guanosine-triphosphate and Wiskott-Aldrich-Syndrome

We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends.

Topics: Actins; Adult; cdc42 GTP-Binding Protein; Cell Membrane; Cell Membrane Permeability; Cell Size; Glucosides; Guanosine Triphosphate; Humans; In Vitro Techniques; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Nerve Tissue Proteins; Neutrophils; Peptide Fragments; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Signal Transduction; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein, Neuronal

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

Topics: Actins; Amino Acid Sequence; Animals; Binding Sites; cdc42 GTP-Binding Protein; Cell Cycle Proteins; Cell Line; Chlorocebus aethiops; Consensus Sequence; Cytosol; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Biological; Molecular Sequence Data; Neutrophils; Protein Biosynthesis; Proteins; Recombinant Proteins; Sequence Homology, Amino Acid; Sequence Tagged Sites; Transfection; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein

The Wiskott-Aldrich syndrome (WAS) is a disease of profound thrombocytopenia and severe immune defects caused by an unidentified defective X chromosome gene. In this study, T lymphocyte function is examined using a panel of allospecific WAS patient T cell lines, previously found to express the abnormal disease gene and the cytoarchitectural defect characteristic of the disease. Although T cell lines from normal individuals proliferate vigorously in response to immobilized anti-CD3 mAb OKT3 and SPV-T3b, five of seven WAS patient T cell lines failed to proliferate and two lines showed significantly decreased proliferation when challenged with the immobilized anti-CD3 mAb. The deficient responsiveness of the WAS T cell lines to immobilized anti-CD3 mAb is a restricted defect, because the cells proliferate normally when challenged with allospecific Ag, PHA, or PMA plus ionomycin. Addition of anti-CD28 mAb did not correct the deficient proliferation of the WAS cells challenged with immobilized anti-CD3. Deficient response of the WAS T cell lines to immobilized anti-CD3 was detected also when earlier events of the proliferation process, IL-2 production and up-regulation of activation Ag CD69 and CD28, were measured. On the other hand, WAS cell lines did not differ from normal cell lines in binding of anti-CD3 mAb, mobilization of Ca2+ in response to soluble OKT3, and tyrosine phosphorylation and GTP binding of the CD3 zeta-chain in response to OKT3. Cumulatively, these findings demonstrate a striking restricted defect in the proliferative response of WAS T cells, which because it is found in cell lines free of secondary changes that occur in the patient circulation must be a reflection of the inherited defective disease gene product.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Calcium; CD28 Antigens; CD3 Complex; Cell Line; Guanosine Triphosphate; Humans; Interleukin-2; Lymphocyte Activation; Molecular Sequence Data; T-Lymphocytes; Tyrosine; Wiskott-Aldrich Syndrome