U.S. patent application number 10/607037 was filed with the patent office on 2004-02-12 for membrane molecule indicator compositions and methods.
This patent application is currently assigned to The Netherlands Cancer Institute. Invention is credited to Jalink, Kees.
Application Number | 20040029206 10/607037 |
Document ID | / |
Family ID | 26941058 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029206 |
Kind Code |
A1 |
Jalink, Kees |
February 12, 2004 |
Membrane molecule indicator compositions and methods
Abstract
The invention provides membrane molecule indicators, including
polypeptides, encoding nucleic acid molecules and cells containing
such polypeptides and nucleic acid molecules. The invention
membrane molecule indicators are characterized in that fluorescence
resonance energy transfer (FRET) between a donor fluorescent domain
and an acceptor fluorescent domain indicates a property of the
membrane molecule. Also provided are methods of using the invention
membrane molecule indicators to determine a property of a membrane
molecule, and to identify compounds that modulates a property of a
membrane molecule.
Inventors: |
Jalink, Kees; (Heemstede,
NL) |
Correspondence
Address: |
Cathryn Campbell
McDERMOTT, WILL & EMERY
7th Floor
4370 La Jolla Village Drive
San Diego
CA
92122
US
|
Assignee: |
The Netherlands Cancer
Institute
|
Family ID: |
26941058 |
Appl. No.: |
10/607037 |
Filed: |
June 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10607037 |
Jun 25, 2003 |
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09997956 |
Nov 29, 2001 |
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6596499 |
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60250679 |
Nov 30, 2000 |
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60256559 |
Dec 18, 2000 |
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Current U.S.
Class: |
435/21 ; 435/196;
435/287.2; 435/325 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 33/5041 20130101; G01N 33/5091 20130101; G01N 33/502 20130101;
G01N 2500/00 20130101; G01N 33/5076 20130101; G01N 33/5008
20130101; G01N 2333/726 20130101; G01N 33/5005 20130101; G01N
33/542 20130101 |
Class at
Publication: |
435/21 ; 435/196;
435/325; 435/287.2 |
International
Class: |
C12Q 001/42; C12N
009/16; C12M 001/34 |
Claims
What is claimed is:
1. A phosphatidylinositol 4,5-bisphosphate (PIP2) indicator, said
indicator comprising: (a) a first polypeptide comprising: (i) a
pleckstrin homology (PH) domain; and (ii) a donor fluorescent
domain (b) a second polypeptide comprising: (i) a pleckstrin
homology (PH) domain; and (ii) an acceptor fluorescent domain;
wherein fluorescence resonance energy transfer (FRET) between said
donor domain and said acceptor domain indicates PIP2 levels.
2. The indicator of claim 1, wherein said PH domain is a
PLC.delta.1 or PLC.beta. PH domain.
3. The indicator of claim 1, wherein said donor fluorescent domain
is selected from the group consisting of a GFP and a dsRED.
4. The indicator of claim 1, wherein said donor fluorescent domain
is a CFP.
5. The indicator of claim 1, wherein said acceptor fluorescent
domain is selected from the group consisting of a GFP and a
dsRED.
6. The indicator of claim 1, wherein said acceptor fluorescent
domain is a YFP.
7. A cell comprising the indicator of claim 1.
8. A nucleic acid kit, the nucleic acid molecule components of
which encode a PIP2 indicator, said indicator comprising: (a) a
first polypeptide comprising: (i) a PH domain; and (ii) a donor
fluorescent domain (b) a second polypeptide comprising: (i) a PH
domain; and (ii) an acceptor fluorescent domain; wherein
fluorescence resonance energy transfer (FRET) between said donor
domain and said acceptor domain indicates PIP2 levels.
9. The kit of claim 8, wherein said PH domain is a PLC.beta.1 or
PLC.beta. PH domain.
10. The kit of claim 8, wherein said donor fluorescent domain is
selected from the group consisting of a GFP and a dsRED.
11. The kit of claim 8, wherein said donor fluorescent domain is a
CFP.
12. The kit of claim 8, wherein said acceptor fluorescent domain is
selected from the group consisting of a GFP and a dsRED.
13. The kit of claim 8, wherein said acceptor fluorescent domain is
a YFP.
14. A cell expressing the nucleic acid molecule components of the
kit of claim 8.
15. A method of indicating PIP2 levels in a cell, comprising: (a)
providing a cell containing the PIP2 indicator of claim 1; and (b)
determining FRET between said donor fluorescent domain and said
acceptor fluorescent domain, wherein FRET between said donor domain
and said acceptor domain indicates PIP2 levels in the cell.
16. The method of claim 15, wherein said PH domain is a PLC.delta.1
or PLC.beta. PH domain.
17. The method of claim 15, wherein said donor fluorescent domain
is selected from the group consisting of a GFP and a dsRED.
18. The method of claim 15, wherein said donor fluorescent domain
is a CFP.
19. The method of claim 15, wherein said acceptor fluorescent
domain is selected from the group consisting of a GFP and a
dsRED.
20. The method of claim 15, wherein said acceptor fluorescent
domain is a YFP.
21. The method of claim 15, wherein said cell recombinantly
expresses a G-protein coupled receptor.
22. A method of identifying a compound that modulates PIP2 levels
in a cell, comprising: (a) contacting a cell containing the PIP2
indicator of claim 1 with one or more test compounds; and (b)
determining FRET between said donor fluorescent domain and said
acceptor fluorescent domain following said contacting, wherein
increased or decreased FRET following said contacting indicates
that said test compound is a compound that modulates PIP2 levels in
the cell.
23. The method of claim 22, wherein said PH domain is a PLC.delta.1
or PLC.beta. PH domain.
24. The method of claim 22, wherein said donor fluorescent domain
is selected from the group consisting of a GFP and a dsRED.
25. The method of claim 22, wherein said donor fluorescent domain
is a CFP.
26. The method of claim 22, wherein said acceptor fluorescent
domain is selected from the group consisting of a GFP and a
dsRED.
27. The method of claim 22, wherein said acceptor fluorescent
domain is a YFP.
28. The method of claim 22, wherein said contacting is by
administration of said test compound to the exterior of said
cell.
29. The method of claim 22, wherein said contacting is by
recombinant expression of said test compound in said cell.
30. The method of claim 22, wherein said cell recombinantly
expresses a G-protein coupled receptor.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/250,679, filed Nov. 30, 2000, and U.S.
