signals from cell surface receptors trigger reorganization of the actin cytoskeleton is poorly understood. Both tyrosine kinase and G protein– coupled receptors are known to transduce signals that cause changes in cell motility, cell shape, and cell attachment (reviewed in Zigmond, 1996
). These changes are brought about, at least in part, by a reordering of actin in the cell, and form the basis of such higher order processes as morphogenesis and cell migration. One of the most useful tools in the study of any complex biological problem is an organism amenable to genetic analysis. Dictyostelium discoideum
, an amoeboid eukaryotic microorganism, has been an excellent genetic model for studying how extracellular signals are translated into dynamic changes in the actin cytoskeleton.
, extracellular cAMP functions as a chemoattractant and morphogenetic signal (reviewed in Devreotes, 1994
). Four receptors for this ligand have been identified (cAR1–4), and each shows a unique pattern of spatial and temporal expression (Ginsburg et al., 1995
; Parent and Devreotes, 1996
). These receptors are highly related to each other, and fall into the seven-transmembrane G protein–coupled class. One of the four subtypes, the cAR2 receptor, is expressed in a subset of prestalk cells that form the presumptive tip structure (Saxe et al., 1996
). Disruption of the gene encoding this receptor (the carB
gene) causes a morphogenetic arrest before tip formation (Saxe et al., 1993
). This phenotype implies that the cAR2 receptor triggers a signaling cascade required for normal tip formation and elongation. The nature of this signaling cascade is unknown, but the more thoroughly studied cAR1 receptor pathway provides some clues as to the possible events involved.
Responses known to be mediated by cAR1 include stimulation of actin polymerization and cross-linking (Caterina and Devreotes, 1991
; Dharmawardhane et al., 1989
). These are the first steps in the formation of pseudopods and subsequent chemotactic movement. Disruption of the gene encoding the cAR1 receptor leads to a block at the aggregation stage of development, consistent with its crucial role in chemotactic signaling. It is unclear precisely which second messenger pathways triggered by cAR1 are involved in this response, but a heterotrimeric G-protein is clearly required (Chen et al., 1996
). Whether or not cAR2 stimulates a similar pathway in the nascent tip cells is a question that remains unanswered. The involvement of the actin cytoskeleton in tip formation is strongly implied by the existence of several mutations in known components of the actin cytoskeleton that block or alter this process (see Discussion). The goal of this study was to help identify components of the signaling cascades (cytoskeletal or otherwise) that cAR2 stimulates in the tip cells, and that cause the dramatic morphogenetic changes associated with tip formation.
To understand the downstream signaling events from the cAR2 receptor, a genetic approach was used. Such approaches to G-protein–coupled signaling in other organisms have revealed both positive and negative regulators of downstream responses. Some of the positively acting regulators that have been found in other systems include components of heterotrimeric G proteins as well as various target effectors (reviewed in Bardwell et al., 1994
). More recently, examples of negative regulators were found independently in C
and yeast in genetic screens for negatively acting components of egg-laying and mating pheromone-signaling pathways, respectively (Koelle and Horvitz, 1996
; Dohlman et al., 1996
). These components, SST2 in the case of yeast and EGL-10 in the case of Caenorhabditis elegans
, helped to define a new class of GTPase-accelerating proteins known as the regulators of G-protein signaling (RGSs). Based on these results in other systems, our screen began with the assumption that negative regulators of cAR2 function could be eliminated by mutation, which might lead to a partial or complete restoration of morphogenesis and development.
In this paper we describe the isolation and initial characterization of one of these suppressors. The protein encoded by the disrupted gene shows areas of striking homology to the Wiskott-Aldrich Syndrome protein (WASP).1 The protein, which we call suppressor of cAR (SCAR), does not appear to be the Dictyostelium homologue of WASP, but instead seems to define a new family of evolutionarily conserved, WASP-related proteins (see below).
Wiskott-Aldrich syndrome is an X-linked human genetic disorder that leads to a variety of defects in the immune system, including thrombocytopenia, compromised immune function, and susceptibility to leukemias and lymphomas (Kirchhausen and Rosen, 1996
). Patients with this disorder fail to produce normal antibody responses to pneumococcal polysaccharide injections, and in in vitro assays, neutrophils from WAS patients have major chemotactic defects (Ochs et al., 1980
). When the morphology of these immune system cells is closely examined, defects in the organization of the actin cytoskeleton are found (Kenney et al., 1986
; Molina et al., 1992
; Gallego et al., 1997). In 1994, the gene responsible for Wiskott-Aldrich Syndrome was identified by positional cloning (Derry et al., 1994
). The protein encoded by the Wiscott-Aldrich Syndrome gene initially failed to reveal much about its function, but was recently identified in screens for Cdc42-interacting proteins by three independent labs (Aspenström et al., 1996
; Kolluri et al., 1996
; Symons et al., 1996
). A close relative of WASP, the N-WASP protein, has been shown to potentiate filopodia formation induced by an activated Cdc42 mutant (Miki et al., 1998a
). More confirmatory evidence for WASP's role in organizing the cytoskeleton was presented in transient overexpression experiments in which accumulation of massive F-actin aggregates occurred in the cytoplasm of the transfected cells (Symons et al., 1996
). In the present paper we identify a new family of WASP-related proteins, and provide genetic evidence for its involvement in G protein–coupled signaling. This work also provides further evidence for the involvement of WASP and WASP-related proteins in regulating the actin cytoskeleton.