Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2.
The objective of this investigation was to determine the amino acid residues of the human neutrophil CXC chemokine receptor-2 (CXCR2) that are critical for binding the ligands interleukin 8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and growth-related protein alpha (GROalpha) and critical for receptor-mediated signal transduction. Charged residues of the amino terminus and the first extracellular loop of CXCR2 were targeted for point mutagenesis studies. Seven separate CXCR2 mutants (Glu7, Asp9, Glu12, Asp13, Lys108, Asn110, and Lys120, all to Ala) were generated. Based on the Scatchard analysis of radioligand binding studies, the following amino acids were deemed critical for ligand binding: (i) Asp9, Glu12, Lys108, and Lys120 for IL-8 and (ii) Glu7, Asp9, and Glu12 for GROalpha. Point mutations in the amino terminus domain (Asp9 and Glu12) and the first extracellular loop (Lys108, Asn110, and Lys120) of CXCR2 reduced cell activation to all three ligands as measured by changes in intracellular calcium concentration. In conclusion, high-affinity binding of IL-8, NAP-2, and GROalpha to CXCR2 involves interaction with specific and different amino acid residues of CXCR2. Furthermore, we propose that the CXCR2 amino acid residues required for cell activation are not necessarily the same residues required for ligand binding.
Katancik
J A
JA
Department of Periodontology, University of Tennessee, Memphis, TN, USA.
Sharma
A
A
de Nardin
E
E
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D023063:Receptors, Interleukin-8B / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D001620:beta-Thromboglobulin
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D008024:Ligands; C061615:PPBP protein, human; D010455:Peptides; D023063:Receptors, Interleukin-8B; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide; D002118:Calcium