Provisional Application No. 60/256,559, filed Dec. 18, 2000, which
are both incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates generally to the field of signal
transduction and, more specifically, to compositions and methods
for indicating properties of membrane molecules using fluorescence
resonance energy transfer (FRET).
[0003] The transduction of signals from the outside to the inside
of a cell underlies most cellular processes, including
proliferation, differentiation, apoptosis, motility and invasion.
Therefore, there is considerable interest in developing improved
methods of monitoring signal transduction in response to normal and
abnormal stimuli. Methods of monitoring signal transduction have
numerous applications, such as in identifying or improving
modulators of signal transduction pathways, which are candidate
therapeutic drugs or therapeutic targets, and in detecting
pathological alterations in cells.
[0004] Some of the earliest and most sensitive signals transduced
in response to stimuli involve changes in properties of membrane
molecules, including membrane lipids and polypeptides, such as
changes in location, abundance, conformation or post-translational
modification state. Accordingly, there exists a need to develop
compositions and methods suitable for indicating changes in
properties of membrane molecule.
[0005] An early response to agonist stimulation of many tyrosine
kinase and G-protein coupled receptors is the activation of the
enzyme phospholipase C, which cleaves the lipid
phosphatidylinositol 4,5-bisphosphate (PIP2) to generate second
messengers that increase cytosolic free Ca.sup.2+ concentration.
Although Ca.sup.2+ indicators and methods have been described that
allow monitoring of Ca.sup.2+ concentration in single living cells
with high spatial and temporal resolution, Ca.sup.2+ fluxes, being
more distal to receptor activation, may not as faithfully report
receptor activation levels as changes in PIP2 levels.
[0006] In a recently developed method for detecting PIP2 dynamics
in living cells, a pleckstrin homology (PH) domain tagged with a
green fluorescent protein (GFP) has been used. Detection of PIP2
hydrolysis was by in vivo visualization, such as by confocal
imaging and post acquisition image analysis, of translocation of
the fluorescence from the membrane to the cytosol. However, this
method suffers from several disadvantages. First, it is hard to
obtain quantitative data using confocal microscopy, since even
minor focal drift and changes in cell morphology that often occur
after stimulation render quantitative measurements unreliable.
Second, it is difficult to visualize translocation in very flat
cells or in cellular subregions. Third, at fast imaging rates,
confocal imaging requires high excitation intensities that can
cause severe cell damage in minutes. Fourth, the imaging approach
is not easily extended to cell populations. Therefore, there exists
a need to develop improved methods for detecting PIP2 dynamics in
cells, and particulary methods amenable to high-throughput
screening.
[0007] The present invention satisfies these needs and provides
related advantages as well.
SUMMARY OF THE INVENTION
[0008] The invention provides a phosphatidylinositol
4,5-bisphosphate (PIP2) indicator. The indicator contains:
[0009] (a) a first polypeptide having:
[0010] (i) a pleckstrin homology (PH) domain; and
[0011] (ii) a donor fluorescent domain
[0012] (b) a second polypeptide having:
[0013] (i) a pleckstrin homology (PH) domain; and
[0014] (ii) an acceptor fluorescent domain;
[0015] wherein fluorescence resonance energy transfer (FRET)
between the donor domain and the acceptor domain indicates PIP2
levels.
[0016] Also provided is a nucleic acid kit, the nucleic acid
molecule components of which encode a PIP2 indicator, the indicator
containing:
[0017] (a) a first polypeptide having:
[0018] (i) a PH domain; and
[0019] (ii) a donor fluorescent domain
[0020] (b) a second polypeptide having:
[0021] (i) a PH domain; and
[0022] (ii) an acceptor fluorescent domain;
[0023] wherein fluorescence resonance energy transfer (FRET)
between the donor domain and the acceptor domain indicates PIP2
levels.
[0024] Further provided is a method of indicating PIP2 levels in a
cell. The method includes the steps of:
[0025] (a) providing a cell containing a PIP2 indicator; and
[0026] (b) determining FRET between the donor fluorescent domain
and the acceptor fluorescent domain,
[0027] wherein FRET between the donor domain and the acceptor
domain indicates PIP2 levels in the cell.
[0028] The invention also provides a method of identifying a
compound that modulates PIP2 levels in a cell. The method includes
the steps of:
[0029] (a) contacting a cell containing a PIP2 indicator with one
or more test compounds; and
[0030] (b) determining FRET between the donor fluorescent domain
and the acceptor fluorescent domain following the contacting,
[0031] wherein increased or decreased FRET following contacting
indicates that the test compound is a compound that modulates PIP2
levels in the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows four exemplary membrane molecule indicator
compositions. Solid bar: membrane anchoring domain. Hatched and
open boxes: fluorescent donor domain or fluorescent acceptor
domain. Thick semi-circle: MMID. Thin semi-circle: linker. Solid
circle: membrane molecule. Solid triangle: represents an altered
property of membrane molecule. (A-D): FRET is high due to
association between membrane molecule indicator domain (MMID) and
membrane molecule at the membrane. (E-H): FRET is low due to
dissociation between MMID and membrane molecule, as a result of an
altered property of membrane molecule. (I-L): FRET is low due to
altered localization of membrane molecule.
[0033] FIG. 2 shows fluorescence resonance detection of PH domain
translocation. (A) Schematic representation of FRET occurring
between CFP-PH and YFP-PH bound to the membrane. Upon hydrolysis of
PI(4,5)P2, PH domains translocate to the cytosol and FRET ceases.
(B) Emission signals of CFP and YFP, collected at 475 and 530 nm
respectively, and the ratio of 530/475, recorded from a single
N1E-115 cell stimulated with bradykinin (BK, 1 .mu.M). Signals were
low-pass filtered at 2 Hz and sampled at 3 Hz. Scale bar for ratio
signal shows percent deviation from baseline. (C) Confocal
detection of GFP-PH translocation, depicted on the same scale.
Images were collected once per 10 seconds, and the ratio of
fluorescence intensities in membrane and cytosol (PM/Cyt) was
deduced for each image by post-acquisition automated image
analysis.
[0034] FIG. 3 shows characterization of fluorescence emission.
Cells expressing constructs as indicated were stimulated with 1
.mu.M bradykinin and fluorescence emission was detected at the
indicated wavelength.
