Differential impact of substitution of amino acids 9-13 and 91-101 of human CD14 on soluble CD14-dependent activation of cells by lipopolysaccharide

The soluble form of the endotoxin receptor CD14 is required for the LPS-ind uced activation of cells lacking membrane-bound CD14, It has been shown tha t a deletion mutant of human CD14 consisting of the N-terminal 152 amino ac ids has the capacity to mediate the stimulation of different cell types by LPS, To identify the structural domains of the molecule related to this fun ctional property, we screened a set of alanine substitution mutants using C D14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants o f human CD14 failed to mediate the LPS-induced IL-6 production in U373 cell s. These mutants were located in two regions of the molecule (aa 9-13 and 9 1-101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vasc ular endothelial and SMC, In contrast, the potential Of sCD14((91-94,96)A), and sCD14((97-101)A) to signal LPS-induced activation of human PBMC was no t significantly reduced. These results show that the regions 9-13 and 91-10 1 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be ident ical.