Solubilization of a functionally active platelet-activating factor receptor from rabbit platelets

Abstract

Binding of platelet-activating factor (PAF) to a specific high-affinity membrane receptor has been demonstrated in numerous cell types, but very little is known about the molecular nature of this receptor. The receptor from rabbit platelets was solubilized using CHAPS, digitonin, octyl glucoside, Nonidet P-40 or sodium cholate, either with pre-bound [3H]PAF or in the absence of ligand. We have been able to demonstrate for the first time that the receptor solubilized with CHAPS, in the absence of ligand, could retain its binding activity. It migrated as a high molecular mass complex (greater than 350 kDa) on a Bio-Gel A-0.5 m gel filtration column. Binding to solubilized receptor rapidly reached an equilibrium at room temperature, but was much slower at 0 degrees C. Scatchard plots were used to calculate the number (approx. 100 per cell) and the affinity (Kd 2.5 +/- 1.4 nM) of the solubilized receptors. These values were comparable with those obtained from whole-cell binding experiments. Competition by PAF antagonists also verified that the assay was measuring PAF receptor binding activity. The presence of a protein in the receptor complex was demonstrated by heat and trypsin inactivation of binding activity. Trypsin had no effect on binding of PAF to whole cells, but was able to decrease binding activity in solubilized receptor preparations. Attempts to demonstrate the involvement of a glycoprotein by use of various lectin columns proved unsuccessful. The latter results are consistent with findings suggesting that the binding site of the PAF receptor may not be exposed at the cell surface.

Autocrine enhancement of leukotriene synthesis by endogenous leukotriene B4 and platelet‐activating factor in human neutrophils

“…Indeed, it was reported that most of the PAF generated upon A23187 challenge remains intracellular, whereas newlysynthesized PAF is mostly released upon activation of GM-CSF-primed PMN by chemoattractants (Riches et al, 1990;DeNichilo et al, 1991;Stewart & Harris, 1991b). In light of many reports which suggest that the PAF receptors are located intracellularly (Hwang & Lam, 1991;Rogers et al, 1991), the intracellular retention of endogenous PAF occurring in A23187-stimulated PMN could facilitate its interaction with its receptors, thereby providing a basis for the autocrine effects of PAF on LT synthesis under such conditions.…”