Background: NKp30 is a major activating receptor of natural killer (NK) cells. Results:The stalk domain of NKp30 increases ligand binding affinity, which is modulated by glycosylation of the ectodomain of NKp30. Conclusion:The stalk domain and the glycosylation status of NKp30 are critical for NK cell killing. Significance: This is the first hint for a novel mode of receptor regulation.

Previously it was shown that the type 1 deiodinase (D1) is subject to substrate dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity we found that while considerable D1-mediated catalysis of rT3 is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT3-mediated loss of D1 activity occurs despite no change in D1mRNA levels, and is blocked by PTU treatment, suggesting a requirement for catalysis. Endogenous D1 activity in sonicates was inactivated in a dose dependent manner in HepG2 cells, with a ~50% decrease after 10 nM rT3 treatment. Inactivation of D1 was rapid, occurring after only ½ hour of rT3 treatment. D1 expressed in HEK293 cells was inactivated by rT3 in a similar manner. 75Se labeling of the D1 selenoprotein indicated that after 4 hours rT3-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT3 treatment causes a loss of D1 protein after 8-24 hours. Bioluminescence Resonance Energy Transfer (BRET) studies indicate that rT3 exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase (D2) are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.