Identification and target-size analysis of the leukotriene D4 receptor in the human THP-1 cell line.
The human acute monocytic leukemia cell line THP-1 has been identified, by radioligand binding, as expressing the leukotriene D4 receptor at a high level (4000 binding sites per cell), without the need for further cell differentiation. [3H]Leukotriene D4-specific binding to THP-1 cell membranes was of high affinity (KD = 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 D4-specific binding with the following rank order of potency: leukotriene D4 >> leukotriene E4 > leukotriene C4. In addition, leukotriene D4-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 D4 competition assays. These data show that [3H]leukotriene D4-specific binding to THP-1 cell membranes fulfils the criteria for binding to a leukotriene D4 receptor regulated through interaction with a G protein. Several novel features of the THP-1 leukotriene D4 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 D4-specific binding sites. Target-size analysis by radiation inactivation estimated a molecular mass of 65 kDa for the [3H]leukotriene D4 specific binding site(s) present in THP-1 cell membranes. Together, these results suggest that the human THP-1 cell leukotriene D4 receptor is a glycosylated protein with a molecular mass of approx. 65 kDa within the membrane environment.
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Dorval, Québec, Canada.
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D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D006801:Humans; D007948:Leukemia, Monocytic, Acute / Q000378:metabolism*; D015289:Leukotrienes / Q000494:pharmacology; D008970:Molecular Weight; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D018077:Receptors, Leukotriene
D015289:Leukotrienes; D011422:Propionates; D011804:Quinolines; D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; C059141:verlukast; D006160:Guanosine Triphosphate