Gerard N P, Gerard C
Department of Medicine, Beth Israel Hospital, Boston, MA 02215.
J Immunol. 1994 Jan 15;152(2):793-800.
A human neutrophil platelet-activating factor (PAF) receptor expressed in transfected cells was utilized to study receptor-dependent interactions with the ligand. This receptor displays ligand-binding properties comparable with those observed with naturally occurring receptor-positive cells when binding experiments are performed using COS-7 cells at 4 degrees C. The ligand-receptor interaction is markedly temperature dependent, with approximately 10-fold more [3H]PAF specifically associated with the cells at 37 degrees C than at 4 degrees C. Such temperature dependence is not observed with other ligand-receptor pairs. At 37 degrees C essentially all the cell-associated PAF appears to be internalized and incorporated into a phospholipid pool. In the absence of transfected receptor no specific binding is detected in COS-7 cells at 4 degrees C, and at 37 degrees C, no significant ligand internalization is observed. The epitope-tagged fusion protein, Flag-PAF receptor, was used to track the receptor independently of ligand binding, and showed that the receptor protein expressed on the cell surface is not influenced by temperature alone. Incubation with PAF at 37 degrees C resulted in the disappearance of approximately 20% of the Flag-PAF receptor epitope from the cell surface relative to cells incubated without PAF or cells preincubated with or without PAF at 4 degrees C. Ligand internalization in PAF receptor-transfected cells is inhibited by the receptor antagonist, WEB 2086, further supporting the receptor-dependence of this action. Stimulation of untransfected cells with phorbol ester, or C5a receptor-transfected cells with C5a, shows minimal increase in PAF internalization relative to PAF receptor-transfected cells, another feature that distinguishes the receptor-dependent pathway. These data suggest that in the COS-7 cell system PAF internalization is dependent on expression of the PAF receptor, and that the apparently nonspecific uptake observed in human neutrophils and other cells may involve receptor-independent mechanisms.
