In addition to diverging in intrinsic activity with its differing
isoforms, PLD is extensively
regulated by
hormones,
neurotransmitters,
lipids,
small monomeric GTPases, and other small molecules that
bind to their corresponding
domains on the enzyme. PLDs regulated by these
phospholipids are commonly involved in
intracellular signal transduction. Their
activity is dependent upon the binding of these
phosphoinositides near the
active site. In plants and animals, this binding site is characterized by the presence of a
conserved sequence of
basic and
aromatic amino acids. In plants such as
Arabidopsis thaliana, this
sequence is constituted by a
RxxxxxKxR motif together with its
inverted repeat, where
R is
arginine and
K is
lysine. Its
proximity to the
active site ensures high level of
PLD1 and
PLD2 activity, and promotes the
translocation of PLD1 to
target membranes in response to
extracellular signals.
Basal regulation The intrinsic activity of PLD differs between its known isoforms. Mammalian
PLD1 and
PLD2, for example, exhibit different basal activities despite high
sequence conservation. PLD1 requires activation by protein factors such as
Arf,
Rho, or
protein kinase C, whereas PLD2 is
constitutively active. Structural differences near the active site tunnel account for this disparity: in PLD2, a segment corresponding to residues 687–696 folds into an alpha helix, widening the substrate entrance; in PLD1, the homologous residues form a flexible loop occluding the tunnel. The displacement of a partially conserved adjacent loop by the PLD2 helix may further contribute to isoform-specific regulation. In plants (e.g.
PLDα), similar conformational gating of the active site by an autoinhibitory helix is observed.
C2 domain Calcium acts as a
cofactor in PLD
isoforms that contain the
C2 domain. Binding of
Ca2+ to the
C2 domain leads to
conformational changes in the enzyme that strengthen
enzyme-substrate binding, while weakening the
association with
phosphoinositides. In some plant
isoenzymes, such as
PLDβ,
Ca2+ may bind directly to the
active site, indirectly increasing its
affinity for the
substrate by strengthening the binding of the activator
PIP2.
PX domain The
pbox consensus sequence (PX) is thought to mediate the binding of additional phosphatidylinositol phosphates, in particular,
phosphatidylinositol 5-phosphate (PtdIns5P), a lipid thought to be required for
endocytosis, may help facilitate the reinternalization of
PLD1 from the
plasma membrane.
PH domain The highly conserved
Pleckstrin homology domain (PH) is a
structural domain approximately 120
amino acids in length. It binds
phosphatidylinositides such as
phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and
phosphatidylinositol (4,5)-bisphosphate (PIP2). It may also bind
heterotrimeric G proteins via their
βγ-subunit. Binding to this
domain is also thought to facilitate the
re-internalization of the protein by increasing its
affinity to
endocytotic lipid rafts.
Interactions with small GTPases In
animal cells, small protein
factors are important additional
regulators of PLD activity. These
small monomeric GTPases are
members of the
Rho and
ARF families of the
Ras superfamily. Some of these proteins, such as
Rac1,
Cdc42, and
RhoA,
allosterically activate mammalian PLD1, directly increasing its activity. In particular, the
translocation of
cytosolic
ADP-ribosylation factor (ARF) to the
plasma membrane is essential for PLD activation. ==Physiological and pathophysiological roles==