Figure 6.

Cys¹⁸ and Cys¹⁹ residues in the hGPR109A atypical N-glycosylation motif are essential for nicotinic acid-induced intracellular signaling. A) Effects of 100 μM nicotinic acid on forskolin-induced cAMP accumulation in CHO-K1 cells transiently expressing hGPR109A/WT or Ala substitution mutants (amino acids proximal to Asn¹⁷), or empty vector-transfected cells (mock) are shown. **P < 0.01, ***P < 0.001 vs. hGPR109A/WT; ANOVA with Tukey post hoc pairwise comparisons. B) Effects of nicotinic acid on forskolin-induced cAMP accumulation in CHO-K1 cells transiently expressing hGPR109A/WT (full circles), /C19A (open circles), or those transfected with empty vector (mock, full squares). C) Stimulatory effects of 100 μM nicotinic acid on the forskolin-induced cAMP accumulation in CHO-K1 cells transiently expressing hGPR109A/WT or Cys¹⁹ substitution hGPR109A mutants, or those transfected with empty vector (mock). ***P < 0.001 vs. hGPR109A/WT; ANOVA with Tukey post hoc pairwise comparisons. D) Effects of nicotinic acid on forskolin-induced cAMP accumulation in CHO-K1 cells transiently expressing WT (full circles), hGPR109A/C19S (full triangles), or /C19T (open circles), or those transfected with empty vector (mock, full squares). E) Effects of nicotinic acid on intracellular [Ca²⁺] levels in CHO-K1 cells stably expressing hGPR109A/WT (blue), /N17A (red), /C18A (aqua), /C19A (green), /C19S (purple), or /C19T (orange), or those transfected with empty vector (mock, gray). All data are represented as means ± se (n = 3). Data are representative of 3 independent experiments yielding similar results. Average EC₅₀ values for hGPR109A/WT, /N17A, /C18A, /C19A, /C19S, and /C19T in 3 independent experiments were 2.5 nM (WT), 3.5 nM (N17A), 1.9 μM (C18A), 4.6 μM (C19A), 71.3 μM (C19S), and 4.1 μM (C19T).