THE HUMAN ERYTHROCYTE INFLAMMATORY PEPTIDE (CHEMOKINE) RECEPTOR - BIOCHEMICAL-CHARACTERIZATION, SOLUBILIZATION, AND DEVELOPMENT OF A BINDING ASSAY FOR THE SOLUBLE RECEPTOR

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 I-125-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 I-125-IL-8 for binding to the CK receptor in erythrocyte ghosts. Scatchard analysis of I-125-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 K(D) 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.