Table 1 Cell-surface expression and signaling data for hBLT1 mutants evaluated in IP1 production assays.

From: Structural insights on ligand recognition at the human leukotriene B4 receptor 1

Category

Mutation

Cell-surface expression, % of WT ± SEM (n)

EC50 LTB4, nM ± SEM (n)

Emax, % ± SEM (n)

IC50 MK-D-046, nM ± SEM (n)

Imax, % ± SEM (n)

Wild type

hBLT1-WT

100 ± 8 (5)

0.64 ± 0.11 (7)

100.0 ± 0.1 (7)

9 ± 2 (5)

92 ± 5 (5)

Mutants of hBLT1-CC

hBLT1-CC

114 ± 8 (3)

N/D

N/D

N/A

N/A

5 mut

92 ± 7 (3)

6.1 ± 0.2 (3)

70 ± 7 (3)

5 ± 2 (3)

71 ± 8 (3)

ICL3-flav

81 ± 9 (3)

N/D

N/D

N/A

N/A

Δ 311–352

111 ± 10 (3)

1.6 ± 0.3 (3)

102 ± 13 (3)

20 ± 3 (3)

94 ± 4 (3)

L1063.41W

125 ± 13 (3)

1.16 ± 0.12 (3)

101 ± 10 (3)

18 ± 3 (3)

86 ± 6 (3)

S1163.51Y

106 ± 9 (3)

4.3 ± 0.3 (3)

75 ± 3 (3)

10.8 ± 1.9 (3)

92 ± 9 (3)

A1965.53I

127 ± 13 (3)

0.35 ± 0.14 (3)

99 ± 5 (3)

30 ± 5 (3)

82 ± 5 (3)

C2877.55F

89 ± 12 (3)

0.52 ± 0.19 (3)

102 ± 3 (3)

28 ± 6 (3)

74 ± 4 (3)

S310A

110 ± 16 (3)

0.22 ± 0.16 (3)

105 ± 6 (3)

36 ± 6 (3)

84 ± 5 (3)

Ligand-interacting residues

H943.29F

139 ± 13 (3)

13.7 ± 0.4 (3)

77 ± 11 (3)

N/D

N/D

C973.32A

142 ± 12 (3)

0.4 ± 0.3 (3)

82 ± 13 (3)

22 ± 10 (3)

45 ± 7 (3)

R1564.64K

118 ± 13 (3)

20.7 ± 0.3 (3)

80 ± 11 (3)

90 ± 60 (3)

36 ± 6 (3)

Y2376.51A

108 ± 12 (3)

109.7 ± 0.2 (3)

66 ± 5 (3)a

16 ± 8 (3)b

69 ± 5 (3)b

I2717.39A

136 ± 14 (3)

218.8 ± 0.1 (3)

54 ± 10 (3)a

4 ± 2 (3)b

60 ± 12 (3)b

Membrane channel

H1815.38W

122 ± 12 (3)

11.2 ± 0.4 (3)

91 ± 12 (3)

8 ± 2 (3)

75 ± 3 (3)

hBLT1 vs. hBLT2

H943.29Y

112 ± 7 (3)

7.33 ± 0.16 (3)

91 ± 12 (3)

N/D

N/D

G983.33A

111 ± 11 (3)

3.7 ± 0.2 (3)

73 ± 10 (3)

5.7 ± 1.5 (3)

84 ± 5 (3)

I2717.39T

122 ± 13 (3)

126.5 ± 0.3 (3)

74 ± 12 (3)a

8 ± 3 (3)b

90 ± 20 (3)b

hBLT1 vs. gpBLT1

F169ECL2L

112 ± 14 (3)

3.1 ± 0.2 (3)

92 ± 6 (3)

19 ± 3 (3)

76 ± 5 (3)

P170ECL2A

119 ± 15 (3)

2.1 ± 0.3 (3)

94 ± 8 (3)

20 ± 3 (3)

61 ± 4 (3)

S2647.32R

110 ± 20 (3)

1.08 ± 0.14 (3)

89 ± 2 (3)

10 ± 2 (3)

75 ± 4 (3)

N2687.36K

116 ± 13 (3)

0.59 ± 0.15 (3)

100 ± 5 (3)

20 ± 4 (3)

80 ± 4 (3)

4 mut

84 ± 15 (3)

1.0 ± 0.3 (3)

101 ± 10 (3)

5.8 ± 1.2 (3)

109 ± 6 (3)

gpBLT1-WT

60 ± 8 (3)

0.29 ± 0.08 (3)

107 ± 16 (3)

132 ± 5 (3)

58 ± 10 (3)

  1. Results are expressed as mean ± SEM from at least three independent experiments carried out in triplicate (cell-surface expression data) or quadruplicate (signaling data). The number of independent experiments (n) is shown in parenthesis. Cell-surface expression values of mutants are reported as % of hBLT1-WT.
  2. hBLT1 or hBLT2 human leukotriene B4 receptor 1 or 2, gpBLT1 guinea pig BLT1, IP1 myo-inositol 1 phosphate, LTB4 leukotriene B4, WT wild type, EC50 and Emax potency and efficacy of LTB4, IC50 and Imax potency and efficacy of MK-D-046 inhibition of LTB4-induced IP1 production, N/D not determined, N/A not available, CC crystallization construct, 5 mut 5 mutations from hBLT1-CC (L1063.41W, S1163.51Y, A1965.53I, C2877.55F, and S310A), ICL3-flav ICL3-flavodoxin, ∆ 311–352 truncation of hBLT1 residues 311–352, 4 mut 4 non-conserved residues in the hBLT1 binding pocket mutated to their gpBLT1 equivalents (F169ECL2L, P170ECL2A, S2647.32R, N2687.36K).
  3. aMaximal efficacy at 1 μM.
  4. bTested with 1 µM of LTB4 as EC80 was >1 µM.
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