Photoaffinity labelling and radiation inactivation of the leukotriene B4 receptor in human myeloid cells.
The leukotriene (LT) B4 receptor has been characterized in the human monocyte leukemia THP-1 cell line. Scatchard analysis of [3H]LTB4 specific binding to THP-1 cell membranes revealed a single population of high affinity (KD = 56 pM) and saturable (2000 receptors/cell) binding sites. [3H]LTB4 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 (KD = 1250 pM). In contrast, Scatchard analysis with human polymorphonuclear leukocyte (PMN) membranes consistently revealed two populations of LTB4 receptors (KD = 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 LTB4 receptor in both THP-1 cell and PMN membranes exists in interconverting affinity states modulated by G-protein coupling. The similarity between the LTB4 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 LTB4 receptors, respectively. Finally, the presence of a single LTB4 receptor in PMN was demonstrated by direct photolabelling. Irradiation of frozen [3H]LTB4 equilibrium binding assay incubations resulted in complete photolysis of [3H]LTB4. 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 LTB4 receptor since radiolabelling could be completely inhibited by the presence of excess unlabelled LTB4 or the LTB4-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 LTB4 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.
Slipetz
D M
DM
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Dorval, Que., Canada.
Scoggan
K A
KA
Nicholson
D W
DW
Metters
K M
KM
D000345:Affinity Labels; D002412:Cations / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005720:Gamma Rays; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007951:Leukemia, Myeloid / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010777:Photochemistry; D011506:Proteins / Q000378:metabolism; D011869:Radioligand Assay; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D018102:Receptors, Leukotriene B4; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D002412:Cations; D011506:Proteins; D011971:Receptors, Immunologic; D018102:Receptors, Leukotriene B4; D006160:Guanosine Triphosphate