Identification and functional characterization of nadrin variants, a novel family of GTPase activating protein for rho GTPases

Abstract

Nadrin is a GTPase-activating protein (GAP) for the rho family of GTPases that controls Ca 2+ -dependent exocytosis in nerve endings. In this study, three novel splice variants of nadrin were identified and the variants were designated as nadrin-102, -104, -116 and -126 according to their relative molecular masses. All nadrin variants share the GAP domain, coiled-coil domain, serine/threonine/proline-rich domain, SH3-binding motif, and a successive repeat of 29 glutamines. Tissue distribution analyses using polyclonal antibodies that can discriminate each variant showed that the expression of nadrin-102, -104 and -116 was dominant in neuronal tissues and correlates well with the differentiation of neurons while nadrin-126 was strongly expressed in embryonic brain. Expression of nadrin-116 in PC12 cells strongly inhibited NGF-dependent neurite outgrowth and this effect was dependent on its GAP activity. In contrast, no significant effect on either cell morphology or neurite outgrowth was observed with other variants. All variants showed punctate appearance throughout the cytoplasm, while the 66-kDa carboxyl-terminal fragment of nadrin-102 and/or nadrin-116 was localized to the nucleus and its nuclear translocation was accelerated by NGF-induced differentiation of the cells. These results suggested that nadrin variants are different in their ability to regulate rho-mediated signaling and that, in addition to being a GTPase-activating protein, nadrin-102 and -116 have other distinct functions in the nucleus of the cell, implying a possible role in the cross-talk between the cytoskeleton and the nucleus.

Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation

“…Because PACSIN proteins lack a RhoGAP domain we asked whether the presence of a GTPase-activating protein (GAP) may be required in the PACSIN 1-Rac1 complex to mediate Rac1 activity. A previous study identified NADRIN (also named RICH1) as a Rac1 GAP, which also interacts with the SH3 domain of PACSIN 1 (32,33). We confirmed the binding of PACSIN 1 to NADRIN (Fig.…”

Characterization of a Novel GTPase-activating Protein Associated with Focal Adhesions and the Actin Cytoskeleton

“…Transfected cells lost actin stress fibers, retracted from the coverslip, and extended long filopodia, ruffles, and lamellipodia. Such a destructive effect on the actin cytoskeleton has been shown for several RhoGAP proteins (11,12,48,53,55). One of the accepted explanations for this phenomenon is inactivation of Rho family GTPases by GAP proteins, based on the established physiological roles of Rho GTPases in the regulation of the actin cytoskeleton.…”

“…tion. For example, diffuse cytoplasmic localization has been reported for RICH-1 (25); ␤-chimaerin, ARHGAP4, and IQGAP are associated with the Golgi apparatus (45)(46)(47); IQGAP and nadrins have been observed in the nucleus (47,48); and ARH-GAP4 and PSGAP show perinuclear localization (46,49). Some RhoGAP proteins co-localize with different structures of the actin cytoskeleton, which is their primary cytoskeletal target.…”

“…Some RhoGAP proteins co-localize with different structures of the actin cytoskeleton, which is their primary cytoskeletal target. ArhGAP15, GRAF, and Grit/RICS/p200RhoGAP are associated with cortical actin (14,18,50), p190RhoGAP, ARHGAP6, CdGAP, and IQGAP colocalize with filopodia and lamellipodia (12,33,35,51), and Grit/RICS/p200RhoGAP, nadrins, and oligophrenin are associated with neurite tips (48,50,52). The co-localization of RhoGAP proteins to stress fibers has been reported for N-chimaerin, oligophrenin1, and GRAF (14,52,53).…”

The role of the Rho GTPases in neuronal development

“…NGF-induced Rac and Cdc42 activation and neurite formation may be further modulated by a specific splice variant of the RhoGAP Nadrin, Nadrin-116. Nadrin-116 inhibits NGF-induced neurite outgrowth in PC12 cells, which is dependent on Nadrin's GAP activity, suggesting a Rac-or Cdc42-directed preference for this molecule in relation to neurite formation (Furuta et al 2002).…”