Genetic dissection of plexin signaling in vivo

Abstract

Mammalian plexins constitute a family of transmembrane receptors for semaphorins and represent critical regulators of various processes during development of the nervous, cardiovascular, skeletal, and renal system. In vitro studies have shown that plexins exert their effects via an intracellular R-Ras/M-Ras GTPase-activating protein (GAP) domain or by activation of RhoA through interaction with Rho guanine nucleotide exchange factor proteins. However, which of these signaling pathways are relevant for plexin functions in vivo is largely unknown. Using an allelic series of transgenic mice, we show that the GAP domain of plexins constitutes their key signaling module during development. Mice in which endogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versions harboring mutations in the GAP domain recapitulate the phenotypes of the respective null mutants in the developing nervous, vascular, and skeletal system. We further provide genetic evidence that, unexpectedly, the GAP domain-mediated developmental functions of plexins are not brought about via R-Ras and M-Ras inactivation. In contrast to the GAP domain mutants, Plexin-B2 transgenic mice defective in Rho guanine nucleotide exchange factor binding are viable and fertile but exhibit abnormal development of the liver vasculature. Our genetic analyses uncover the in vivo contextdependence and functional specificity of individual plexin-mediated signaling pathways during development.neural tube | cerebellum | outflow tract P lexins constitute a family of transmembrane proteins that serve as receptors for semaphorins (1). They function as key regulators of a multitude of developmental processes, including axon guidance, pattern and synapse formation in the nervous system (2), vasculogenesis and angiogenesis (3, 4), and morphogenesis of the heart, kidney, and skeletal system (5). In the adult organism, plexins play crucial roles in the physiology and pathophysiology of the immune and cardiovascular system, as well as in bone homeostasis and in cancer (6-9). Nine plexins have been identified in the mammalian system, which are grouped into four subfamilies, A-D, according to sequence homologies.The activation of plexins by their semaphorin ligands triggers several intracellular signaling cascades, most of which modulate the activity of small GTPases (10). The intracellular domain of all plexins shares homology with GTPase-activating proteins (GAPs) and confers the deactivation of R-Ras, M-Ras, and Rap1 (11-17). The GAP activity toward R-Ras and M-Ras, but not toward Rap1, requires binding of Rnd GTPases to the plexin receptor (11,12,15,16). Plexins of the B-subfamily differ from all other plexins in that they carry a C-terminal PDZ domain interaction motif that mediates a stable interaction with the Rho guanine nucleotide exchange factor (RhoGEF) proteins . Activation of B-plexins by semaphorin ligands results in activation of the RhoGEF proteins and subsequent activation of . This process and the GAP function of B-plexins are independent of each other, becau...

Sema3e/Plexin D1 Modulates Immunological Synapse and Migration of Thymocytes by Rap1 Inhibition

“…However, recent biochemical and structural studies demonstrated that the recombinant GAP domain of plexins promoted GTP hydrolysis of recombinant Rap1 but not R-Ras (20). A functional GAP domain and GAP activity were required for proper early embryogenesis in BAC transgenic mice that expressed plexin B1 and D1 (21). In this study, we demonstrated sema3e-mediated inhibition of Rap1 activation and LFA-1-dependent adhesion directly through the Rap1 GAP activity of plexin D1.…”

“…A recent in vitro biochemical study demonstrated the selective GAP activity of plexin D1 for Rap1 rather than for R-Ras/M-Ras (20). Interestingly, the GAP activity of plexin D1 functioned independently of R-Ras and M-Ras activity during cardiovascular development and skeletal morphogenesis throughout embryogenesis (21). Sema3e/plexin D1 was also reported to be important for thymocyte positioning via regulation of b 1 integrin activation (22)(23)(24).…”

GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis

“…Functionally, sinusoidal dilatation and enhanced angiogenesis as seen in endothelial Notch signaling-deficient LSECs may promote hepatic metastasis (38). Notably, sinusoidal dilatation is a common end point of several signaling defects in LSECs; for example, plexin B2-transgenic mice defective in Rho guanine nucleotide exchange factor binding show sinusoidal dilatation and further abnormalities in the architecture of the liver vasculature (39). Recently, LXRα has This GATA4-dependent transcriptional program determines the functional competence of the hepatic sinusoidal endothelium.…”

The role of the semaphorins in cancer

“…[25][26][27] In the cases of plexin-D1 and plexin-B1 it was demonstrated that most of the developmental effects of these plexins are lost if the function of this GAP domain is compromised. 28 Type-A plexins associate spontaneously to form homodimers 11,12 or heterodimers. 29 Recent data indicates that activation of plexin signaling by semaphorins that bind directly to plexins such as sema6A is likely to be associated with a change in the spatial organization of plexin dimers, shifting the conformation from the inactive to the active form.…”