Receptor Allostery Promotes Sonic Hedgehog Signaling During Embryonic Development

Sonic Hedgehog (SHH) signaling functions in temporal and context-dependent manners to pattern diverse tissues during embryogenesis. The signal transducer Smoothened (SMO) is regulated by sterols, oxysterols, and arachidonic acid (AA) through binding pockets in its extracellular cysteine rich domain (CRD) and 7-transmembrane (7TM) bundle. In vitro analyses suggest SMO signaling is allosterically enhanced by combinatorial ligand binding to the CRD and 7TM pockets but in vivo evidence of SMO allostery is lacking. Herein, we demonstrate that disruption of AA binding to a pocket at the top of the 7TM attenuates SHH and sterol-stimulated SMO activity. We generated a knockin mouse model of compromised AA binding and show that homozygous mutants are cyanotic with high perinatal lethality. Surviving animals fail to thrive due to cardiopulmonary mispatterning. Neurodevelopment is unaltered in these mice, suggesting context-dependent allosteric regulation of SMO signaling allows for precise tuning of pathway activity for heart and lung development. Mature SmoK399A/+ females were crossed with SmoK399A/+ males to generate litters of E9.5 Smo+/+ and SmoK399A/K399A embryos. Multiple litters were harvested, and expression profiles of wild type and mutant were compared.