[0035] FIG. 4 shows Fluorescence Recovery After Photobleaching
(FRAP) to reveal dynamic movements of GFP-PH between cytosol and
membrane. Spots (approx. 1.3 .mu.m full-width half maximum) were
completely bleached in the basal membrane (or in the cytosol for B)
with a 30 m-s pulse of 488 nm laser light, and recovery was
monitored in line-scan mode in a confocal microscope. (A) FRAP of
membrane-delimited YFP-CAAX; (B) cytosolic PLC.delta.1PH(R40L)-GFP
mutant that cannot bind PI(4,5)P2; (C) PLC.delta.1PH-GFP in a
resting cell; (D) PLC.delta.1PH-GFP in a cell that has
agonist-induced partial translocation of fluorescence. Insets show
confocal images for the distribution of these constructs, taken
from distinct cells.
[0036] FIG. 5 shows PLC activation in single cells, neurites or in
cell populations recorded by FRET. (A) A single N1E-115 cell was
stimulated repeatedly with neurokinin A (NKA) as indicated by the
lines (dashes, 10 s pulses of 100 .mu.M NKA from a puffer pipette;
solid line, addition of 1 .mu.M final concentration to the culture
dish). The response shows repeated PLC activation and partial
desensitization. PLC activation induced by subsequently added
bradykinin (BK, 1 .mu.M) was not desensitized by NKA pretreatment.
For calibration, maximal translocation was induced by adding 5
.mu.M ionomycin+2 mM additional Ca.sup.2+. (B) PLC activation in a
single neurite of a neuroblastoma cell, differentiated by culturing
in serum-free medium for 48 hours. Area of measurement (2.5.times.9
.mu.m) is indicated in the micrograph. Excitation bandwidth was
increased to 20 nm. (C) FRET recording from a cluster of about 15
transfected cells demonstrates improved signal-to-noise ratio and
averaged kinetics (note the same scale for B and C).
[0037] FIG. 6 shows that the PH domain of PLC.delta.1 reports
changes in PI(4,5)P2 rather than in IP3 in N1E-115 cells. (A) Cells
expressing GFP-PH were loaded with both Fura-Red (20 FM) and caged
IP3 (100 PM) by in-situ high frequency electroporation. Shown is
the response of a single cell, assayed simultaneously for GFP
translocation and Ca.sup.2+ mobilization induced by flash
photolysis of caged IP3. Arrows indicate photolysis of 1 .mu.M, 10
.mu.M and 90 .mu.M. For comparison, bradykinin (1 .mu.M) was added
afterwards. Representative trace from 16 similar experiments. (B)
FRET response to bradykinin detected in a single cell, pretreated
with 5 .mu.M of phenyl arsine oxide for 10 minutes. (C-D), time
course of Ins(1,4,5)P3 and Ins(1,3,4)P3 formation in adrenal
glomerulosa cells prelabeled with [.sup.3H]inositol, after
stimulation with angiotensin II (Ang, 1 .mu.M) in the presence of 2
mM Sr.sup.2+ or Ca.sup.2+. (E) Angiotensin II-induced translocation
as quantitated by analysis of serial confocal images of glomerulosa
cells in the presence of Sr.sup.2+ or Ca.sup.2+. Data points
represent means.+-.S.E.M., n=5. (F) Bradykinin-induced
translocation, with and without Sr.sup.2+, as detected by FRET in
N1E-115 cells.
[0038] FIG. 7 shows heterogeneity of PLC activation responses to
different GPCR agonists. Single N1E-115 cells expressing CFP-PH and
YFP-PH were stimulated with 1 .mu.M bradykinin (BK), 1 .mu.M
neurokinin A (NKA), 50 .mu.M thrombin-receptor activating peptide
(TRP), 1 .mu.M lysophosphatidate (LPA) or 10 .mu.M histamine (HIS).
PLC activation as assayed by FRET, and intracellular Ca.sup.2+
recordings for these agonists detected ratiometrically using Yellow
Cameleon 2.1 in separate experiments, are shown. Changes in
fluorescence ratio are expressed as percent of resting values.
Shown are representative examples of experiments performed at least
10 times.
[0039] FIG. 8 shows that PLC inactivation kinetics mirror receptor
inactivation. (A) FRET recording from a single N1E-115 cell
stimulated with neurokinin A (NKA) and with 1 mM aluminum fluoride
(AlF.sup.4-). (B) Confocal micrographs of cells, taken 56 hours
after transfection with PLC.delta.1PH-GFP (5 .mu.g DNA/well)
together with different amounts of constitutively active G.alpha.q
subunit (Gq*, 0.8 .mu.g/well, and Gq* 1:10, 0.08 .mu.g/well) or
with constitutively active Ga12 at 0.8 .mu.g/well (G12*). (C) PLC
activation detected by FRET in single neuroblastoma cells (left
panel), expressing wild-type NKA receptors, stimulated with 10
second pulse from a puffer pipette with 100 .mu.g NKA; and cells
stimulated by prolonged addition of NKA (1 .mu.M) to the medium,
expressing either wild-type receptors (middle panel) or a mutant
truncated at its C-terminus (right panel). Recordings are all to
the same scale. (D) Kinetics of PLC activation by NKA in a N1E-115
cell transfected with the C terminally truncated NK2 receptors on
an extended time scale.
[0040] FIG. 9 shows an exemplary membrane molecule indicator. Oval:
membrane molecule. Trapezoid: MMID. The donor and acceptor
fluorescent domains are indicated. Top: FRET is high due to
association between MMID and the membrane molecule at the membrane
and proximity of the donor and acceptor. Bottom: FRET is low due to
relocalization of membrane molecule and resulting separation of the
donor and acceptor.
[0041] FIG. 10 shows an exemplary membrane molecule indicator.
Oval: membrane molecule. Trapezoid: MMID. The donor and acceptor
fluorescent domains are indicated. Top: FRET is low due to
association between MMID and the membrane molecule at the membrane
and separation of the donor and acceptor. Bottom: FRET is high due
to relocalization of membrane molecule and resulting proximity of
the donor and acceptor.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention provides membrane molecule indicator
compositions, including polypeptides, encoding nucleic acid
molecules, and cells, as well as related methods for determining
properties of a membrane molecule and for identifying modulatory
compounds.
[0043] The membrane molecule indicator compositions of the
invention are characterized by a membrane molecule indicator
domain, a donor fluorescent domain and an acceptor fluorescent
domain. The donor fluorescent domain and acceptor fluorescent
domain exhibit a characteristic fluorescence resonance energy
transfer (FRET) when the membrane molecule indicator domain is
associated with a membrane molecule at a membrane. This
characteristic FRET observed when the membrane molecule indicator
domain and membrane molecule are associated at the membrane differs
from FRET observed when the membrane molecule indicator domain
dissociates from the membrane molecule, or when the membrane
molecule is no longer localized to the membrane. Therefore, FRET
between the donor and acceptor fluorescent domains serves as an
indicator of association at the membrane between the membrane
molecule indicator domain and the membrane molecule, and thus
serves as an indicator of a property of the membrane molecule.
