Cloning of rat ARHGAP4/C1, a RhoGAP family member expressed in the nervous system that colocalizes with the Golgi complex and microtubules

Abstract

The Rho GTPase family of intracellular molecular switches control multiple cellular functions via the regulation of the actin cytoskeleton. Increasing evidence implicates a critical involvement of these molecules in the nervous system, particularly during neuronal migration and polarity, axon and growth cone guidance, dendritic arborization and synaptic formation. However, the molecules regulating Rho GTPase activities in the nervous system are less known. Here, we present the cloning of rat ARHGAP4, a member of the Rho GTPase activating protein family, and also demonstrate its close linkage to the vasopressin 2 receptor gene. In vitro, recombinant ARHGAP4 stimulated the GTPase activity of three members of Rho GTPases, Rac1, Cdc42 and RhoA. ARHGAP4 mRNA expression was observed in multiple tissues with marked expression throughout the developing and adult nervous systems. On closer analysis of protein levels, ARHGAP4 was significantly restricted to specific regions in the nervous system. These included the stratum lucidem in the CA3 area of the hippocampus, neuronal fibers in the ventral region of the brainstem and striatum, and in the cerebellar granule cells. Subcellularly, endogenous ARHGAP4 expression localized to the Golgi complex and could redistribute to the microtubules, for example during mitosis. In addition, distinct protein expression was observed in the tips of differentiating neurites of PC12 cells. Collectively, these results demonstrate that ARHGAP4 is more widely expressed than previously thought but potentially possesses specialized activity in regulating members of the Rho GTPase family in specific cellular compartments of the nervous system. Published by Elsevier Science B.V. Theme: Cellular and molecular biologyTopic: Gene structure and function Keywords: RhoGAP; Nervous system; Golgi network; Microtubulin; cDNA cloning; In situ hybridization histochemistry and immunofluorescence . Introductionidentified thus far, Rho, Rac and Cdc42 remain the best characterized and are increasingly being shown to mediate Rho GTPases are members of the Ras superfamily of morphological changes during neuronal development and small GTP-binding molecules that regulate diverse cellular synaptic plasticity via the actin cytoskeleton (reviewed in processes including cell movement, growth and differentia- [29]). However, neuronal expression analyses of various tion (reviewed in [54]

ARHGAP4 is a novel RhoGAP that mediates inhibition of cell motility and axon outgrowth

“…The significant enhancement of outgrowth produced by the dominant-negative R562A mutant protein also suggests that the GAP domain of ARHGAP4 plays an important role in its regulation of motility. This inhibition of motility is consistent with the ability of ARHGAP4 to inhibit the function of Rac1 and Cdc42 (Foletta et al, 2002), which are RhoGTPases that are typically associated with enhancing axon outgrowth and cell motility (Govek et al, 2005;Negishi and Katoh, 2002).…”

“…4) and as previously reported (Foletta et al, 2002). However, it is unknown if its GAP function is important for the regulation of axon outgrowth or cell migration.…”

“…Endogenous ARHGAP4 has been shown to be expressed in NIH/3T3 fibroblasts, NRK epithelial cells and PC12 cells (Foletta et al, 2002). These data showed ARHGAP4 was localized to the golgi, along microtubules in NRK cells, and at the tips of extending neurites of NGF-treated (neuronally differentiated) PC12 cells.…”

“…ARHGAP4 cDNA was generated by polymerase chain reaction (PCR) using the pεMTH-ARHGAP4 plasmid as a template (Foletta et al 2002). Specific primers (5′-end primer: 5′-CTAGGCGGCCGCATGGCGGCGCACGGGAAGTTGCGG-3′; 3′-end primer: 5′-GCTAGAATTCGACTGGCTTGCGAGTTGAATCTGG-3′) were used to insert Not I and Eco RI restriction sites into the 5′ and 3′ ends, respectively, of the ARHGAP4 cDNA.…”

“…each with PBS. Primary antibodies used in this study were anti-GFAP (1:200, MP Biomedicals, Irvine, CA; or 1:1,000, Sigma, St. Louis, MO), rabbit polyclonal antibody anti-ARHGAP4 (1:1,000, (Foletta et al, 2002)), anti-β-Tubulin III (1:1,000, Sigma), M2 anti-FLAG (1:250-1:1000, Sigma), and JL8 anti-GFP (1: 1,000, BD Clontech). The cell adhesion molecule L1 was identified using a rabbit polyclonal antibody (1:1,000, gift from Dr. Vance.…”