Responses to various pruritogens in Mrgpr-transfected cell lines. (a) Representative traces of the rise in intracellular calcium evoked by Mrgpr agonists in transfected HEK cells. Cells were loaded with the calcium indicator Fura-2 and imaged at 340- and 380-nm excitation. The 340/380 ratio is used to establish relative calcium levels. Traces of different colors are from different cells on the same cover slips. Left panel, MrgprA3-transfected cells respond to chloroquine (CQ, 1 mM) but not to histamine (His, 50 μM). Middle panel, MrgprC11-transfected cells respond to BAM8-22 (2 μM) but not CQ (1 mM). Right panel, MrgprX1-transfected cells respond to CQ (1 mM) and BAM8-22 (2 μM). (b) Structure of chloroquine and related compounds. (c) Dose-response curves in MrgprA3 and MrgprX1 stable HEK cell lines to CQ and related compounds shown in (b). Response (%) refers to the number of cells responding to the agonist. Each data point, including standard error, is taken from ≥3 experiments and over 50 cells. (d) Representative traces in cells transfected with the indicated Mrgprs to the mouse PAR-2 agonist SLIGRL-NH2 (100 μM). Experiments were performed similarly to a, except that CHO cells were used instead of HEK cells. (e) Dose-response curve to SLIGRL-NH2 and SLIGR-NH2 in MrgprC11 stably transfected cells. Note that cells respond to SLIGRL-NH2, the prototypical PAR-2 agonist, while they do not respond to SLIGR-NH2, another PAR-2 agonist. Because all responses to SLIGRL-NH2 have been ascribed solely to PAR-2, this experiment demonstrates that SLIGR-NH2 is a more selective PAR-2 agonist and that some effects of SLIGRL-NH2 may be due to MrgprC11 activation. Each data point, including standard error, is taken from ≥3 experiments and over 100 cells. (Sections a to c from Liu, Q. et al., Cell, 139, 1353–1365, 2009. Sections d and e from Liu, Q. et al., Sci. Signal, 4, ra45, 2011.)