Phosphatidylinositols are important in intracellular
signaling. In response to extracellular signals, these molecules undergo
rapid turnover and generate second messengers including diacylglycerol,
inositol 1,4,5trisphosphate, phosphatidylinositol 3,4bisphosphate
(PtdIns 3,4-P2) and phosphatidylinositol 3,4,5trisphosphate
(PtdIns 3,4,5P3).
In addition to the phosphatidylinositols from liver
and soybean, Avanti now produces the ammonium salt of the phosphorylated
phosphatidylinositols, PtdIns 4-P and PtdIns 4,5-P2,
from swine brain. The specifications for these products are >99%
purity by HPLC and <2000ppm Ca+2 and Na+1
ions.
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The NMR peak assignment was based on 1H-NMR spectra with and without
31P coupling, 1H-1H COSY and 1H-31P correlation. From the 1H-31P
correlation spectrum, we can assign the up field phosphorus peak
is the phosphate group at 1 position, which bond to the 1 position
of inositol and the 3' position of glycerol. We can also assign
the peak at 3.95 ppm in the proton dimension as the proton at 1
position of inositol and the peak at 4.08 ppm as H-3' on glycerol
from the integration and coupling pattern. In the H-H cosy spectra,
H-1 proton peak was correlated to peak at 3.86 ppm and 4.25 ppm.
The peak at 3.86 ppm was assigned as H-6 because of its coupling
pattern. The peak at 4.25 ppm should be H-2, which coupled with
peak at 3.61 ppm (H-3). The peak at 3.86 ppm (H-6) coupled with
the peak at 3.45 ppm, which was H-5. Both H-5 and H-3 coupled with
the peak at 4.25 ppm. This peak should be H-4 on inositol, which
also showed the correlation with the down field phosphorus peak
in the 1H-31P correlation spectrum. The peak at 5.28 ppm was assigned
as H-2' proton on glycerol according to other known glycerol based
lipids. The peaks for H-1' and H-3' on glycerol were assigned by
their correlation with H-2' and phosphorus peak in COSY and 1H-31P
correlation. It happened the multiplet at 4.25 ppm were three proton
together (H-1', H-4 and H-2). From the above proton and phosphorus
NMR peak assignment, we can conclude the PIP sample is PI(4)P.
PIP NMR A = COSY Spectra (PI4P)
PIP NMR B = HMQC spectra (PI4P)
PIP2 AND PROTEINS:
Interactions, Organization, and Information Flow.
Annual Review of Biophysics and Biomolecular Structure 2002,
Volume 31, Pages 151-175.
By Stuart McLaughlin, Jiyao Wang, Alok Gambhir, and Diana Murray
Production of Second Messengers
Receptor-mediated activation of PLC catalyzes hydrolysis of
PIP2 to produce the second messengers DAG and IP3
(16). IP3 releases CaCC from intracellular stores
(17, 27), whereas DAG remains in the membrane and helps activate
PKC by binding to its C1 domain (54). More recently it has
been recognized that PIP3, which can be formed
from phosphorylation of PIP2 by a PI3 kinase, is
also an authentic second messenger that functions as a membrane
anchor for a number of proteins (32).
It is easy to understand how the products of PI3 kinases,
such as PIP3, function as second messengers. Their
level in a quiescent cell is low (106); signaling to a PI3
kinase can thus produce a large increase in the level of the
3-phosphorylated lipid in the membrane, which can be recognized
by a binding domain with a high specificity for that lipid.
In contrast to DAG, IP3, and PIP3, where
the level of messenger can increase dramatically, the overall
concentration of PIP2 in the plasma membrane is
unlikely to increase significantly, making it less clear how
PIP2 itself acts as a second messenger (53) to
activate actin-binding proteins such as the WASP and ERM families,
and enzymes such as PLD.
Membrane Targeting
In some cases it is not necessary for the concentration of
PIP2 to increase for it to function effectively
as a membrane anchor. The PH domain of PLC± was the
first PIP2-binding domain to be understood in atomic
detail, and the local concentration effect (cheap trick #1)
explains why targeting is important with this enzyme. The
enzyme is activated not by translocation to the membrane but
by an increase in the intracellular [CaCC]: Membrane anchoring
simply facilitates interaction of the catalytic domain of
PLC± with its membrane-bound substrate PIP2
(91). TUBBY, in contrast, is apparently anchored to the plasma
membrane to prevent it from interacting with its target molecules
in the nucleus: Hydrolysis of PIP2 produces translocation
of TUBBY from membrane, allowing it to diffuse to the nucleus
(24, 93). In many other cases, PIP2 acts as a second
messenger and activates proteins.
Enzyme Activation
We consider only one example, a major PIP2 synthesis
pathway in mammalian cells. The PI4P 5-kinases (PIP kinases)
produce PIP2 mainly by phosphorylating PI4P; they
are strongly activated by phosphatidic acid (PA) (5, 40, 49,
52). PA is produced by PLD, which requires PIP2
for activation (68). Thus the potential exists for a positive
feedback loop. Actually, several complicated positive and
negative feedback loops involving PIP2 control
the activation of PIP kinases (32, 34, 89). There is also
evidence the enzymes involved in these control mechanisms
may be concentrated together in specific regions of the plasma
membrane, such as membrane ruffles (52).
Cytoskeletal Attachment
Many actin-binding proteins bind to PIP2 and are
activated by this lipid. We mentioned that PIP2
induces conformational changes in N-WASP and the ERMproteins
[more extensive lists are considered elsewhere (49, 97)].
The possibility that a local increase in the free concentration
of PIP2 acts as a signal for anchoring actin has
been discussed widely, and the importance of PIP2
in cytoskeletal attachment was demonstrated directly by elegant
experiments using laser tweezers (90). Decreasing the level
of PIP2 produced a dramatic release of the cytoskeleton
from the membrane (90).
Exocytosis, Endocytosis, and Membrane Traffic
This topic has been reviewed recently (30, 70, 97a) and there
is no lack of candidate PIP2-binding molecules
that could be involved in exocytosis and clathrin-mediated
endocytosis. Several investigators have stressed the importance
of understanding the lateral organization of PIP2
in the membrane and the role it might play in these functions.
With respect to exocytosis, Martin notes that immunocytochemical
studies from his lab using PC12 cells reveal "plasma
membrane rafts of PIP2 that colocalize with secretory
granules" (70). With respect to endocytosis, "the
focal assembly of clathrin lattices implies that there may
be PIP2 rich patches in the plasma membrane"
(45). The mechanism(s) by which these putative PIP2-enriched
rafts or patches are assembled and maintained is unclear.
Other Functions
Space limitations prevent us from discussing the many other
functions of PIP2 [e.g., regulation of ion channels
(48a), binding of scaffolding proteins]. How does one simple
lipid do all these different jobs? We consider the possibility
there are separate pools of PIP2 in the plasma
membrane.
Working With PIP2
PIP2 is more tricky to work with than other more conventional
lipids (e.g. POPC, POPS).
If you mix it in chloroform with say POPC, then dry it down
slowly and add water to hydrate,
you wind up with very nonuniform distribution of PIP2 between
the vesicles. You have to dry it
down rapidly (to prevent the PIP2 from coming out of the chloroform
before the POPC).
One also has to be cautious about sonicating the vesicles: PIP2
beaks down much more easily
than conventional lipids. It is important to hard dry the chloroform
solution under hard vacuum for
2 hours (to get rid of all traces of chloroform), use a short
duty cycle
(say 5 seconds on, 30 seconds off for sonicator, to let tip
cool) and keep the solution cool (say 10° C).
