Association of Rho(D) polypeptides with the membrane skeleton in Rho(D)-positive human red cells

The Rho(D) antigen was recently identified as a 28,000 to 33,000 m.w. polypeptide expressed on the surface of human Rho(D)+ cells. We now show that 70 to 80% of the Rho(D) polypeptides remain firmly associated with the membrane skeleton (detergent-insoluble matrix) obtained after treatment of isolated membranes with Triton X-100. The same treatment solubilized most of the major sialoglycoprotein, glycophorin A. The membrane skeleton-bound Rho(D) polypeptides were not solubilized by procedures that dissociated spectrin, actin, and glyceraldehyde-3-phosphate dehydrogenase from the membrane. Affinity-purified 125I-labeled anti-Rho(D) antibodies bound to intact Rho(D)+ cells, Rho(D)+ membranes, and isolated membrane skeletons from Rho(D)+ cells, but not to Rho(D)- cells. The binding to Rho(D)+ cells was competitively inhibited efficiently by Rho(D)+ membranes and weakly by Rho(D)- membranes. When isolated unsealed Rho(D)+ and Rho(D)- membranes were labeled by lactoperoxidase-catalyzed iodination and solubilized in Triton X-100, Rho(D) polypeptides were immune precipitated only from Rho(D)+ membranes.     

Molecular characterization of the human red cell Rho(D) antigen

Human red cells of Rh blood groups -D-/-D- ('super-D'), -/- (Rhnull) and normal Rho(D)+ cells were radioactively surface-labeled using the lactoperoxidase 125I method. Polyacrylamide gel electrophoresis in the presence of SDS followed by fluorography showed a strong enrichment of a polypeptide with an apparent mol. wt. of 28,0000-33,000 in the 125I-labeled -D-/-D- membranes. This polypeptide was specifically immune precipitated with anti-Rho(D) antiserum. Treatment of intact cells with trypsin or Pronase did not digest the protein. The Rho polypeptide migrated identically on polyacrylamide gel electrophoresis under reducing and non-reducing conditions. It was not phosphorylated after in vitro incubation of red cells with 32P. When whole labeled membranes were solubilized in neutral detergent and applied to lectin-Sepharose columns the Rho(D) polypeptide adsorbed to Ricinus communis lectin but not to wheat germ lectin or Lens culinaris lectin. The purified molecule did not adsorb to R. communis lectin-Sepharose. Treatment of the Rho(D) antigen with endo-N-acetyl glucosaminidase H, endo-beta-galactosidase or mild alkali did not lower its apparent mol. wt.     

Studies on the characterization of the Rho(D) antigen.

The Rho(D) antigen of red cell membranes was solubilized using ethylene-diamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, and pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rho(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rho(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rho(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rho(D) molecule in its natural environment. The solubilized Rho(D) molecules are apparently not dependent on lipid for their Rho(D) activity.     

Effects of modulating erythrocyte membrane cholesterol on Rho(D) antigen expression

Alteration of Rh_o(D)-antigen expression in Rh-positive erythrocytes after modification of membrane fluidity has recently been demonstrated by indirect fluorescence staining. The isolation of D-antigen from Rh-negative erythrocyte membranes has also recently been claimed. We therefore attempted quantification of D-antigen sites in modified Rh-positive and Rh-negative cells by the direct radiolabelled (¹²⁵I) antibody uptake technique. The cholesterol/phospholipid molar ratio of erythrocytes was modified with resulting membrane-cholesterol enrichment or depletion. ¹²⁵I-anti-D uptake doubled in enriched Rh-positive erythrocytes, and was decreased in depleted Rh-positive erythrocytes. No change in anti-D uptake could be shown for Rh-negative erythrocytes similarly modified. If the mechanism for enhanced D-antigen expression in cholesterol enriched erythrocytes is vertical displacement of antigen, our inability to unmask the D-antigen in Rh-negative erythrocytes suggests that it is deeply buried in the lipid matrix.     

Interactions between Rho GTPases and Rho GDP dissociation inhibitor (Rho-GDI)

The Rho-GDP dissociation inhibitor (Rho-GDI) was used as bait in a two-hybrid screen of a human leucocyte cDNA library. Most of the isolated cDNAs encoded GTPases of the Rho subfamily: RhoA, B, C, Rac1, 2, CDC42 and RhoG. The newly discovered RhoH interacted very poorly with Rho-GDI. Another protein partner shared a homology with RhoA that points to Asp67(RhoA)-Arg68(RhoA)-Leu69(RhoA) as critical for interaction with Rho-GDI. A second screen with RhoA as bait led to the isolation of GDI only. In order to investigate the relative role of protein-protein and protein-lipid interactions between Rho GTPases and Rho-GDI, CAAX box mutants of RhoA were produced. They were found to interact with Rho-GDI as efficiently as wild type RhoA, indicating that protein-protein interactions alone lead to strong binding of the two proteins. The C-terminal polybasic region of RhoA was also shown to be a site of protein-protein interaction with Rho-GDI.     

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.     

