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Type: Journal article
Title: Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops
Author: Damaj, B.
McColl, S.
Neote, K.
Songqing, N.
Ogborn, K.
Hebert, C.
Naccache, P.
Citation: FASEB Journal, 1996; 10(12):1426-1434
Issue Date: 1996
ISSN: 0892-6638
Statement of
Bassam B. Damaj, Shaun R. Mccoll, Kuldeep Neote, Na Songqing, Kevin T. Ogborn, Caroline A. Hebert, and Paul H. Naccache
Abstract: Interleukin 8 (IL-8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL-8 receptors and the alpha subunit of guanine nucleotide regulatory protein (Gi(alpha)2) upon stimulation of human neutrophils by IL-8. In the present study, we identified by site-directed mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with Gi(alpha)2. We first systematically mutated, in groups of two to four, all the residues in the three intracellular loops of the IL-8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 293 cells. Four residues in the second intracellular loop (Y136, L137, I139, V140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL-8. Other residues in the second and third intracellular loops were also found to affect IL-8RA-mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL-8 binding affinities to the mutated receptors. Mutagenesis of the residues in the first intracellular loop had only weak effects on the mobilization of calcium induced by IL-8. We then used a coimmunoprecipitation protocol with anti-Gi(alpha)2 antibodies to determine the involvement of the two regions defined above in Gi(alpha)2 coupling to IL-8 type A receptors. Whereas the anti-Gi(alpha)2 antibodies coimmunoprecipitated IL-8 receptors in the wild-type cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for Gi(alpha)2 coupling and induction of intracellular calcium mobilization were next introduced into cells expressing wild-type IL-8RA or IL-8RB to assess their role in coupling Gi(alpha)2 to both IL-8 receptors. The results obtained in the latter experiments suggest that the same regions of the second intracellular loop (Y136, L137, I139, V140) and of the third intracellular loop (M241) are critically involved in the coupling of both IL-8RA and IL-8 RB to Gi(alpha)2 as well as to a downstream effector (or effectors) involved in calcium mobilization.
Keywords: Cell Line, Transformed; Humans; GTP-Binding Proteins; Receptors, Interleukin-8A; Receptors, Interleukin; Antigens, CD; Precipitin Tests; Transfection; Mutagenesis; Binding Sites; Amino Acid Sequence; Molecular Sequence Data; Adult
Rights: © 1996 by The Federation of American Societies for Experimental Biology
RMID: 0030004240
DOI: 10.1096/fasebj.10.12.8903513
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Appears in Collections:Microbiology and Immunology publications

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