FIGURE 5.
A lipidated, stable chemerin analog (s-Stable Chem) has higher potency than the corresponding soluble peptide. Activities were compared at both the human (a) and mouse (c) receptors. Signaling of this analog persists despite serial washes, whereas activity of the soluble counterpart (s-Stable Chem) is markedly diminished at both the human (b) and mouse (d) receptors. Structures of s-Stable Chem and l-Stable Chem are shown in Table 1. HEK293 cells were transiently transfected with cDNAs encoding: (i) human or mouse CMKLR1, (ii) a SRE5x-Luc-PEST reporter gene, (iii) a Gαq5i66V chimera, and (iv) a β-galactosidase control. Twenty-four hours after transfection, cells were stimulated with increasing concentrations of the indicated ligands for 15 min. Selected wells were then washed three times with serum-free media and plates were further incubated for 4 h. Luciferase activity, determined as described under “Experimental Procedures,” was normalized relative to the maximal value observed using saturating concentrations of s-Stable Chem in cells that were unwashed (=100%). Data points represent the mean ± S.E. from at least three independent experiments, each performed in triplicate.