Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor.
Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor. In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR). Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott-Aldrich syndrome protein (WASP) as a potential partner of C5aR. WASP is known to have an essential function in regulating actin dynamics at the cell leading edge. The interaction was detected with both the fragment of WASP containing amino acids 1-321 (WASP.321) and WASP with its actin-nucleation-promoting domain [verprolin-like, central and acidic (VCA) domain] deleted. The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments. In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR. This is most probably due to an auto-inhibited conformation imposed by the VCA domain. In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor. However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active 'open conformation', the amount of WASP associated with the receptor was markedly increased. We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation. This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.
Tardif
Marianne
M
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Commissariat à l'Energie Atomique (CEA)/Centre National de la Recherche Scientifique (CNRS)/Université Joseph Fourier, CEA-Grenoble, France. tardif@dsvsud.cea.fr
Brouchon
Laurence
L
Rabiet
Marie-Josèphe
MJ
Boulay
François
F
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells / Q000166:cytology; D002462:Cell Membrane; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015936:Complement C5a / Q000378:metabolism; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
D000199:Actins; D015703:Antigens, CD; D010446:Peptide Fragments; D011506:Proteins; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D015936:Complement C5a; D005982:Glutathione Transferase; D020764:cdc42 GTP-Binding Protein