Characterization of platelet-activating factor (PAF) receptor by specific binding of [3H]L-659,989, a PAF receptor antagonist, to rabbit platelet membranes: possible multiple conformational states of a single type of PAF receptors

SB Hwang, MH Lam and AH Hsu

Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065- 0900.

(trans)-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989) is a potent and orally active platelet-activating factor (PAF)-specific and competitive receptor antagonist. The 2,5-tritium-labeled L-659,989 ([3H]L-659,989) specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (KD) of 1.60 +/- 0.20 nM in 10 mM MgCl2. Several selected PAF analogs and PAF receptor antagonists show equilibrium inhibition constants roughly similar to those found in the specific [3H]PAF binding assay. Other pharmacological agents with no PAF antagonistic activities do not inhibit the specific binding of [3H]L- 659,989. K+ and divalent cations such as Mg2+, Ca2+, and Mn2+ potentiate the specific [3H]L-659,989 binding. Na+ and Li+ also enhance but GTP shows no effect on the specific binding of [3H]L-659,989. However, Ni2+ inhibits the specific binding. Scatchard analysis demonstrates that the potentiating effect of these cations is due to an increase in the detectable receptor number for L-659,989. In 10 mM MgCl2 [3H]L-659,989 shows higher receptor number than [3H]PAF. Under various ionic conditions with or without GTP, in which [3H] L-659,989 binding remains approximately the same, C16-PAF shows different potencies in competing against the specific [3H] L-659,989 binding. These results demonstrate the existence of multiple conformational states of the PAF-specific receptor. The variation in the detectable receptor number under different conditions is due to the coexistence of the high and low affinity states and the fact that the low affinity state(s) of the receptor with KD value(s) possibly in the micromolar range cannot be detected in the Scatchard analysis with the radioligand at nanomolar concentrations. In the presence of 150 mM NaCl and 1 mM GTP, receptors exist in a single conformational state with an equilibrium dissociation constant (KB) of 0.931 microM for PAF.

Volume 35, Issue 1, pp. 48-58, 01/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics