TABLE 1

Nicotinic acid and acifran evoked Ca2+ responses (EC50) and nicotinic acid binding affinity (Kd) for GPR109A mutants Shown are data for GPR109A and GPR109B wild-type receptors as well as 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. Saturation binding results are the mean ± S.D. of at least three experiments; shown are the KD and Bmax values determined by Scatchard analysis of [3H]nicotinic acid binding saturation isotherms. 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).

EC50 [3H]Nicotinic Acid Binding Surface Expression
Nicotinic Acid Acifran KdBmax
μM nM pmol/mg RLU
WT receptors
   GPR109A 0.7 ± 0.2 1.9 ± 0.4 60 ± 8 3.36 ± 0.45 39 ± 2.21
   GPR109B Inactive 90 ± 12 >500 42.4 ± 5.09
GPR109A mutants
   L83V 3 ± 0.5 2 ± 0.3
   N86Y >100 88 ± 20 >500 45.27 ± 1.91
   W91S >100 96.8 ± 10 >500 51.24 ± 4.9
   K94N 1.4 ± 0.8 4 ± 0.7
   M103V 5.3 ± 1.1 2.2 ± 0.5
   L107F 3.1 ± 0.5 2.9 ± 0.3
   R142W 1 ± 0.3 4.3 ± 0.5
   I156V 0.5 ± 0.1 2.8 ± 0.4
   M167L 2.3 ± 0.3 5.6 ± 0.2
   P168L 2.3 ± 0.4 4.7 ± 0.6
   G173P 1.1 ± 0.2 2.4 ± 0.3
   L176V 3 ± 0.4 18.7 ± 2
   S178I >100 93.1 ± 15 >500 49 ± 2.8
   F198L 0.7 ± 0.1 2 ± 0.1
   R111A Inactive Inactive >500 39.27 ± 3.01
   R210A 0.8 ± 0.2 6 ± 1.5
   R251A 70 ± 12 80 ± 14 351 ± 22 4.1 ± 0.21 40.2 ± 3.4
   R253A 0.9 ± 0.1 1.9 ± 0.4
   C100A Low expression Low expression Low expression 3.5 ± 0.27
   C177A Low expression Low expression Low expression 0.51 ± 0.02
   C183A >100 >100 >500 20.37 ± 1.7
   C266A 20 ± 3 40 ± 9 160 ± 13 3.03 ± 0.25 39.87 ± 3.1