[0044] In one embodiment, FRET is high when the membrane molecule
indicator domain and membrane molecule are associated at the plasma
membrane (e.g. FIGS. 1A-D and FIG. 9, top), and low when the
membrane molecule indicator domain dissociates from the membrane
molecule (e.g. FIGS. 1E-H), or when the membrane molecule
relocalizes (e.g. FIGS. 1I-L and FIG. 9, bottom).
[0045] In another embodiment, FRET is low when the membrane
molecule indicator domain and membrane molecule are associated at
the plasma membrane (e.g. FIG. 10, top), and high when the membrane
molecule indicator domain dissociates from the membrane molecule,
or when the membrane molecule relocalizes (e.g. FIG. 10,
bottom).
[0046] Properties of a membrane molecule that can affect its
ability to associate at the membrane with an indicator domain
include, for example, its localization, abundance, conformation and
post-translational modifications. These properties of membrane
molecules are of considerable interest, as they often reflect
changes that occur as a result of activation or inactivation of
cellular signaling pathways that regulate fundamental cellular
processes, including growth, differentiation, apoptosis, motility
and invasion. Therefore, the invention compositions and methods can
be used to identify and determine the function of modulators of
cellular signaling pathways, and thus have important therapeutic,
diagnostic and research applications.
[0047] In one embodiment, the membrane molecule indicator
compositions of the invention contain (or encode) a single
polypeptide that contains a membrane molecule indicator domain, a
membrane anchor, a donor fluorescent domain and an acceptor
fluorescent domain (shown schematically in FIG. 1A).
[0048] In an alternative embodiment, the membrane molecule
indicator compositions of the invention contain (or encode) two
polypeptides, one containing a membrane molecule indicator domain,
the other containing a membrane anchor domain, one of which further
contains a donor fluorescent domain, the other of which further
contains an acceptor fluorescent domain (shown schematically in
FIG. 1B).
[0049] In another embodiment, the membrane molecule indicator
compositions of the invention contain (or encode) a single
polypeptide that contains two membrane molecule indicator domains,
a donor fluorescent domain and an acceptor fluorescent domain
(shown schematically in FIG. 1C).
[0050] In yet another embodiment, the membrane molecule indicator
compositions of the invention contain (or encode) two polypeptides,
each containing a membrane molecule indicator domain, one of which
contains a donor fluorescent domain and the other of which contains
an acceptor fluorescent domain (shown schematically in FIG.
1D).
[0051] In a further embodiment, the membrane molecule indicator
compositions of the invention contain (or encode) one polypeptide,
containing a central membrane molecule indicator domain, with a
donor fluorescent domain and an acceptor fluorescent domain at the
termini (shown schematically in FIGS. 9 and 10).
[0052] It will be appreciated by the skilled person that the
membrane molecule indicators shown in FIGS. 1, 9 and 10 can be
modified in a variety of ways, so long as the donor and fluorescent
domains are operably positioned so as to exhibit a characteristic
FRET when the membrane molecule indicator domain and membrane
molecule are associated at the membrane, which differs from FRET
observed when the membrane molecule indicator domain dissociates
from the membrane molecule, or when the membrane molecule is no
longer localized to the membrane.
[0053] For example, the relative locations of the donor fluorescent
domain and acceptor fluorescent domain with respect to a membrane
anchoring domain can be reversed in the compositions shown in FIGS.
1A and B. The membrane molecule indicator compositions can also
contain additional peptide or non-peptide domains, such as linker
sequences between the donor fluorescent domain and acceptor
fluorescent domain, or between a fluorescent domain and either the
MMID or the membrane anchor. Likewise, either the donor or acceptor
fluorescent domains shown in FIGS. 9 and 10 can optionally contain
membrane anchor domains.
[0054] When two MMIDs are present, the MMIDs can each associate
with the same type of membrane molecule. In such applications, the
MMIDs can be identical, or different, so long as they associate
with the same type of membrane molecule. For other applications, it
may be preferable that the MMIDs associate with different types of
membrane molecules, which are commonly or differentially regulated.
Thus, such the membrane molecule indicator compositions can
simultaneously, or alternatively, report the properties of two
different membrane molecules.
[0055] As used herein, the term "membrane molecule" refers to a
molecule that transiently, or permanently, resides at, partially or
completely within, or across, a lipid bilayer of a cell. A membrane
molecule can thus be an integral membrane molecule, such as a lipid
bilayer component or an integral membrane protein. Alternatively, a
membrane molecule can be a peripheral membrane molecule that
directly associates with the lipid bilayer, or indirectly
associates with the lipid bilayer by virtue of interaction with an
integral membrane molecule.
[0056] A membrane molecule useful in the methods of the invention
is a molecule that as a direct or indirect response to a normal or
pathological stimulus, exhibits a change in a property that results
in an increased or decreased association at the membrane between
the membrane molecule and the particular membrane molecule
indicator domain.
[0057] Exemplary properties of a membrane molecule that can change
in response to a stimulus, and which can result in an increased or
decreased association at the membrane between the membrane molecule
and the MMID, include location (e.g. translocation of the membrane
molecule from its membrane location to a different cellular
location, or vice versa), abundance (e.g. local, or overall,
increase or decrease in abundance of the membrane molecule at the
membrane), conformation (e.g. tertiary or quaternary structure,
which can reflect activation state), and post-translational
modification state (e.g. acylation, biotinylation, mannosylation,
farnesylation, formylation, geranyl-geranylation, hydroxylation,
methylation, myristoylation, palmitoylation, phosphorylation,
sulphation and the like). Therefore, such properties of a membrane
molecule, as indicated by its relative ability to associate with a
membrane molecule indicator domain, reflect the presence and nature
of the stimulus. The appropriate property which changes in response
to a stimulus, will depend on the nature of the membrane molecule
and the stimulus.
[0058] As an example of a class of membrane molecules that exhibit
changes in properties in response to stimuli, it is well known in
the art that tyrosine kinase receptors often exhibit changes in
location and abundance at the membrane (e.g. by becoming
internalized), conformation (e.g. by adopting an activated tertiary
conformation, dimerizing, or associating with effector molecules),
and/or post-translational state (e.g. by becoming tyrosine
phosphorylated) in response to ligands. Certain phospholipids
exhibit changes in abundance (e.g. by becoming hydrolyzed or
produced) in response to agonist activation of receptors. Other
examples of membrane molecules and changes in their properties in
response to stimuli, which can be detected using the methods and
compositions of the invention, are known in the art and described
further below.
