Abstract 524: Identification of Roles for the Rho-Specific Guanine Nucleotide Dissociation Inhibitor (RhoGDI) Ly-Gdi in Platelet Function

Introduction: Upon activation, platelets undergo specific morphological alterations critical to hemostatic plug and thrombus formation via actin cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in regulating platelet function. Methods and Hypothesis: Through an approach combining pharmacology, cell biology and systems biology methods we assessed the hypothesis that RhoGDI proteins regulate Rho GTPase-driven platelet functions downstream of platelet integrin and glycoprotein receptors. Results: We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular staining and super resolution microscopy assays find that Ly-GDI displays an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI in platelets, as Ly-GDI is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Notably, inhibition of PKC diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Conclusion: In conclusion, our results support roles for Ly-GDI as a localized regulator of Rho GTPases in platelets and link PKC and Rho GTPase signaling systems to platelet function.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI–specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

Distinct expression patterns of the GDP dissociation inhibitor protein gene (OsRhoGDI2) from Oryza sativa during development and abiotic stresses

Guanine nucleotide dissociation inhibitor (GDI) plays an essential role in regulating the forms of Rac/Rop between GDP-bound inactivity and GTP-bound activity in plants. In this paper, we reported a stress-responsive GDI gene (

Loss of Rho-GDIα sensitizes podocytes to lipopolysaccharide-mediated injury

Nephrotic syndrome is a disease of glomerular permselectivity that can arise as a consequence of heritable or acquired changes to the integrity of the glomerular filtration barrier. We recently reported two siblings with heritable nephrotic syndrome caused by a loss of function mutation in the gene ARHGDIA, which encodes for Rho guanine nucleotide dissociation inhibitor-α (GDIα). GDIs are known to negatively regulate Rho-GTPase signaling. We hypothesized that loss of GDIα sensitizes podocytes to external injury via hyperactivation of Rho-GTPases and p38 MAPK. We examined the response of cultured podocytes with and without knockdown of GDIα to LPS injury by assessing the levels of phospho-p38 as well as the degree of synaptopodin loss. GDIα knockdown podocytes showed more pronounced and sustained p38 phosphorylation in response to LPS compared with control podocytes, and this was blunted significantly by the Rac1 inhibitor. In LPS-treated control podocytes, synaptopodin degradation occurred, and this was dependent on p38, the proteasome, and cathepsin L. In GDIα knockdown podocytes, the same events were triggered, but the levels of synaptopodin after LPS treatment were significantly lower than in control podocytes. These experiments reveal a common pathway by which heritable and environmental risk factors converge to injure podocytes, from Rac1 hyperactivation to p38 phosphorylation and synaptopodin degradation via the ubiquitin-proteasome pathway and cathepsin L.