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Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors

Academic Article
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Overview

authors

  • Schraufstatter, I. U.
  • Barritt, D. S.
  • Ma, M.
  • Oades, Z. G.
  • Cochrane, Charles

publication date

  • 1993

journal

  • Journal of Immunology  Journal

abstract

  • To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.

subject areas

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Calcium
  • Chemokine CXCL1
  • Chemokines, CXC
  • Chemotactic Factors
  • Growth Substances
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Interleukin-8
  • Molecular Sequence Data
  • Mutation
  • Neoplasm Proteins
  • Receptors, Interleukin
  • Receptors, Interleukin-8A
  • Structure-Activity Relationship
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Identity

International Standard Serial Number (ISSN)

  • 0022-1767

PubMed ID

  • 8245475
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Additional Document Info

start page

  • 6418

end page

  • 6428

volume

  • 151

issue

  • 11

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