[0059] As used herein, the term "membrane," with respect to the
location of a membrane molecule detected by the indicator
compositions of the invention, refers to any lipid bilayer of a
cell, including, but not limited to, the plasma membrane, Golgi
membrane, endoplasmic reticulum (ER) membrane, mitochondrial
membrane, endosomal membrane, peroxisomal membrane, lysosomal or
vacuolar membrane, and nuclear membrane.
[0060] A membrane molecule can be of any nature, such as a lipid,
protein, saccharide, or any combination thereof. In one embodiment,
the membrane molecule is a membrane lipid. Exemplary membrane
lipids include cholesterol, sphingolipids, polyisoprenoids, mono-,
di- and triacylglycerols, acyl chains and their derivatives (e.g.
arachadonic acid and its metabolites, such as prostaglandins), and
phospholipids (e.g. phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, phosphatidic acid,
phosphaytidlyglycerols, lyso-derivatives thereof and
phosphatidylinositols. Exemplary phosphatidylinositols include
PtdIns(4,5)P.sub.2 (also referred to as PIP2), PtdIns(3,4)P.sub.2,
PtdIns(3,4,5)P.sub.31 PtdIns, PtdIns(3)P, PtdIns(4)P, as well as
D-enantiomers (e.g. D-Ins(1,4,5)P.sub.3), di-carboxy derivatives
(e.g. DiC.sub.8-PtdIns(4,5)P.sub.2) and glycerophosphoryl
derivatives (e.g. g-PtdIns(4,5)P.sub.2)of these molecules.
[0061] The structural and regulatory function of membrane lipids in
normal and abnormal biological processes, as well as the changes in
properties of lipids (e.g. abundance, localization, conformation
and post-translation modifications) that occur in response to
normal and pathological stimuli, are well known in the art.
[0062] For example, a variety of sphingolipids have roles in
signaling, such as sphingosine in inhibiting PKC, ceramide in
modulating arachidonic acid (AA) release, and
sphingosine-l-phosphate in mobilizing calcium (reviewed in Shayman,
Kidney International 58:11-26 (2000). As other examples of the role
of membrane lipids in signaling, diacylglycerol (DAG) activates
protein kinase C (PKC); phosphatidic acid (PA) activates certain
kinases; and phosphatidyl choline serves as a substrate for
phospholipase D to generate PA and then DAG, as well as a substrate
for phospholipase A2 to generate AA, which is the precursor for
eicosanoids and prostaglandins.
[0063] Phosphatidylinositols are particularly important signaling
molecules. For example, many cell surface receptors are coupled to
phospholipase C activation. PLC activation cleaves the
phosphatidylinositol PIP2 to produce the second messengers inositol
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These second
messengers increase intracellular Ca.sup.2+ concentration and
activate the serine/threonine specific protein kinase C (PKC),
respectively. PIP2 also serves as a substrate for phosphatidyl
inositol 3-kinase (PI3K), producing the second messenger
phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP2 also is
implicated in the regulation of the actin cytoskeleton, based on
its ability to bind to and regulate the function of a number of
actin severing, capping and bundling proteins. Additionally, PIP2
modulates the activity of phospholipase D (PLD), which catalyzes
the hydrolysis of phosphatidylcholine to phosphatidic acid and
choline.
[0064] PIP2 resides at the plasma membrane of resting cells. Upon
agonist stimulation of a receptor coupled to PLC, such as a
tyrosine kinase receptor, or a G-protein coupled receptor (GPCR)
that acts through a G.alpha.q-containing effector G protein, PIP2
is hydrolyzed to yield soluble IP3 and membrane bound DAG. PIP2 is
then resynthesized and returns to the membrane. Accordingly, the
abundance of PIP2 at the plasma membrane reports the activation
state of a PLC-coupled receptor, in that high abundance of PIP2 at
the plasma membrane indicates the resting state, and low abundance
indicates agonistic activity through the receptor.
[0065] In an alternative embodiment, a membrane molecule is a
membrane protein. Exemplary membrane proteins include integral
membrane proteins such as cell surface receptors (e.g. G-protein
coupled receptors (GPCRs), tyrosine kinase receptors, integrins and
the like) and ion channels; and proteins that shuttle between the
membrane and cytosol in response to signaling (e.g. Ras, Rac, Raf,
G.alpha. subunits, arresting, Src and other effector proteins). In
certain embodiments, when specifically indicated, excluded from the
scope of the invention is a membrane molecule that is a GPCR.
[0066] The structural and regulatory function of membrane proteins
in normal and abnormal biological processes, as well as the changes
in their properties (e.g. abundance, localization, conformation and
post-translation modifications) that occur in response to normal
and pathological stimuli, are well known in the art.
[0067] As used herein, a "membrane molecule indicator domain" or
"MMID" refers to a domain that associates with a membrane molecule
with sufficient affinity and selectivity to report a property of
the membrane molecule. The choice of membrane molecule indicator
domain will depend on the particular membrane molecule. MMIDs for
the membrane molecules described above are known in the art, or can
be readily determined. Suitable MMIDs include, for example, domains
that mediate interaction with the membrane molecule that are
present in its naturally occurring oligomeric partner(s),
regulators and effectors, as well as functional variants of such
domains. Thus, for example, MMIDs that bind to membrane molecules
can consist of SH2, SH3, PH, PTB, EH, PDZ, EVH1 and WW domains that
bind the membrane molecule in vivo, as well as functional variants
of such domains.
[0068] In certain embodiments, such as when the membrane molecule
indicator is designed to indicate activation state of a GPCR, the
MMID can comprise a G-protein subunit, such as a G.alpha., G.beta.
or G.gamma. subunit. For example, high FRET between a G.alpha.
subunit linked to a donor fluorescent domain and a G.beta. and/or
G.gamma. subunit linked to an acceptor fluorescent domain (or vice
versa) can indicate the inactive state of the GPCR, in which the
trimeric G-protein complex is present at the membrane. In contrast,
low FRET can indicate activation of the GPCR and dissociation of
the G-protein complex. In other embodiments, when specifically
indicated, excluded from the scope of the invention is an MMID
which comprises a G-protein subunit.
