Exchanging Interleukin-8 and Melanoma Growth-stimulating Activity Receptor Binding Specificities

Abstract

Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B. The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils. In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity. Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al. (1994) J. Mol. Biol. 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors. The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand. When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced. However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced. Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity. Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8. Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity.

Identification of the Extracellular Loop 2 as the Point of Interaction between the N Terminus of the Chemokine MIP-1α and Its CCR1 Receptor

“…Receptor recognition is mainly driven by an extended hydrophobic groove delineated by the second and third ␤-sheet strands (Clubb et al, 1994;Mizoue et al, 1999;Mayer and Stone, 2000;Ye et al, 2000) as well as the N-terminal end and the N-loop (Lowman et al, 1996) of the chemokine. On the receptor side, the N terminus has been identified as being counterpart in the interaction.…”

Monomeric variants of IL-8: Effects of side chain substitutions and solution conditions upon dimer formation

“…However, estimates of dimerization affinity in vitro under conditions approaching physiological have previously been reported as Kdd = Lowman et al (1996).…”

“…The primary functional determinant for binding of IL-8 to its neutrophil receptors is a short peptide motif corresponding to the peptide sequence GluLeu-Arg ("ELR') near the amino terminus of IL-8 (Clark- Lewis et al, 1991Lewis et al, , 1993HCbert et al, 1991). Additionally, a region of more hydrophobic character has been identified in the residues 12-2 1 region of IL-8 (the N-loop) that clearly contributes to highaffinity receptor binding (Clubb et al, 1994;Schraufstatter et al, 1995;Lowman et al, 1996;Williams et al, 1996). Both of these regions, however, are rather distal to the dimer interface, and well exposed to solvent in either dimeric (Baldwin et al, 1991;Clore & Gronenborn, 1991) or monomeric forms of the protein.…”

Conformational Constraining of Inactive and Active States of a Seven Transmembrane Receptor by Metal Ion Site Engineering in the Extracellular End of Transmembrane Segment V

“…6) (Fairbrother et al, 1994). The N-loop is directly involved in receptor binding, whereas the second region is located distant from the presumed receptor binding site as well as distant from the region being important for the glycosaminoglycan interaction (i.e., between the second and third ␤-sheets) (Lowman et al, 1996;Fernandez and Lolis, 2002). The fact that both analogs ([H19A] and [H34A]) acted very similarly to the wt CXCL1/GRO␣ excludes a putative, activating metal ion site within the ligand or between the ligand and the receptor.…”