Mutational Analysis Reveals Distinct Features of the Nox4-p22^phox Complex^*

Abstract

The integral membrane protein p22^phox forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22^phox constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22^phox essential for formation and localization of the Nox4-p22^phox complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22^phox-deficient and promotes cell surface expression of the Nox4-p22^phox heterodimer. Expression of p22^phox truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22^phox interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22^phox, which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22^phox mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22^phox association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22^phox subunit with Nox4, a feature which could provide the basis for selective inhibitor development.