[0069] MMIDs also include domains which do not normally interact
with the membrane molecule in the cell, but are determined, by
methods known in the art, to have sufficient affinity and
selectivity to report a property of the membrane molecule.
[0070] Where the membrane molecule is a phosphatidylinositol, a
suitable membrane molecule indicator domain is a
phosphatidylinositol binding domain. Phosphatidylinositol binding
domains include, for example, "pleckstrin homology" or "PH"
domains, "FYVE" domains, "C2" domains, "SH2" domains,
PtdIns-binding domains of actin-binding proteins, PtdIns-binding
domains of clathrin adaptor proteins, and START domains (reviewed
in Bottomley et al., Bioc. Biophys. Acta 1436:165-183 (1998);
Stenmark et al., J. Cell Science 112:4175-4183 (1999); Janmey,
Chem. Biol. 2:61-65 (1995); and Ponting et al., TIBS 24:130-132
(1999)).
[0071] In one embodiment, the phosphatidylinositol indicator domain
is a pleckstrin homology (PH) domain. PH domains are generally
around 120 amino acids long and share characteristic structural
features that include two orthogonal .beta.-sheets of three and
four anti-parallel .beta.-strands, which sandwich an .alpha.-helix
at the C-terminus. PH domains also contain clusters of lysine and
arginine residues distal to the C-terminal .alpha.-helix that
create a highly charged surface, and an almost invariant tryptophan
residue near the C-terminus. PH domains have been found in more
than 100 different proteins, including mammalian, Drosophila, C.
elegans and yeast proteins. Many PH domain containing proteins are
involved in intracellular signaling and cytoskeletal
organization.
[0072] Examples of PH domain containing proteins include protein
kinases (e.g. Btk, .beta.-ARK and Akt), all phospholipase C (PLC)
isoforms (e.g. PLC.beta., .gamma. and .delta.), insulin receptor
substrates (IRS-1 and IRS-2), phosphoinositide 3-kinase (PI3
kinase) p110.gamma. subunit, the guanine nucleotide release factor
SOS, rasGAP, dynamin, CDC25, Tiam-1, Vav, guanine nucleotide
exchange factors (e.g. GRP-1, ARNO, cytohesin) and .beta.-spectrin.
The sequences, ligands and relative binding affinities of a variety
of PH domains are known in the art (see, for example, Bottomley et
al., supra (1998)).
[0073] A preferred PH domain is a PH domain of a PLC, such as the
PIP2-indicator PH domain of PLC.delta.1. The cloning and expression
of the PH domain of PLC.delta.1, and its use in membrane molecule
indicator polypeptides, is described in the Example, below.
[0074] An alternative PH domain of a PLC is the PH domain of
PLC.beta.. PLC.beta. is responsible for physically cleaving PIP2,
and thus the PH domain therefrom can be used to determine
tranlocation or disassociation from the membrane of the actual PIP2
lipids cleaved by PLC.beta.. The PLC.beta. PH domain sequence is
known in the art (e.g. Rebecchi et al., Annu. Rev. Biophys. Biomol.
Struct. 27:503-528 (1998)).
[0075] In another embodiment, the phosphatidylinositol indicator
domain is an FYVE domain. FYVE domains have been demonstrated to
specifically bind to PtdIns(3)P. FYVE domains generally contain
eight conserved cysteines, which coordinate two Zn.sup.2+ ions in a
cross-braced topology, a conserved R(R/K)HHCRXCG (SEQ ID NO:1)
motif surrounding the third and fourth cysteine residues, and
several highly conserved hydrophobic residues (see, for example,
Stenmark et al., supra (1999), and Gaullier et al., Chem. Phys.
Lipids 98:87-94 (1999)). FYVE domains have been found in mammalian,
yeast and C. elegans proteins. Exemplary FYVE domain containing
proteins include EEA1, Fab1p, YOTB, Vac1p, Vps27p, Hrs, Smad anchor
for receptor activation (SARA), Fgd1, and have also been described
in a large number of proteins of unknown function whose sequences
are available in public databases (Stenmark et al., supra
(1999)).
[0076] In another embodiment, the phospholipid indicator domain is
a C2 domain. C2 domains are about 130 amino acids in length, and
have been found in single or multiple copies in over 60 proteins.
C2 domains bind a variety of ligands and substrates, including
Ca.sup.2+, phospholipids, inositol polyphosphates and intracellular
proteins. C2 domains are found, for example, in synaptotagmin
I-VIII, rabphilin, phosphatidylserine decarboxylase, protein kinase
C, GAPs, perforin, PLC family members, BCR, ABR, PI3-kinase,
cytosolic phospholipase A2, and have also been described in a large
number of proteins of unknown function whose sequences are
available in public databases (reviewed in Nalefski et al., Protein
Science 5:2375-2390 (1996).
[0077] In yet another embodiment, the phospholipid indicator domain
is an SH2 domain. SH2 domains are well-characterized mediators of
protein-protein interactions, but in addition certain SH2 domains
bind phosphoinositides. For example, the SH2 domains from
p85.alpha. and c-Src have been shown to directly and selectively
bind PtdIns(3,4,5) P.sub.3 (Bottomley et al., supra (1998)). The
sequences of a variety of SH2 domains are known in the art.
[0078] In a further embodiment, the phospholipid indicator domain
is a lipid binding domain of an actin binding protein, such as the
lipid binding domain of the actin monomer sequestering protein
profilin; the actin filament severing proteins gelsolin, villin,
severin, adseverin, destrin and cofilin; the protein gCap39, which
blocks the ends of actin filaments; and the actin filament
cross-linking protein .alpha.-actinin (reviewed in Janmey, supra
(1995)). The sequences of a variety of lipid binding domains of
actin binding proteins are known in the art.
[0079] In another embodiment, the phospholipid indicator domain is
a lipid binding domain of a clathrin adaptor protein, such as
residues 5-80 of AP-2 (.alpha.-subunit), which specifically
associates with PtdIns(3,4,5) P.sub.3, or residues 1-304 of AP-3,
which specifically associates with pyrophosphate(PP)-InsP.sub.5
(reviewed in Bottomley et al., supra (1998)). The sequences of a
variety of lipid binding domains of clathrin adaptor proteins are
known in the art.
[0080] Other membrane molecule indicator domains can be readily
identified, for example, by database searching and by structural
predictions based on sequence or structural homology to known
membrane molecule indicator domains, as described above.
