Mutagenesis of the RXFP1 and RXFP2 receptors did not affect binding, cell surface expression, or functional responses
All receptor constructs were expressed at the cell surface, bound relaxin (B29) with correct affinity, and stimulated cAMP accumulation with correct potency, as determined by FLAG-tag binding (cpm/3.5 × 105 cells), competition binding with 100 pM [33P]relaxin (B33) (pIC50 values) and cAMP accumulation to increasing concentrations of relaxin (pEC50 values), respectively. There was no significant difference between receptor mutants and the appropriate receptor control (RXFP1 or RXFP2) in terms of pIC50 and pEC50 values, except for the chimeric receptors as described previously (Halls et al., 2005). Values are expressed as mean ± S.E.M. of (n) experiments.
Construct | Description | Relative Expression | pIC50 | pEC50 |
|---|---|---|---|---|
| RXFP1 | RXFP1 | 1039.43 ± 279.67 (3) | 9.23 ± 0.17 (4) | 10.01 ± 0.95 (4) |
| RXFP2 | RXFP2 | 1255.07 ± 440.04 (3) | 8.39 ± 0.18 (4) | 9.49 ± 0.56 (5) |
| wtRXFP1 | RXFP1 with extra Bam site removed | 1115.98 ± 404.61 (3) | 8.90 ± 0.24 (4) | 10.81 ± 0.52 (4) |
| tRXFP1-703 | RXFP1 with C-terminal truncated at residue 703 | 876.45 ± 354.33 (3) | 9.40 ± 0.19 (5) | 9.89 ± 0.76 (5) |
| tRXFP1-747 | wtRXFP1 with C-terminal truncated at residue 747 | 952.32 ± 87.84 (3) | 9.27 ± 0.43 (4) | 9.90 ± 0.65 (4) |
| RXFP1 S704A | wtRXFP1 with Ser704 mutated to Ala | 1359.39 ± 228.00 (4) | 9.53 ± 0.33 (4) | 10.95 ± 0.35 (4) |
| RXFP1 R752A | wtRXFP1 with Arg752 mutated to Ala | 1263.80 ± 296.90 (3) | 9.26 ± 0.26 (4) | 10.85 ± 0.45 (4) |
| RXFP1 S755A | wtRXFP1 with Ser755 mutated to Ala | 773.70 ± 92.48 (3) | 9.00 ± 0.31 (4) | 10.82 ± 0.48 (4) |
| RXFP1/2 | RXFP1 ectodomain with RXFP2 transmembrane region | 1247.52 ± 167.38 (3) | 8.97 ± 0.07 (4)* | 10.37 ± 0.53 (4) |
| RXFP2/1 | RXFP2 ectodomain with RXFP1 transmembrane region | 1148.48 ± 100.33 (3) | 8.84 ± 0.11 (4)† | 10.18 ± 0.84 (4) |
| RXFP2-10ct1 | Final 10 amino acids of RXFP1 C-terminus added to C-terminus of RXFP2 | 491.79 ± 40.08 (4) | 8.70 ± 0.33 (4) | 9.47 ± 0.92 (4) |