Photoaffinity labelling and radiation inactivation of the leukotriene B 4 receptor in human myeloid cells

Abstract

The leukotriene (LT) B 4 receptor has been characterized in the human monocyte leukaemia THP-1 cell line. Scatchard analysis of [ 3H]LTB 4 specific binding to THP-1 cell membranes revealed a single population of high affinity (K D = 56 pM) and saturable (2000 receptors / cell) binding sites. [ 3H]LTB 4 specific binding was enhanced by divalent cations, but inhibited by both monovalent cations and a non-hydrolysable GTP analogue. Treatment with GTP analogue resulted in a concentration-dependent reduction in the number of high affinity binding sites, accompanied by the appearance of an equal number of binding sites of lower affinity (K D = 1250 pM). In contrast, Scatchard analysis with human polymorphonuclear leukocyte (PMN) membranes consistently revealed two populations of LTB 4 receptors (K D = 48 pM and 270 pM). Treatment with GTP analogue, however, converted all these detectable binding sites to the lower affinity state. These data suggest that the LTB 4 receptor in both THP-1 cell and PMN membranes exists in interconverting affinity states modulated by G-protein coupling. The similarity between the LTB 4 receptors present in these two cell types was also substantiated by target-size analysis by radiation inactivation, which estimated a comparable molecular mass of 56.5 kDa and 52.8 kDa for the THP-1 cell and PMN LTB 4 receptors, respectively. Finally, the presence of a single LTB 4 receptor in PMN was demonstrated by direct photolabelling. Irradiation of frozen [ 3H]LTB 4 equilibrium binding assay incubations resulted in complete photolysis of [ 3H]LTB 4. Subsequent resolution of the tritiated PMN proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed one major radioactive peak migrating with an apparent molecular weight of 61,000. This peak was identified as the LTB 4 receptor since radiolabelling could be completely inhibited by the presence of excess unlabelled LTB 4 or the LTB 4-receptor antagonist, L-662,328. Photolabelling was also partially inhibited by pretreatment with GTP analogue, consistent with G-protein uncoupling reagents reducing receptor affinity without complete inhibition. In summary, the LTB 4 receptor identified in human myeloid cells is a G-protein coupled receptor with interconvertible high and low affinity states, having a molecular mass of 53–61 kDa.