Mutational analysis reveals distinct features of the Nox4-p22 phox complex.
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.
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
Katharina
K
von Löhneysen
Deborah
D
Noack
Algirdas J
AJ
Jesaitis
Mary C
MC
Dinauer
Ulla G
UG
Knaus
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004252:DNA Mutational Analysis; D019281:Dimerization; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D051379:Mice; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism*
D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human