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.
Sai
Jiqing
J
Department of Veteran Affairs, Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Fan
Guo-Huang
GH
Wang
Dingzhi
D
Richmond
Ann
A
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