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 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.
Horuk
R
R
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
Colby
T J
TJ
Darbonne
W C
WC
Schall
T J
TJ
Neote
K
K
D000581:Amidohydrolases / Q000494:pharmacology; D001667:Binding, Competitive; D018932:Chemokine CCL2; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000097:blood* / Q000378:metabolism; D003902:Detergents; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000097:blood*; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D012995:Solubility; D014162:Transfection
C475829:CXCL1 protein, human; D018932:Chemokine CCL2; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D003902:Detergents; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase