title: L-659,989: a useful probe in the detection of multiple conformational states of PAF receptors.
abstract: L-659,989 is a potent, specific and competitive platelet-activating factor (PAF) receptor antagonist. The 2,5-tritium labeled L-659,989, similar to [3H]PAF, specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (KD) of 1.60 (+/- 0.20) nM in 10 mM MgCl2. However, guanosine 5'-triphosphate (GTP) and several cations affect the specific binding of [3H]PAF and of [3H]L-659,989 to rabbit platelet membranes in different ways. K+, Mg2+, Ca2+ and Mn2+ potentiate the specific binding of both ligands. Na+ and Li+ inhibit the specific [3H]PAF binding, but enhance the binding of [3H]L-659,989; GTP reduces the [3H]PAF binding but has no effect on the binding of [3H]-L-659,989. Ni2+ inhibits the [3H]L-659-989 binding, but has no effect on the binding of [3H]PAF. In the presence of 150 mM NaCl, [3H]L-659,989 exhibits identical KD and detectable binding sites (Bmax) values as those in the presence of 10 mM MgCl2, while KD And Bmax values of [3H]PAF are dramatically reduced in the presence of 150 mM NaCl compared to those in 10 mM MgCl2. These results suggest the existence of multiple conformational states of the PAF specific receptor and that PAF and L-659,989 bind differently to those states. In the presence of 150 mM NaCl and 1 mM GTP, receptors appear to exist in a single conformational state with an equilibrium dissociation constant (KB) of 0.93 microM for PAF as derived from the Schild plot. In isolated rabbit platelets pretreated with 10 microM ETH 227, a Na(+)-specific ionophore, the detectable [3H]PAF binding sites drop from 260 to 100 binding sites per platelet, but the binding sites for [3H]L-659,989 remain roughly the same. The Na+ binding sites which modulate the conformation of PAF receptors are therefore protected from extracellular Na+ until ionophore is added, and are probably located on the cytoplasmic side of the plasma membrane.
authors.affiliation: Merck Sharp & Dohme Research Laboratories, Department of Biochemical Regulation, Rahway, New Jersey 07065-0900.
authors.forename: S B
authors.initials: SB
authors.lastname: Hwang
authors.forename: M H
authors.initials: MH
authors.lastname: Lam
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005663:Furans / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins; D011487:Protein Conformation; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled
chemical_list: D005663:Furans; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; C054893:L 659989; C037145:1-hexadecyl-2-acetyl-glycero-3-phosphocholine