Identification and target-size analysis of the leukotriene D 4 receptor in the human THP-1 cell line

Abstract

The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D 4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [ 3H]Leukotriene D 4-specific binding to THP-1 cell membranes was of high affinity ( K D = 0.47 nM) and saturable, enhanced by divalent cations but inhibited by both monovalent cations and non-hydrolyzable GTP analogs. The cysteinyl leukotrienes competed for [ 3H]leukotriene D 4-specific binding with the following rank order of potency: leukotriene D 4 ⪢ leukotriene E 4 〉 leukotriene C 4 In addition, leukotriene D 4-receptor antagonists from two structural classes, the quinolines MK-571 and L-697,008, and the indole ICI 204,219, displayed nanomolar potency in [ 3H]leukotriene D 4 competition assays. These data show that [ 3H]leukotriene D 4-specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D 4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D 4 receptor were investigated. Culture of THP-1 cells in the presence of tunicamycin, an inhibitor of N-glycosylation, resulted in a 6-fold decrease in the number of detectable [ 3H]leukotriene D 4-specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [ 3H]leukotriene D 4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D 4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.