Physical association of Gi2alpha with interleukin-8 receptors.
Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (neutrophil-activating peptide-2) and Gro-alpha 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-alpha. 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 alpha-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Galpha immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2alpha (raised against amino acids 159-168 (LERIAQSDYI) of Gi2alpha) and anti-Gtalpha (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtalpha), 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-Gtalpha and anti-Gi2alpha antibodies. On the other hand, the Gi2alpha peptide only inhibited the immunoprecipitation induced by the anti-Gi2alpha antibody. Peptides derived from Gi1alpha or Gi3alpha had no effect in this assay. The introduction of the anti-Gi2alpha or anti-Gtalpha antibodies or their neutralizing peptides, but not the Gi1alpha or Gi3alpha 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 alpha-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Gialpha, one involved in receptor/Gialpha 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.
Damaj
B B
BB
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Université Laval, Sainte-Foy, Québec G1V 4G2, Canada.
McColl
S R
SR
Mahana
W
W
Crouch
M F
MF
Naccache
P H
PH
D000328:Adult; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D008969:Molecular Sequence Data; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015703:Antigens, CD; D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D010566:Virulence Factors, Bordetella; D019204:GTP-Binding Proteins; D002118:Calcium