[0081] Association between a MMID and a membrane molecule can be
determined by binding assays known in the art. For example,
association can be determined by co-immunoprecipitation assays,
sedimentation assays, affinity chromatography, two-hybrid assays,
gel-overlay assays, radiolabeled ligand binding assays, and the
like. Association between molecules can also be determined by
surface plasmon resonance (SPR) on BIAcore, nuclear magnetic
resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy,
and mass spectroscopy. Association between a MMID and lipid
membrane molecule can conveniently be determined by adsorbing the
MMID to a vesicle containing the lipid, and sedimenting the
vesicle-bound protein by centrifugation. Such methods are reviewed,
for example, in Winzor, J. Mol. Recognit. 13:279-298 (2000); and
Bottomley et al., supra (1998).
[0082] The membrane molecule indicator domains described herein
need not have the exact sequence of a domain found in a native
sequence, so long as the domain retains the membrane molecule
indicator function of the native sequence. Thus, a membrane
molecule indicator domain can be a variant sequence having one or
several amino acid additions, deletions or substitutions compared
with a native amino acid sequence. Such modifications can be
advantageous, for example, in enhancing the stability, expression
level, or binding specificity of the domain, as well as for
facilitating chimeric polypeptide construction. The function of a
variant MMID can be confirmed by the binding assays described
above.
[0083] Modifications to the amino acid sequence of a MMID can be
randomly generated, such as by random insertions, deletions or
substitutions of nucleotides in a native MMID nucleic acid
molecule. Alternatively, modifications can be directed, such as by
site-directed mutagenesis of a nucleic acid molecule encoding a
native MMID.
[0084] The skilled person appreciates that extensive guidance in
predicting which amino acid residues of a MMID can be modified,
while retaining membrane molecule indicator ability, is provided by
examining alignments between orthologs and other members of a
particular MMID family. It is well known in the art that
evolutionarily conserved amino acid residues and motifs are more
likely to be important for maintaining biological activity than
less well-conserved residues and domains. Thus, it would be
expected that substituting a residue that is highly conserved among
MMIDs within a family or across species with a non-conserved
residue may be deleterious, whereas making the same substitution at
a residue which varies widely would likely not have a significant
effect on biological activity. These guiding principles have been
confirmed for a variety of MMID containing proteins by mutagenesis
studies. In general, a variant MMID will have at least 70%
identity, more preferably at least 75% identity, including at least
80%, 85%, 90%, 95%, 98%, 99% or greater identity to the native
domain to which the variant domain is most closely related.
[0085] Thus, as a non-limiting example, a PIP2 indicator domain can
be a domain that has at least 70% identity, more preferably at
least 75% identity, including at least 80%, 85%, 90%, 95%, 98% or
greater identity to amino acids 1-174 of the human PLC.delta.-1
sequence (GenBank Accession No. NM.sub.--006225).
[0086] As used herein, the term "membrane anchoring domain" refers
to the portion of a membrane molecule indicator polypeptide that
localizes the polypeptide to a particular membrane. Membrane
anchoring domains suitable for localizing polypeptides to membranes
of interest are known in the art.
[0087] For example, a membrane anchoring domain suitable for
localizing a polypeptide to the plasma membrane is the C-terminal
sequence CaaX (where "a" is an aliphatic residue, and "X" is any
residue, generally L). An exemplary membrane anchoring domain
suitable for localizing a polypeptide to the endoplasmic reticulum
is the C-terminal sequence KDEL (SEQ ID NO:2), assuming a signal
sequence present at the N-terminus. Additionally, membrane
anchoring domains can be small proteins, and portions of proteins,
that confer appropriate localization to the membrane molecule
indicator polypeptide when present in a chimera.
[0088] Optionally, the membrane anchoring domain can be a second
membrane molecule indicator domain that associates with a different
membrane molecule than the first membrane molecule indicator
domain, and that is not co-regulated with the first membrane
molecule. For example, in order to determine membrane abundance of
PIP2, an appropriate indicator composition can include a membrane
molecule indicator domain that associates with PIP2 (e.g. a PH
domain) fused to a donor fluorescent domain, and a membrane
molecule indicator domain that associates with a different membrane
molecule that is not co-regulated with PIP2 fused to an acceptor
domain, which thus serves to anchor the acceptor domain to the
plasma membrane.
[0089] As used herein, the terms "donor fluorescent domain" and
"acceptor fluorescent domain" refer to a pair of moieties selected
so as to exhibit fluorescence resonance energy transfer (FRET) when
the donor moiety is excited with appropriate electromagnetic
radiation or becomes luminescent.
[0090] The donor fluorescent domain is excited by light of
appropriate intensity within its excitation spectrum, and emits the
absorbed energy as fluorescent light. When the acceptor fluorescent
domain is positioned to quench the donor fluorescent domain in the
excited state, the fluorescence energy is transferred to the
acceptor fluorescent domain, which can emit fluorescent light. FRET
can be manifested as a reduction in the intensity of the
fluorescent signal emitted from the donor fluorescent domain, by
reduction in the lifetime of the excited state of the donor
fluorescent domain, or by emission of fluorescent light at the
longer wavelengths (lower energies) characteristic of the acceptor
fluorescent domain. When the association between the MMID and the
corresponding membrane molecule changes, the donor and acceptor
fluorescent domains physically separate (or come closer together),
and FRET is decreased (or increased) accordingly (see FIG. 1).
[0091] One factor to be considered in choosing the fluorescent
domain pair is the efficiency of fluorescence resonance energy
transfer between them. Preferably, the efficiency of FRET between
the donor and acceptor moieties is at least 10%, more preferably at
least 50% and even more preferably at least 80%. The efficiency of
FRET can easily be empirically tested using the methods described
herein and known in the art.
[0092] The efficiency and detectability of FRET also depend on the
separation distance and the orientation of the donor and acceptor
fluorescent domains, as well as the choice of fluorescent domains.
Considerations for the choice of fluorescent domains are well known
in the art, and described, for example, in U.S. Pat. Nos. 5,998,204
and 5,981,200. For example, it is preferred that the emission
spectrum of the donor fluorescent domain overlap as much as
possible with the excitation spectrum of the acceptor fluorescent
domain. In addition, the excitation spectra of the donor and
acceptor fluorescent domains should overlap as little as possible
so that a wavelength region can be found at which the donor
fluorescent domain can be excited selectively and efficiently
without directly exciting the acceptor moiety. Likewise, the
emission spectra of the donor and acceptor fluorescent domains
should have minimal overlap so that the two emissions can be
distinguished. Furthermore, it is desirable that the quantum yield
of the donor fluorescent domain, the extinction coefficient of the
acceptor fluorescent domain, and the quantum yield of the acceptor
fluorescent domain be as large as possible.
