Table 4.
Summary of the Functional Characteristics of LHR IL3 Mutants after Ala-Scanning Mutagenesis
| LHRa | n | Bmax (%)a | IC50 (nm) | EC50 (nm) | Rmax(%)a | Qmut/Qwta |
|---|---|---|---|---|---|---|
| Wild type | 19 | 100 | 1.00 ± 0.12 | 0.35 ± 0.04 | 100 | 1.00 |
| K548(5.60)A | 3 | 117 ± 17 | 0.91 ± 0.15 | 0.47 ± 0.19 | 123 ± 14 | 1.00 ± 0.02 |
| I549(5.61)A | 3 | 13 ± 2b | 0.92 ± 0.13 | 1.55 ± 0.34b | 13.6 ± 2.6b | 0.50 ± 0.11b |
| Y550(5.62)A | 3 | 84 ± 9 | 0.91 ± 0.11 | 0.93 ± 0.33c | 82 ± 8 | 0.64 ± 0.07b |
| F551(5.63)A | 4 | 155 ± 29 | 0.66 ± 0.03 | 0.20 ± 0.05 | 131 ± 15 | 1.03 ± 0.06 |
| V553A | 3 | 130 ± 28 | 0.80 ± 0.12 | 0.22 ± 0.10 | 96 ± 12 | 0.87 ± 0.05 |
| R554A | 3 | 56 ± 4 | 0.87 ± 0.08 | 0.42 ± 0.07 | 66 ± 3 | 0.97 ± 0.09 |
| N555A | 3 | 140 ± 36 | 0.72 ± 0.18 | 0.17 ± 0.07 | 107 ± 27 | 1.16 ± 0.18 |
| P556A | 4 | 157 ± 51 | 0.75 ± 0.12 | 0.42 ±0.09c | 87 ± 12 | 0.49 ± 0.03b |
| E557A | 3 | 175 ± 26 | 0.74 ± 0.01 | 0.19 ± 0.07 | 126 ± 6 | 0.91 ± 0.02 |
| L558A | 3 | 133 ± 10 | 1.64 ± 0.10 | 0.25 ± 0.01 | 94 ± 0.4 | 0.97 ± 0.08 |
| M559A | 3 | 99 ± 25 | 1.92 ± 0.07 | 0.43 ± 0.06 | 117 ± 23 | 1.08 ± 0.08 |
| T561(6.27)A | 3 | 170 ± 25 | 1.60 ± 0.10 | 0.39 ± 0.04 | 108 ± 9 | 0.91 ± 0.03 |
| N562(6.28)A | 3 | 34 ± 9b | 1.34 ± 0.10 | 0.69 ± 0.10 | 66 ± 25 | 0.95 ± 0.07 |
| K563(6.29)A | 3 | 125 ± 36 | 1.00 ± 0.18 | 0.56 ± 0.24 | 95 ± 7 | 0.96 ± 0.10 |
| D564(6.30)A | 3 | 87 ± 8 | 1.62 ± 0.25 | 0.70 ± 0.10b | 84 ± 3 | 0.60 ± 0.03b |
| D564(6.30)R | 5 | 156 ± 36 | 0.81 ± 0.10 | 1.44 ± 0.09b | 27 ± 5b | 0.14 ± 0.05b |
| K570(6.36)N | 3 | 92 ± 36 | 1.87 ± 0.10 | 0.68 ± 0.06c | 77 ± 27 | 0.58 ± 0.05b |
WT LHR and the receptor mutants were expressed in HEK 293 cells and characterized via binding and signaling studies (see additional information in Table 2).
The Bmax, Rmax, and Q values for WT LHR are 34.7 ± 9.5 fmol/well, 394 ± 57 pmol/well, and 44.6 ± 6.1, respectively. Basal cAMP levels for WT LHR were 4.3 ± 0.5 pmol/well. None of the mutants in IL3 exhibited increased basal cAMP values except the D564(6.30)A and D564(6.30)R mutants that had basal cAMP values of 20.6 ± 6.3 (n = 3) and 77.1 ± 10.0 (n = 5) pmol/well, respectively.
Significantly different from WT LHR, P < 0.05 (see footnote b in Table 1).
The EC50 values were shifted somewhat to higher values, but they were not significantly different from WT LHR.