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Max-Planck-Institut für Experimentelle Medizin

3.         Diacylglycerol Signaling


The second messenger diacylglycerol (DAG), which is generated by members of the phospholipase C superfamily, does not only bind to and activate protein kinase C enzymes but also regulates a variety of other, as yet not well characterized target proteins (Brose and Rosenmund, 2002; Brose et al., 2004). The aim of our research is the characterization of neuronal non-PKC DAG receptors. Our main focus is on the functional characterization of neuronal Chimaerin proteins (RacGAP molecules) and members of the RasGRP family (RasGEF molecules). With our studies, we hope to contribute to a better understanding of DAG mediated cellular effects in neurons.

C1 domain proteins (Brose et al., 2004).



Chimaerin proteins constitute a family of Rac-specific GAP molecules, which are encoded in the mouse genome by two genes (a-Chimaerin, chn1; b-Chimaerin, chn2), each of which gives rise to the expression of at least two isoforms, a1/a2 and b1/b2. All Chimaerins contain a RacGAP domain and a regulatory DAG-binding C1 domain, while only a2- and b2-chimaerin contain an additional SH2 domain at their N termini. To elucidate the site, time point, and significance of Chimaerin-mediated Rac regulation in the mouse nervous system, we generated, in collaboration with the group of A. Betz at our institute, Chimaerin knock-out (pan-a, pan-b? and b2) and C1 domain knock-in mutant mice (a and b).

The analysis of a-Chimaerin mutant mice revealed that both, homozygous pan-a-Chimaerin knock-out and a-Chimaerin knock-in mice display a rabbit-like hopping gait upon the onset of locomotion. Instead of a normal alternating gait, these animals move first their forelimbs in parallel and then their hindlimbs in parallel. In a modified yeast two-hybrid (YTH) screen we identified the kinase domain of EphB1 as an interactor of a2-Chimaerin. Eph receptors constitute the largest family of receptor tyrosine kinases and signal upon activation to Rho family GTPases. This typically triggers cell-cell detachment and reorganization of the actin cytoskeleton, which ultimately results in growth cone collapse and axon retraction. Interestingly, mouse mutants with deficient EphA4-dependent forward signaling are characterized by aberrant wiring of neuronal networks that control coordinated limb movements, which in turn causes an unusual hopping gait. We therefore performed further YTH, in vitro binding, and neuronal overexpression assays and found that activated EphA4 binds to and phosphorylates a2-Chimaerin. Encouraged by these results we analyzed a-Chimaerin mutant mice for defects in EphA4-downstream signaling in collaboration with the laboratories of R. Klein (Martinsried, Germany) and K. Kullander (Uppsala, Sweden). In situ hybridization experiments revealed that a2-Chimaerin is spatially and temporally expressed in the same regions of the spinal cord and motor cortex as EphA4. Similar to EphA4 mutant mice with defective forward signaling, a-Chimaerin mutants show perturbations of the spinal cord architecture and dysfunction of the central pattern generator for hindlimb movement. Additionally, cultured cortical neurons from a-Chimaerin mutants exhibit a significantly reduced number of collapsed growth cones after EphA4 stimulation. With these studies we showed that a2-Chimaerin is a downstream effector of activated EphA4 and is essential for EphA4-dependent axon navigation during neuronal network formation in the mouse brain and spinal cord (Wegmeyer et al., 2007).

Hop gait and aberrant midline crossing of axons in alpha-Chimaerin knock-out mice (Wegmeyer et al., 2007).



RasGRP proteins display GEF-activity towards small GTPases of the Ras family. RasGRPs consist of a catalytic domain composed of a REM and a CDC25-like GEF domain, a DAG-binding C1 domain, and a pair of Ca2+-binding EF-hands. Despite the similarities in their protein structure, RasGRPs show differences in their tissue distributions and substrate specificities towards RasGTPases. Of the four identified RasGRP isoforms, only RasGRP1 and RasGRP2 (also called CalDAG-GEFI) are expressed in neurons of the murine central nervous system. Until now, the mode of action of RasGRP1 and RasGRP2 was analyzed only in non-neuronal tissues. RasGRP1 is known to couple T- and B-cell receptor stimulation and phospholipase C activation to Ras signaling, and is important for the proper development of T- and B-cells. RasGRP2 on the other hand, acts as an activator of Rap and is essential for platelet aggregation in mice. In order to analyze the function of neuronal RasGRPs we generated and are currently analyzing RasGRP1 and RasGRP2 mouse knock-out lines as well as RasGRP1 and RasGRP2 mouse C1 domain knock-in lines with mutations disrupting DAG-binding. These studies are being performed in collaboration with the group of A. Betz at our institute.

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