Table 1 Activity data for the mutant RXFP1 constructs used in this study compared with wild-type RXFP1.

From: The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1

Receptor construct

Cell surface exp (% RXFP1)

K d nM

H2 relaxin E max

H2 relaxin pEC 50

ML290 E max

ML290 pEC 50

RXFP1

100±4.58

0.57±0.06 (4)

116.3±4.2 (6)

10.97±0.05 (6)

91.1±6.3 (5)

6.82±0.15 (5)

G41D42-AA

107.48±13.69 (4)

>20

106.8±46.67 (3)

7.26±0.32 (3)***

81.9±3.9 (3)

6.96±0.04 (3)

N43N44-AA

108.97±12.03 (4)

3.28±0.57 (5)***

118.90±12.81 (3)

8.29±0.63 (3)**

84.51±7.6 (3)

6.69±0.02 (3)

G45W46-AA

96.76±13.37 (4)

>20

—

<6 (3)

85.4±16.5 (4)

7.29±0.17 (4)*

G41A

130.5±8.05 (3)

0.95±0.13 (4)

104.7±1.65 (3)

11.13±0.1 (3)

ND

ND

D42A

106.27±8.18 (3)

20.7±3.5 (3)***

—

<6 (4)

ND

ND

N43A

81.78±8.45 (3)

2.55±0.55 (3)***

120.23±7.81 (4)

9.06±0.48 (4)***

ND

ND

N44A

83.7±6.88 (3)

0.81±0.17 (4)

92.16±6.98 (4)

11.0±0.1 (4)

ND

ND

G45A

118.64±5.04 (3)

19.1±3.07 (3)***

135.94±31.68 (4)

7.48±0.57 (4)****

ND

ND

W46A

112.30±5.90 (3)

12.26±1.36 (4)***

109.6±13.69 (3)

7.02±0.18 (3)****

ND

ND

S47L48-AA

76.95±4.47 (3)

0.43±0.01 (3)

120.9±10.9 (3)

11.07±0.17 (3)

84.7±8.1 (4)

6.48±0.11 (4)

F50A

101.09±7.03 (4)

6.48±0.76 (4)***

91.9±2.1 (4)*

9.45±0.07 (4)***

65.6±5.6 (6)**

6.60±0.15 (6)

Q49A/F50A

99.44±8.76 (3)

6.74±1.66 (4)***

98.2±8.8 (3)

9.17±0.04 (3)***

69.7±4.48 (5)*

6.87±0.03 (5)

D51A

89.77±8.97 (4)

0.73±0.11 (3)

96.5±4.0 (3)

11.01±0.56 (3)

79.4±3.8 (4)

6.67±0.11 (4)

K52A

90.25±15.21 (4)

ND

154.63±33.73 (5)

11.06±0.11

ND

ND

F54A

118.21±12.8 (3)

2.43±0.46 (5)***

104.89±10.48 (4)

10.23±0.13 (4)***

89.68±11.93 (4)

6.72±0.03 (4)

A55L

126.58±19.22 (4)

1.54±0.22 (4)*

123.59±8.51 (5)

10.75±0.17 (5)*

111.9±16.4 (3)

6.71±0.03 (3)

A55S

137.4±18.75 (4)**

0.80±0.09 (3)

154.43±39.77 (5)

11.39±0.13 (5)

ND

ND

Y57A

138.6±7.34 (5)**

0.81±0.22 (4)

126.7±8.46 (5)

10.93±0.10 (5)

85.5±6.4 (3)

6.76±0.04 (3)

Y58A

91.88±8.46 (3)

1.56±0.09 (4)***

89.14±7.93 (3)

10.88±0.09 (3)

108.3±10.4 (3)

6.78±0.01 (3)

F54A/Y58A

95.09±10.55 (3)

6.51±1.33 (5)***,#,ф

99.94±2.19 (5)

9.87±0.08 (5)***,#,θ

72.26±3.24 (4)*

6.60±0.08 (4)

M60A

93.79±16.66 (3)

1.08±0.21 (5)

118.2±6.51 (4)

11.20±0.03 (4)

104.8±11.05 (4)

6.73±0.04(4)

T61A

127.22±8.88 (5)*

0.69±0.08 (3)

142.58±15.97 (4)

11.15±0.17 (5)

ND

ND

F66A

81.6±7.45 (3)

ND

88.29±3.52 (3)

11.20±0.08

ND

ND

E67A

91.14±7.86 (3)

ND

141.74±27.08 (5)

11.13±0.13

ND

ND

Insertion/deletion mutants

 C40Ains

97.10±7.68 (4)

3.92±0.82 (3)***

No activity

No activity

94.1±7.2 (3)

6.77±0.03 (3)

 F50Ains

135.36±12.81 (4)**

16.7±5.8 (4)***

93.62±5.34 (3)

9.41±0.13 (3)***

82.8±7.8 (3)

6.85±0.10 (3)

 E67Ains

128.19±18.28 (3)

ND

110.15±6.43(3)

11.32±0.04 (3)

ND

ND

 A68Δ

113.97±14.5 (4)

ND

93.78±3.19 (3)

11.18±0.02 (3)

ND

ND

  1. ND, not determined.
  2. Kd values from Eu-H2 relaxin saturation binding experiments; cell surface expression data from receptor expression ELISA experiments; pEC50 and maximum activity (Emax) values for both H2 relaxin and ML290 stimulated cAMP activity assays. The number of replicates of each experiment for each individual construct are shown in parentheses with data reported as±s.e.m. Significance is calculated using one-way analysis of variance (ANOVA) and uncorrected Fisher’s least squares difference multiple comparison test.
  3. *P<0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001 versus RXFP1.
  4. #P<0.05 versus F54A, фP<0.05 versus Y58A, θP≤0.001 versus Y58A.
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