Androgens such as testosterone typically act through nuclear androgen receptors (AR), but they can also bind to membrane proteins to activate non-genomic signaling, independent of transcription. Here, Daniel Gorelick and colleagues find that testosterone acts through GPRC6A, a G-protein-coupled receptor, during zebrafish embryonic development. First, the authors establish an in vivo screening system to identify membrane androgen receptors that mediate androgen-dependent developmental phenotypes in zebrafish embryos. Combining chemical exposure with CRISPR/Cas9 gene editing, they observe that three androgens (androstanedione, DHT and testosterone) cause cardiac edema in embryos. Both wild-type embryos and nuclear androgen receptor mutants display cardiac developmental defects when exposed to androgens, suggesting this is mediated by pathways that are independent of nuclear androgen receptors. Next, the authors mutate putative membrane androgen receptors and find that, of the three androgens tested, only testosterone acts through GPRC6A to cause cardiac phenotypes. Exposing wild-type embryos to GPRC6A antagonists leads to reduced testosterone-dependent cardiac edema. Mechanistically, the authors find that testosterone-mediated activation of GPRC6A causes cardiac phenotypes by reducing signaling through Pak1, a serine/threonine kinase. Overall, the findings uncover a new link between testosterone and cardiac developmental defects. The study also acts as a proof-of-principal for the in vivo chemical-genetic screening approach to identify various ligand-receptor interactions.
