cDNA cloning and characterization of guinea-pig leukotriene B4 receptor.
The cDNA for leukotriene B(4) (LTB(4)) receptor (BLT) was cloned from a guinea-pig leucocyte cDNA library. The cloned receptor cDNA encodes 348 amino acid residues and shares 73% identity with the amino acid sequence of human BLT. Northern blot analysis showed the highest expression of the receptor mRNA in leucocytes, followed by lung and spleen. The membrane fractions of HEK-293 and Cos-7 cells transfected with the cDNA showed specific LTB(4)-binding activities, with K(d) values of 0.27 and 0.17 nM respectively. Xenopus laevis oocytes injected with the cRNA of guinea-pig BLT showed LTB(4)-induced Cl(-) currents, indicating that the cloned receptor is functional. LTB(4) is metabolized to 20-hydroxy-LTB(4) and then to 20-carboxy-LTB(4), a transformation considered as a major inactivation pathway of the compound. Using the cloned receptor, we analysed the agonistic effects of LTB(4) and these two metabolites. 20-Carboxy-LTB(4) is a much weaker agonist, with a K(d) value higher than that of LTB(4) by three orders of magnitude, corresponding to a much weaker chemotactic activity. Although 20-hydroxy-LTB(4) is as potent as LTB(4) in inhibiting [(3)H]LTB(4) binding and cAMP formation, it is less potent than LTB(4) in the mobilization of intracellular Ca(2+) and the chemotaxis of Chinese hamster ovary cells expressing the guinea-pig BLT. The present study demonstrated that although LTB(4) and 20-hydroxy-LTB(4) bind to the receptor with similar affinities, they do differ in activating intracellular signalling.
Masuda
K
K
Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yokomizo
T
T
Izumi
T
T
Shimizu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004553:Electric Conductivity; D006168:Guinea Pigs; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D007975:Leukotriene B4 / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D018102:Receptors, Leukotriene B4 / Q000819:agonists / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D014982:Xenopus laevis
D002712:Chlorides; D012333:RNA, Messenger; D018102:Receptors, Leukotriene B4; D005576:Colforsin; D007975:Leukotriene B4; D000242:Cyclic AMP; D002118:Calcium