On the relevance of PS exposure as a regulatory event forADAM10-mediated substrate release

The “a disintegrin and metalloproteinase 10” (ADAM10) shedding activity is vital for several physiological and pathophysiological processes. Although ADAM10 activity in these processes is comprehensively described, little is known about the mechanism controlling the protease shedding activity. As a transmembrane protease, the plasma membrane itself may play an essential role in the regulation of ADAM10 shedding activity. Phosphatidylserine (PS) exposure was shown before to be the major regulatory mechanism of ADAM17 shedding activity, the structural and functional closest relative to ADAM10. In a series of experiments with different cell types, the headgroup of phosphatidylserine (OPS) showed a competitive inhibitory effect on ADAM10-mediated substrate release, presumably by abolishing ADAM10 interaction with PS. This inhibitory effect of OPS on ADAM10-mediated substrate release was shown for multiple ADAM10 substrates and stimuli, indicating that PS exposure is in general essential for ADAM10-mediated substrate release. Overexpression of phospholipid scramblase TMEM16F/ANO6 contributed to PS exposure and led to enhanced ADAM10-mediated substrate release. Hyperactive TMEM16F/ANO6 mutant (human ANO6 D408G) showed enhanced PS exposure in conjunction with increased ADAM10-mediated substrate release, without any stimulation. B-cells from a patient with the Scott syndrome showed TMEM16F/ANO6-related impaired PS scrambling, resulting in the absence of ADAM10 shedding activity. Furthermore, biophysical experiments with a peptide version of the ADAM10 stalk domain indicated a direct interaction of ADAM10 with PS. The mutation of the positively charged amino acids in the stalk domain of ADAM10 significantly reduced the ability of the proteases to release its substrate betacellulin. These results highlight the importance of the positively charged amino acids in the ADAM10 stalk domain for the interaction with PS and substrate shedding