figure 8B shows the result of sucrose-density gradient centrifugation (sdgc) in order to isolate putative native protein complexes from porcine rod outer segments (ROS). This work by Magdalena Swiatek-de Lange in our lab, provided the first hint that the conserved signaling network RhoA/CRMP2/tubulin may also be existent in ROS. The occurrence of rhodopsin in the same sdgc fractions may indicate that rhodopsin is an integral part of this signaling network, suggesting a possible light-regulation of the RhoA/CRMP2/tubulin that was further investigated in this study.
Figure 8: A) Schematic representation of signal transduction pathways involved in growth cone collapse (Liu and
Strittmatter, 2001). a) Activation of the EphA receptor leads to RhoA activation through the GEF Ephexin, and concomitant downregulation of Rac1 activity. b) The serum factor lysophosphatidic acid (LPA) induces growth cone collapse and neurite retraction. LPA signals trough a seven transmembrane receptor that triggers activation of RhoA, leading to activation of Rho kinase, phosphorylation of CRMP2 and growth cone collapse. c) Sema3A signaling initiates clustering of the receptors Plexin and Neurophilin. In a CRMP2-dependent process, this clustering leads to alterations of a Rac1-dependent pathway that modulates the actin filament assembly in the growth cone. B)
SDS-PAGE of fractions from sucrose-density gradient centrifugation of porcine rod outer segments (ROS). Proteins were identified by mass spectrometry. All proteins co-segregating in one fraction may belong to a common protein complex. In ROS, CRMP2, tubulin and RhoA were found in a rhodopsin-associated complex (Swiatek-de Lange in Prep.). This links the light-percepting molecule rhodopsin, a seven transmembrane receptor like the LPA-receptor, with known components responsible for neuronal structure, morphology and polarity
3. PHOSPHODIESTERASES AND PDEδ