The human erythrocyte inflammatory peptide (chemokine) receptor. Biochemical characterization, solubilization, and development of a binding assay for the soluble receptor

• Published in: Biochemistry (Easton) Vol. 32; no. 22; pp. 5733 - 5738
• Main Authors: Horuk, Richard, Colby, Timothy J, Darbonne, Walter C, Schall, Thomas J, Neote, Kuldeep
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 08.06.1993
• Subjects: Amidohydrolases - pharmacology; Binding, Competitive; Biological and medical sciences; Cell receptors; Cell structures and functions; Chemokine CCL2; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors - blood; Chemotactic Factors - metabolism; Detergents; Erythrocyte Membrane - drug effects; Erythrocyte Membrane - metabolism; Fundamental and applied biological sciences. Psychology; Growth Substances - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosine Triphosphate - pharmacology; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8 - blood; Miscellaneous; Molecular and cellular biology; Molecular Weight; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Receptors, Interleukin-8A; Solubility; Transfection; Human; Competitive fixation; Ligand binding; Membrane receptor; High affinity site; Cytokine; Red blood cell; Scatchard plot; Chemotactic peptide; In vitro; Interleukin 8
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00073a002

In addition to the two human interleukin-8 (IL-8) receptors that have been cloned, IL-8RA and IL-8RB, we recently described a binding protein in human erythrocytes that binds IL-8 and monocyte chemotactic peptide-1 (MCP-1), which we have termed the chemokine (CK) receptor. This communication describes the biochemical characterization, detergent solubilization, and development of a solubilized receptor binding assay for the erythrocyte CK receptor. Competitive 125I-IL-8 binding studies in cells transfected with IL-8RA and IL-8RB revealed that only IL-8 and MGSA were able to displace the radiolabeled IL-8 from these cells. In contrast, a whole array of chemokines were able to cross-compete with 125I-IL-8 for binding to the CK receptor in erythrocyte ghosts. Scatchard analysis of 125I-IL-8 binding to erythrocyte membranes and to dodecyl beta-maltoside solubilized CK receptors revealed a single class of high affinity binding sites in both cases with KD values of 9.5 nM +/- 3.6 and 15.4 nM +/- 5.0, respectively. Chemical cross-linking studies with erythrocyte membranes and with solubilized CK receptors indicated that the CK receptor has a lower molecular mass than the cloned IL-8 receptors (39 kDa compared to 57-69 kDa). Treatment of the cross-linked 47-kDA protein with N-glycanase reduced its molecular mass to 42 kDa.