Fig 1.
IP-1 accumulation induced by GPR142 agonists is Gq-dependent and Gi-independent.
IP-1 levels in HEK293 cells expressing (A-B) human GPR142 or (C-D) mouse GPR142 treated with varying concentrations of (A, C) L-Trp or (B, D) Compound A, with or without different G protein inhibitors. Representative data of 3 independent experiments are shown. EC50 values, 95% confidence interval of EC50, and relative Emax are reported. NA: EC50 cannot be determined.
Fig 2.
GPR142 agonists do not induce acute calcium flux.
Calcium flux quantified by FLIPR in HEK293 cells expressing (A) human GPR142 or (B) mouse GPR142 treated with varying concentrations of ATP, L-Trp, or Compound A. Representative data of 3 independent experiments are shown.
Fig 3.
GPR142 agonists suppress cAMP via Gi signaling.
(A-B) cAMP levels in HEK293 cells expressing human GPR142 treated with or without 5μM forskolin and with varying concentrations of L-Trp (A) or CpdA (B). (C-D) cAMP levels in forskolin-stimulated HEK293 cells expressing human GPR142, either pretreated with 100 ng/ml PTX or DMSO control, and treated with varying concentrations of L-Trp (C) or CpdA (D). Data are expressed as percentage normalized to cells treated with 5μM forskolin and no addition of GPR142 agonist compounds or PTX. Representative data of 3 independent experiments are shown. EC50 values, 95% confidence interval of EC50, and relative Emax are reported. NA: EC50 value cannot be determined.
Fig 4.
ERK phosphorylation upon GPR142 activation requires both Gq and Gi-coupled signaling.
Levels of phospho-Thr202/Tyr204 ERK in HEK293 cells expressing (A-B) human GPR142 or (C-D) mouse Gpr142 treated with or without different G protein inhibitors and with varying concentrations of L-Trp (A, C) and CpdA (B, D). Representative data of 3 independent experiments are shown. EC50 values, 95% confidence interval of EC50, and relative Emax are reported. NA: EC50 value cannot be determined.
Fig 5.
DMR signal upon activation of GPR142 is Gq-dependent and largely Gi-independent.
(A) Traces of label-free DMR assays in HEK293 cells expressing human GPR142 incubated in the presence of 0.1μM CpdA and treated with or without G protein inhibitors. Representative data of 3 independent experiments are shown. (B) Concentration response curve of CpdA in HEK293 cells expressing human GPR142 treated with or without G protein inhibitors. The maximal DMR signal reached within 20000 cycles at each concentration was used to determine the concentration response curve. Data are mean ± SEM of four replicate wells. Representative data of 3 independent experiments are shown. EC50 values, 95% confidence interval of EC50, Emax (in arbitrary units), and 95% confidence interval of Emax are reported.
Fig 6.
Gq-coupled signaling mediates GPR142 activation induced GSIS in islets.
Insulin secretion was examined in pancreatic islets isolated from normal male C57 mice. GPR142 agonist triggered insulin secretion was assayed by incubating 4 islets per well in Kreb’s buffer in absence or presence of different G protein inhibitors. Data are shown as mean ± s.e.m (n = 6 wells per treatment). *p<0.05, ***p<0.001 for GPR142 agonist stimulated islets compared to 11.1mM glucose group. NS: not significantly different for GPR142 agonist stimulation compared to 11.1mM glucose group. #p<0.05, ###p<0.001 for 11.1mM glucose group compared to low glucose group.
Fig 7.
Stimulation of insulin secretion by GPR142 agonists is glucose-dependent.
Insulin secretion was examined in pancreatic islets isolated from normal male C57 mice. Islets were incubated in the presence of GPR142 agonists L-Trp (A) or CpdA (B) at various ambient glucose concentrations. Data are shown as mean ± SEM (n = 6). **p<0.01, ***p<0.001 for GPR142 agonist stimulated islets compared to the vehicle group at the same glucose concentration.
Fig 8.
Proposed model of GPR142 mediated signaling pathways in β cells.
Stimulation of GPR142 triggers activation of Gq signaling and IP-3 formation, which is critical for enhancement of glucose-dependent insulin secretion. Activation of GPR142 can also result in the activation of Gi and suppression of cAMP, the contribution of which to GPR142 function remains to be determined. Activation of both Gq and Gi signaling by GPR142 agonism result in the phosphorylation of ERK, which may contribute to the previously reported beneficial effects on β cell survival and/or proliferation for GPR142 agonists [17].