Design and synthesis of Rho kinase inhibitors (II)

In a previous study, we identified several structurally unrelated scaffolds of the Rho kinase inhibitor using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. 1H-Indazole is one of the candidate scaffolds on which a new series of potent Rho kinase inhibitors could be developed. In this study, the detailed structure-activity relationship of 1H-indazole analogues was studied. During this study, we found that the cell-free enzyme inhibitory potential of Rho kinase inhibitors having the 1H-indazole scaffold did not necessarily correlate with their inhibitory potential toward the chemotaxis of cultured cells. The choice of the linker substructure was shown to be an important factor for the 1H-indazole analogues to inhibit the chemotaxis of cells. Optimization of the 1H-indazole inhibitors with respect to the in vitro inhibition of monocyte chemotaxis induced by MCP-1 was carried out. The inhibitory potential was improved both in the cell-free enzyme assay and in the chemotaxis assay.     

Design and synthesis of Rho kinase inhibitors (I)

Several structurally unrelated scaffolds of the Rho kinase inhibitor were designed using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. A docking simulation using the ligand-binding pocket of the Rho kinase model helped to comprehensively understand and to predict the structure-activity relationship of the inhibitors. This understanding was useful for developing new Rho kinase inhibitors of higher potency and selectivity. We identified several potent platforms for developing the Rho kinase inhibitors, namely, pyridine, 1H-indazole, isoquinoline, and phthalimide.     

Plexin B regulates Rho through the guanine nucleotide exchange factors leukemia-associated Rho GEF (LARG) and PDZ-RhoGEF

Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. Here we show that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, we show that ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration.     

Protective effect of the membrane skeleton on the immunologic reactivity of the human red cell Rho(D) antigen

It has recently been shown that the 30,000 m.w. Rho(D) protein is associated with the membrane skeleton of the human red cell. We have studied the effects of the membrane skeleton on the immunoreactivity of the Rho(D) antigen present in Rho(D)+ membranes. Solubilization of the membranes with the Triton X-100 detergent and centrifugation of the extracts showed that more than 90% of the immunoreactive Rho(D) antigen sedimented with the membrane skeleton structures. The skeleton-bound Rho(D) antigen could be solubilized by disruption of the skeleton in low ionic strength medium. The removal of the membrane skeleton structure before the solubilization of the membranes with detergent resulted in the inactivation of the majority of the Rho(D) antigen. The effect of the membrane skeleton on the stability of the Rho(D) antigen was additionally studied in detergent extracts prepared from native and skeleton-free membranes. The assay of the Rho(D) antigen activity in the extracts showed that the Rho(D) antigen was 100 times more sensitive to the detergent inactivation in skeleton-free membranes than in native membranes. These results indicate that the membrane skeleton is important for stabilizing the immunoreactive form of the Rho(D) protein on the red cell membrane.     

Quantitative comparison of Rh1 (Rho, D) antigen on D, Du and d red cells by radioimmunoassay using 125I-protein A

Relative Rh1 (Rho, D) antigen contents of the red Rh: 1 (Rh positive, D), Rh: wl (Rh variant, Du) and Rh: -1 (Rh negative, d) cells were estimated from the quantity of 125I-protein A bound to the sensitized red cells. The isotope binding activity to both D and Du cells decreased in parallel with the dilution of anti-D serum. The relative amount of the 125I-protein A bound to Du cells was about one-sixth that of D cells without papain treatment, while no isotope binding was observed in d cells. The Du red cells were quantitatively deficient in Rh1 (Rho, D) antigen activity compared with the D cells. A radioimmunoassay using 125I-protein A was a very useful method for studies regarding measuring the relative amounts of various blood group antigens.     

Specificity of Rho insert-mediated activation of phospholipase D1

Members of the Rho subfamily of GTP-binding proteins regulate phospholipase D1 (PLD1) activity and signaling. In previous work, we demonstrated that binding of the Rho family member Cdc42 to PLD1 and the subsequent stimulation of its enzymatic activity are distinct events. Deletion of the insert helix from Cdc42 does not interfere with its switch I-mediated, GTP-dependent binding to PLD1 but inhibits Cdc42-stimulated PLD1 activity. To understand the mechanism of the insert-mediated activation of PLD1 by Cdc42 and to develop reagents to study Cdc42-activated PLD1 in cellular signaling events, we have undertaken a mutational analysis of the Rho insert region of Cdc42 and examined the specificity of the insert helix requirement in the other Rho family members, RhoA and Rac1. Here, we identify a critical residue, serine 124, in the Cdc42 insert helix central to its activation mechanism. Further, we examine this activation mechanism with respect to other members of the Rho family and demonstrate that each Rho protein activates PLD by distinct mechanisms, potentially allowing for unique signaling outcomes in the cell.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase,yet it is crucial for the faithful division of one cell into two.Cytokinesis failure is often associated with cancer.Cytokinesis can be morphologically divided into four steps:cleavage furrow initiation,cleavage furrow ingression,midbody formation and abscission.Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis.At the same time,Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis,including cytokinesis.Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks.More specifically,Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1,thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains.Ect2 itself can be phosphorylated by Plk1 in vitro.Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity.Once activated,RhoA-GTP will activate downstream effectors,including ROCK1 and ROCK2.ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen,and Plk1 can phosphorylate ROCK2 in vitro.We review current understandings of the interplay between Plk1,RhoA proteins and other proteins (e.g.,NudC,MKLP2,PRC1,CEP55) involved in cytokinesis,with partitular emphasis of its clinical implications in cancer.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase, yet it is crucial for the faithful division of one cell into two. Cytokinesis failure is often associated with cancer. Cytokinesis can be morphologically divided into four steps: cleavage furrow initiation, cleavage furrow ingression, midbody formation and abscission. Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis. At the same time, Polo-like kinase 1 (Plk1) is a...