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Type: Journal article
Title: Physical association of Gi₂α with Interleukin-8 receptors
Other Titles: Physical association of Gi(2)alpha with Interleukin-8 receptors
Author: Damaj, B.
McColl, S.
Mahana, W.
Crouch, M.
Naccache, P.
Citation: Journal of Biological Chemistry, 1996; 271(22):12783-12798
Issue Date: 1996
ISSN: 0021-9258
Statement of
Bassam B. Damaj, Shaun R. McColl, Wahib Mahana, Michael F. Crouch, and Paul H. Naccache
Abstract: Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (Graphiceutrophil-Graphicctivating Graphiceptide-2) and Gro-α and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-α. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R). The formation of a physical coupling between IL-8 receptors and the α-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Gα immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2α (raised against amino acids 159-168 (LERIAQSDYI) of Gi2α) and anti-Gtα (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtα), but not anti-Gq, immunoprecipitates. Similar results were obtained in human 293 cells stably transfected with IL-8RA or IL-8RB. The peptide derived from the COOH-terminal sequence of Gt inhibited the co-immunoprecipitation of IL-8R and Gi observed in response to the anti-Gtα and anti-Gi2α antibodies. On the other hand, the Gi2α peptide only inhibited the immunoprecipitation induced by the anti-Gi2α antibody. Peptides derived from Gi1α or Gi3α had no effect in this assay. The introduction of the anti-Gi2α or anti-Gtα antibodies or their neutralizing peptides, but not the Gi1α or Gi3α peptides, into 293 IL-8RA or 293 IL-8RB cells completely blocked the calcium responses obtained upon stimulation with IL-8. These results demonstrate that the occupation of either type of IL-8 receptor leads to a physical coupling to the α-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Giα, one involved in receptor/Giα interaction (KENLKDCGLF) and the other mediating downstream signal transmission (LERIAQSDYI). Finally, the results of this study also validate the use of the transfected 293 cell line as a model for the study of the signal transduction pathway(s) initiated by IL-8.
Keywords: Cell Line
GTP-Binding Proteins
Receptors, Interleukin-8A
Receptors, Interleukin
Antigens, CD
Virulence Factors, Bordetella
Signal Transduction
Amino Acid Sequence
Protein Binding
Molecular Sequence Data
Rights: © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
DOI: 10.1074/jbc.271.22.12783
Published version:
Appears in Collections:Aurora harvest 2
Microbiology and Immunology publications

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