TABLE 2

Nicotinic acid and acifran evoked Ca2+ responses (EC50) and nicotinic acid binding affinity (Kd) for aromatic residue mutants of GPR109A Shown are data for the indicated GPR109A mutants. The EC50 values of nicotinic acid-induced increases in [Ca2+]i were determined in CHO-K1 cells expressing Gα15 and the indicated wild-type or mutant receptors. The KD and Bmax values were determined by Scatchard analysis of [3H]nicotinic acid binding saturation isotherms and represent the mean ± S.D. of at least three independent experiments. Relative cell surface expression levels of wild-type and GPR109A receptor mutants were determined by ELISA assay in nonpermeabilized CHO-K1 cells (see Materials and Methods).

GPR109A Mutants EC50 [3H] Nicotinic Acid Binding Surface Expression
Nicotinic Acid Acifran KdBmax
μM nM pmol/mg RLU
F180A >100 >100 >500 40.78 ± 2.5
F180L 1.8 ± 0.2 5.4 ± 6
F193A >100 >100 >500 42.2 ± 3.9
F193L 4 ± 0.6 8 ± 2
F276A >100 >100 >500 39.96 ± 4.1
F276L >100 >100 >500 37.88 ± 2.4
Y284A 81 ± 17 >100 300 ± 36 3.60 ± 0.42 39.91 ± 3.7
Y284L 153 ± 10 >100 342 ± 40 3.75 ± 0.39 39.08 ± 4.5