Extended Data Figure 5 |. Cell surface expression and SE301 binding for RXFP1 constructs.

a-d, Flow cytometry cell surface expression tests with HEK293T cells for RXFP1 ECL2 mutants (a), Leu402 and Leu403 hinge region mutants (b), ectodomain truncation constructs (c), and evolutionary coupling analysis Ile396 and Ser397 hinge mutants (d). Data is mean ± s.e.m., n = 3 technical replicates. Cell surface expression was calculated as a percentage of wild type RXFP1 expression level. e-f, Ratio of SE301 (Fc-tagged relaxin-2) binding to receptor expression for flow cytometry binding assays in Expi293F cells¹¹. The ratio is calculated by dividing the mean fluorescence intensity (MFI) of SE301 binding by the MFI of the expression level. Data is mean ± s.e.m., n = 3 technical replicates. Deletion or mutations to the linker region reduce the ratio of binding to expression, while LDLa deletions and ECL2 mutations retain an ability to bind SE301 (e). Ectodomain deletions (7TM + β₂N-term) reduce the ratio of binding to expression, while mutations to the hinge region maintain an ability to bind SE301 (f).