TABLE 1.
RXFP1 variants and their corresponding mutation as well as the template DNA and primers used in QuikChange site-directed mutagenesis
| Receptor construct | Mutation introduced | Template DNA | Sense mutagenic primer (5′–3′ DNA sequence) | Antisense mutagenic primer (5′–3′ DNA sequence) |
|---|---|---|---|---|
| Gain-of-function mutants | ||||
| Single site | ||||
| G12del-RXFP1-LB2 | G12del | RXFP1-LB2 | gctgtggccgtgtcaaccgctgc | ggccacagctgaagtccccggatttgc |
| S34A-RXFP1-LB2 | S34A | RXFP1-LB2 | ggcgcagacgagcaaggctgtggagac | ctcgtctgcgccgttgtcgcagtccacttg |
| SLGYFP-RXFP1-LB2 | K4S, S5L, D7Y, S9P | RXFP1-LB2 | ctgcagtttagggtacttcccctgtgggggccgtgtcaac | cacaggggaagtaccctaaactgcaggtgacggatccacg |
| NITK-RXFP1-LB2 | G12del, R13N, V14I, N15T, R16K | RXFP1-LB2 | ggacttcagctgtgggaacatcacaaagtgcattcctcagttctgg | ccagaactgaggaatgcactttgtgatgttcccacagctgaagtcc |
| LLH-RXFP1-LB2 | F21L, W22L, R23H | RXFP1-LB2 | cgctgcattcctcagctcctgcactgcgatggccaagtgg | ccacttggccatcgcagtgcaggagctgaggaatgcagcg |
| NGVD-RXFP1-LB2 | D25N, Q27V, V28D | RXFP1-LB2 | cctcagttctggaggtgcaacggcgtggacgactgcgacaacggc | gccgttgtcgcagtcgtccacgccgttgcacctccagaactgagg |
| Double site | ||||
| SLGYFP G12del-RXFP1-LB2 | K4S, S5L, D7Y, S9P, G12del | SLGYFP-RXFP1-LB2 | cctgtggccgtgtcaaccgct | cggccacaggggaagtaccctaaactgc |
| SLGYFP NITK-RXFP1-LB2 | K4S, S5L, D7Y, S9P, G12del, R13N, V14I, N15T, R16K | SLGYFP-RXFP1-LB2 | gggaacatcacaaagtgcattcctcagttctggaggtgc | gcactttgtgatgttcccacaggggaagtaccctaaactg |
| Loss-of-function mutants | ||||
| Single residue | ||||
| L7A-RXFP1 | L7A | RXFP1 | ctccgccggctatttcccctgtgggaacatc | gccggcggagcacttgacatcctggtcg |
| L7K-RXFP1 | L7K | RXFP1 | ctccaaaggctatttcccctgtgggaacatc | gcctttggagcacttgacatcctggtcg |
| Y9F-RXFP1 | Y9F | RXFP1 | ggctttttcccctgtgggaacatcacaaag | gaaaaagccaagggagcacttgacatcc |
| Y9M-RXFP1 | Y9M | RXFP1 | ggcatgttcccctgtgggaacatcacaaag | gaacatgccaagggagcacttgacatcc |
| I15A-RXFP1 | I15A | RXFP1 | gaacgccacaaagtgcttgcctcagctc | gtggcgttcccacaggggaaatagccaag |
| T16A-RXFP1 | T16A | RXFP1 | catcgcaaagtgcttgcctcagctcctg | ctttgcgatgttcccacaggggaaatagccaag |
| K17A-RXFP1 | K17A | RXFP1 | cacagcgtgcttgcctcagctcctg | cacgctgtgatgttcccacaggggaaatag |
| K17M-RXFP1 | K17M | RXFP1 | caatgtgcttgcctcagctcctgc | gataaaggggacacccttgtagtgttacac |
| L19A-RXFP1 | L19A | RXFP1 | gtgcgcgcctcagctcctgcactgtaacg | gctgaggcgcgcactttgtgatgttcccacaggg |
| Double residue | ||||
| L7A/L22A-RXFP1 | L7A, L22A | L7A-RXFP1 | caggccctgcactgtaacggtgtggacg | cagggcctgaggcaagcactttgtgatgttcc |
| L7K/L22K-RXFP1 | L7K, L22K | L7K-RXFP1 | cagaaactgcactgtaacggtgtggacg | cagtttctgaggcaagcactttgtgatgttcc |
| L7K/Y9M-RXFP1 | L7K, Y9M | Y9M-RXFP1 | ctccaaaggcatgttcccctgtgggaac | gcctttggagcacttgacatcctggtcg |
| L19A/P20A-RXFP1 | L19A, P20A | RXFP1 | gtgcgcggcacagctcctgcactgtaacggtg | gctgtgccgcgcactttgtgatgttcccacaggg |
| Triple residue | ||||
| L7K/Y9M/K17A-RXFP1 | L7K, Y9M, K17A | L7K/Y9M-RXFP1 | cacagcgtgcttgcctcagctcctg | gcacgctgtgatgttcccacaggggaac |