Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins

Eur J Biochem. 1999 Jan;259(1-2):419-25. doi: 10.1046/j.1432-1327.1999.00054.x.


Leukotriene B4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide-binding regulatory protein (G-protein)-coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G-proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G-proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G-proteins, in combination with the fraction which contained endogenous phospholipids and Gbeta gamma. Kinetic studies of LTB4 were performed to determine the association with G-proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G-proteins showed a lower affinity for LTB4 (Kd = 500 nm), but reconstitution of the BLT fraction with a G-protein-rich fraction (flow-through of an RCA column) increased the affinity for LTB4 10-fold (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant alpha subunits of Gi1, Gi2, Gi3, and Go expressed in Spodoptera frugiperda-9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin-sensitive G-proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G-protein-coupled receptors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Chromatography, Affinity
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism
  • GTP-Binding Proteins / isolation & purification*
  • GTP-Binding Proteins / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Lectins
  • Leukocytes / chemistry*
  • Leukotriene B4 / metabolism*
  • Plant Lectins*
  • Protein Binding
  • Receptors, Leukotriene B4 / isolation & purification*
  • Receptors, Leukotriene B4 / metabolism
  • Swine


  • Lectins
  • Plant Lectins
  • Receptors, Leukotriene B4
  • Ricinus communis agglutinin-1
  • Leukotriene B4
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, Gi-Go