The C-terminal domain LLKIL motif of CXCR2 is required for ligand-mediated polarization of early signals during chemotaxis.
HEK293 cells expressing wild-type CXCR2 recruit PH-Akt-GFP to the leading edge of the cell in response to chemokine. However, in cells expressing mutant CXCR2 defective in AP-2 and HIP binding, i.e. with a mutation in the LLKIL motif, PH-Akt-GFP does not localize to the leading edge in response to ligand. Inhibition of Akt/PKB by transfection of HEK 293 cells with a dominant negative (kinase defective) Akt/PKB inhibits CXCR2 mediated chemotaxis. FRET analysis reveals that membrane-bound activated Cdc42 and Rac1 localize to the leading edge of cells expressing wild-type CXCR2 receptor, but not in cells expressing mutant CXCR2. By contrast, when the activation of Cdc42 and Rac1 are monitored by affinity precipitation assay, cells expressing either wild-type or LLKIL mutant receptors show equivalent ligand induction. Altogether, these data suggest that restricted localized activation of Akt/PKB, Rac1 and Cdc42 is crucial for chemotactic responses and that events mediated by the LLKIL motif are crucial for chemotaxis.
Department of Veteran Affairs, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Jiqing
J
Sai
Guo-Huang
GH
Fan
Dingzhi
D
Wang
Ann
A
Richmond
D020816:Amino Acid Motifs / Q000502:physiology; D001779:Blood Coagulation Factors / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D002633:Chemotaxis / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins; D023063:Receptors, Interleukin-8B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012269:Ribosomal Proteins; D015398:Signal Transduction / Q000502:physiology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D001779:Blood Coagulation Factors; D004268:DNA-Binding Proteins; D008024:Ligands; D011518:Proto-Oncogene Proteins; D016601:RNA-Binding Proteins; C104946:RPL29 protein, human; D023063:Receptors, Interleukin-8B; D012269:Ribosomal Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein