Karen Bedard and Karl-Heinz Krause

258 KAREN BEDARD AND KARL-HEINZ KRAUSE
Given the good in vitro data on the role of p22 phox for
NOX1, NOX3, and NOX4, it is puzzling that the phenotype
of these p22 phox -deficient CGD patients did not show
obvious differences from CGD patients with other underlying
mutations. This may in part be due to the small
number of cases, the young age of the patients, and the
limited scope of the clinical examination performed in
these patients. Thus, at this point, it is unclear whether
p22 phox is indispensable in vivo for the other NOX enzymes.
2. Organizer subunits: NOXO1, p47 phox
Two NOX organizer subunits are known: p47 phox
(alias NOXO2) and NOXO1. p47 phox was discovered in the
1980s together with p67 phox (660, 923). The existence of a
p47 phox homolog was first suggested by Geiszt and Leto at
the first international meeting on NOX family NADPH
oxidases (organizer: H. Schmidt) and subsequently confirmed
by several groups (53, 147, 297, 857).
The genes for human p47 phox and NOXO1 are located
on chromosomes 7 and 16, respectively. To our knowledge,
no splice variants of p47 phox exist. In contrast, a
total of four different splice variants of NOXO1 have been
described (145).
The proteins NOXO1 and p47 phox share �25% sequence
identity to one another and share a high degree of
similarity in their functional domains (Fig. 2A). NOXO1
and p47 phox each have phox (PX) domains that interact
with membrane phospholipids. Both also have two SH3
domains that interact with the proline-rich regions in the
COOH terminal of p22 phox (528, 857). p47 phox has an autoinhibitory
region (AIR) that prevents this interaction
until the protein is phophorylated and undergoes a conformational
change. This AIR is absent in NOXO1. Finally,
both NOXO1 and p47 phox also contain a COOH-terminal
proline-rich region that can interact with SH3 domains in
NOXA1 and p67 phox , respectively (531, 857). The molecular
masses of p47 phox and NOXO1 are 47 and 41 kDa,
respectively. Both are cytosolic proteins and not glycosylated.
p47 phox is highly expressed in myeloid cells (751,
923), although it has also been detected in other tissues,
including the testis (145), inner ear (54, 145), neurons
(866), hepatocytes (739), hepatic stellate cells (9), lung
(23), glomerular mesangial cells (433), endothelial cells
(434), and vascular smooth muscle (61). NOXO1 is highly
expressed in the colon (53, 147, 297, 857), but also found
in other tissues, including testis, small intestine, liver,
kidney, pancreas, uterus, and inner ear (53, 145, 857).
The expression of p47 phox is induced under various
circumstances, including retinoic acid-induced differentiation
of monocytes and granulocytes (751), in the rat
kidney in streptozotocin-induced diabetes (37), and in
thrombin-stimulated smooth muscle cells (61). Helicobac-
Physiol Rev VOL 87 JANUARY 2007 www.prv.org
ter pylori LPS activates transcription of NOXO1 in guinea
pig gastric mucosa (443).
The minimal promoter activity for the p47 phox gene
lies in the first 86 bp of the 5�-flanking region. The myeloid-specific
transcription factor PU.1 is absolutely required
for expression in phagocytes (543). However,
p47 phox has been found in numerous other tissues, where
distinct regulatory elements are likely to play a role. The
NOXO1 promoter has so far not been studied.
The designation of p47 phox and NOXO1 as organizer
subunits comes from observations in the phagocyte
NADPH oxidase: the activator subunit p67 phox , the small
p40 phox subunit, and the GTPase Rac all fail to translocate
to the membrane in neutrophils from patients lacking
p47 phox (235, 374). The organizer role of p47 phox and
NOXO1 is also visible from the motifs found within the
two proteins (Fig. 2A): an NH 2-terminal phox homology
(PX) domain allows interaction with membrane phospholipids,
centrally located tandem SH3 domains allow interaction
with p22 phox , and a COOH-terminally located proline-rich
repeat allows interaction with p67 phox or NOXA1.
The major difference between p47 phox and NOXO1 is the
presence of an autoinhibitory domain in p47 phox , which is
absent in NOXO1. The autoinhibitory domain of p47 phox
binds to the tandem SH3 domain within the same molecule,
preventing association with p22 phox . Upon phosphorylation
of the autoinhibitory domain, the tandem SH3
domain is exposed and allows p47 phox to translocate to
the plasma membrane and bind p22 phox . The lack of an
autoinhibitory domain in NOXO1 might suggest that it is
constitutively active. Indeed, NOXO1 is found localized at
the cell membrane with NOX1 and p22 phox in transfected
HEK293 cells (146), while p47 phox translocates to the
membrane only after its autoinhibition has been released
by phosphorylation (163, 408, 866). However, constitutive
activation of NOX1 by NOXO1/NOXA1 has been observed
only in the mouse system (53, 54), while in the human
system stimulation by the PKC activator PMA was necessary
to get maximal activation (297, 857). Another difference
between these two organizer homologs is the affinity
of the PX domain for phosphatidylinositols. p47 phox binds
to 3�-phosphorylated phosphatidylinositols, the products
of phosphotidylinositol 3-kinase, while NOXO1 binds to
monophosphorylated phosphatidylinositols, which are
abundant in the membranes of nonactivated cells (146).
The physiological consequence of this differential affinity
for phospholipids remains to be determined.
The two organizer proteins NOXO1 and p47 phox can
combine interchangeably with the two activator proteins,
but the degree to which these complexes lead to activation
of NOX1 and NOX2 varies markedly with the specific
combination of subunit used (53, 54, 145, 857).
While there is no doubt that p47 phox and NOXO1 are
crucial for the organization of active NOX1, -2, and -3
complexes, some evidence also points toward a more
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