The Expression Of SrGAPs In Rat Brain

Neuronal migration and axonal guidance are important events in development of the nervous system. It is crucial to identify the associated genes and reveal the signal transduction mechanisms. Slit and its membrane receptor, Robo, are important molecules known to guide neuronal migration and axonal extension. It has been proved that there are multiple intracellular pathways that transduce Slit-Robo signal. Rao et al, using the yeast two-hybrid system screened the mouse brain cDNA library in search for proteins interacting with Robo, and identified a novel family of Slit-Robo GTPase activating proteins(srGAPs). The novel family consists of srGAPl, srGAP2, and srGAP3, corresponding to the human KIAA1304, KIAA0456, KIAA0411, respectively. GAPs can stimulate the small intrinsic GTPase proteins , thereby convert it into inactive GDP-bound form. The results of Rao et al indicate that srGAPl is an intracellular effector of Robol, an important component of the pathway mediating the repulsive signaling of Slit-Robo. The extracellular binding of Slit and Robol increase the interaction of srGAPl with Robol, resulting in activation of the srGAPl which inactivates CDC42, a member of small Rho GTPase family, leading to promotion of actin depolymerization. Asymmetry of actin protein results in changes of direction of theneuronal migration and axonal extension. Besides, the study by Endris et al shows that srGAPs have putative roles in severe mental retardation, indicating the complexity of the roles srGAPs may play in the brain.Rao et al studied with in situ hybridization the distribution of srGAP mRNAs in CNS. The distribution of srGAP proteins in CNS, however, has not been investigated. We, thus raised a rabbit polyclonal antibody against purified GST-srGAPs, and its specificity was characterized with antigen absorption test. We investigated the expression patterns of srGAPs in the normal rat brains at adult and different developmental stages by immunohistochemistry.We found that in adult rat brain the distribution of different members of the srGAP family differed. srGAP 1 and srGAP3 were found to be extensively expressed, whereas no srGAP2 could not be detected. srGAP 1 and srGAP3 were identified only in neurons but not in astrocytes or oligodendrocytes. The subcellular localization of srGAP 1 and srGAP3 was completely different. srGAP 1 was distributed in the cytoplasm, whereas srGAP3 was mainly located in the nucleus.During development, all three molecules of srGAP family were found to be extensively expressed in neurons. Subcellularly, srGAP 1 was distributed in the nucleus at P1 and gradually translocated to the cytoplasm. It appeared in the both cytoplasm and nucleus at P7 and P14. As for srGAP2, it was distributed in the cytoplasm at P1 and P7, and in the cytoplasm and nucleus at P14. The subcellular localization of srGAP3 in developmental process changed obviously, it was distributed mainly in the cytoplasm at P1 and can be detected in the both cytoplasm and nucleus at P7 and P14. The intensity inhippocampus formation in development also changed. The expression of srGAP3 in hippocampus and dentate gyrus at P1 was both obscure. The expression in hippocampus was enhanced markedly at P7 and maintained till adult. The expression in dentate gyrus was enhanced from P14 and highest at adult stage.The results indicate that there may be other important functions of srGAPs in adult rat brain besides guiding axonal extention and neuronal migration in developmental stage, such as synapse plasticity. Different subcellular localization of different members of srGAP family may indicate their diverse functions. Changes of subcellular localization of srGAPs in developmental process indicate that the expression of srGAPs in developmental process is strictly regulated temporally and spatially. srGAPs, together with their downstream specific Rho molecules, form complex signal network that integrate inputs from a number of different signal cues in the surroundings and respond rapidly and accurately, which ensure effi...