[0093] For example, in a suitable pair of fluorescent domains, the
donor fluorescent domain is excited by ultraviolet light (<400
nm) and emits blue light (<500 nm), while the acceptor
fluorescent domain is efficiently excited by blue light (but not by
ultraviolet light) and emits green light (>500 nm). In an
alternative pair of fluorescent domains, the donor fluorescent
domain is excited by violet light (about 400-430 nm) and emits
blue-green light (450-500 nm), while the acceptor fluorescent
domain is efficiently excited by blue-green light (but not by
violet light) and emits yellow-green light (about 520-530 nm).
[0094] Generally, the donor fluorescent domain and acceptor
fluorescent domain will be fluorescent proteins, as described
below. Alternatively, the donor can contain a tag, such as an
artificial tetracysteine-based peptide tag, to which a cell
permeable fluorescent label, such as FLASH-EDT.sub.2, can bind
(e.g. Griffin et al., Science 281:269-272 (1998)).
[0095] Fluorescent proteins suitable for use as donor or acceptor
fluorescent domains in the compositions and methods of the
invention have been isolated from a number of species, including
jellyfish (e.g. Aequorea species) and coral (e.g. Renilla species
and Discosoma species).
[0096] In one embodiment, the donor and/or acceptor fluorescent
domain is a "green fluorescent protein" or "GFP," such as a native
GFP from an Aequorea or Renilla species, an ortholog of a GFP from
another genus, or a variant of a native GFP with optimized
properties. As used herein, the term "GFP variant" is intended to
refer to polypeptides with at least about 70%, more preferably at
least 75% identity, including at least 80%, 90%, 95% or greater
identity to a native GFP, such as Aequorea victoria GFP.
[0097] A variety of GFP variants having useful excitation and
emission spectra, have been engineered by modifying the amino acid
sequence of a naturally occurring Aequorea or Renilla GFP (see, for
example, U.S. Pat. Nos. 5,625,048 and 5,998,204; Miyawaki et al.,
Nature 388:882-887 (1997); Delagrave et al., Biotechnology
13:151-154 (1995); Pollok et al., Trends in Cell Biol. 9:57-60
(1999)). Additionally, a variety of enhanced GFPs (or EGFPs) with
optimized codons for expression in human cells, are known in the
art (e.g. ECFP and EYFP).
[0098] GFP variants with optimized dimerization properties can also
be prepared. It is postulated that the weak dimerization observed
between GFPs (e.g. kD about 100 .mu.M) allows donor and acceptor
fluorescent domains present on separate polypeptide chains (e.g.
FIG. 1B or 1D) to associate at the membrane and exhibit FRET, even
at low expression levels where based simply on polypeptide
concentration at the membrane, FRET would not be expected. The
dimerization is suitably weak so that once dissociated from the
membrane or from the membrane molecule, the donor and acceptor
fluorescent domains separate so as to no longer exhibit FRET. GFP
variants with altered dimerization properties can be selected so as
to optimize the differential in FRET between alternatives
configurations. For example, GFP variants with slightly higher, but
still moderate, dimerization (e.g. kD about 25 .mu.M) are expected
to provide for suitably high FRET at the membrane even at low
polypeptide expression levels, while still separating once
dissociated from the membrane or from the membrane molecule.
[0099] Cyan fluorescent proteins (CFPs) are variant GFPs that
contain the mutation Y66W with respect to Aequorea victoria GFP.
Yellow fluorescent proteins (YFPs) are variant GFPs that contain
aromatic residues at position 203. Blue fluorescent proteins (BFPs)
are variant GFPs that contain a Y66H mutation. A group of GFPs
which lack the near-UV excitation peak, but retain the wild-type
GFP emission peak, have Ser65 substitutions. Other variants of
native GFPs with useful fluorescent properties are known in the
art, or can be readily prepared by random or directed mutagenesis
of a native GFP. Exemplary pairs of donor and acceptor fluorescent
domains include BFP-GFP and CFP-YFP.
[0100] In another embodiment, the donor and/or acceptor fluorescent
domain is a "DsRed," such as a native DsRed from a Discosoma
species, an ortholog of DsRed from another genus, or a variant of a
native DsRed with optimized properties (e.g. a K83M variant or
DsRed2 (available from Clontech)). As used herein, the term "DsRed
variant" is intended to refer to polypeptides with at least about
70%, more preferably at least 75% identity, including at least 80%,
90%, 95% or greater identity to a native DsRed, such as a Discosoma
DsRed. Other variants of native DsReds with useful fluorescent
properties are known in the art, or can be readily prepared by
random or directed mutagenesis of a native DsRed (see, for example,
Fradkov et al., FEBS Lett. 479:127-130 (2000)).
[0101] Other exemplary pairs of donor and acceptor fluorescent
domains, respectively, include GFP-dsRED2 and YFP-dsRED2.
[0102] Included within the term "donor fluorescent domain" is a
bioluminescent domain, such as luciferase from Renilla, related
species, and variants thereof. Renilla luciferase emits blue light
in the presence of an appropriate substrate, such as coelenterazin,
which can be transferred to an appropriate fluorescent acceptor
domain, such as a GFP, in a process called Bioluminescence
Resonance Energy Transfer, or BRET. BRET is described, for example,
in Angers et al., Proc. Natl. Acad. Sci. USA 97:3684-3689 (2000);
Xu et al., Proc. Natl. Acad. Sci. USA 96:151-156 (1999); and
components are commercially available from BioSignal Packard
(Montreal, Canada). Those skilled in the art can readily apply the
compositions and methods described herein with respect to FRET, to
compositions and methods involving BRET.
[0103] In constructs in which the donor fluorescent domain and the
acceptor fluorescent domain are present on the same polypeptide,
the fluorescent domains can optionally be separated by a flexible
"linker sequence." An appropriate linker sequence allows the donor
and acceptor fluorescent domain to be functionally coupled when the
single MMID (FIG. 1A), or pair of MMIDs (FIG. 1B), are associated
with a membrane molecule, such that FRET is high, and functionally
uncoupled when the MMIDs are not associated with the membrane
molecule, such that FRET is low (FIGS. 1D and E). In order to
optimize the FRET effect, the average distance between the donor
and acceptor fluorescent domains should become less than about 10
nm when the MMID is associated with the membrane molecule (e.g.
from 1 nm to 10 nm).
[0104] The linker moiety preferably is between about 1 and 50 amino
acid residues in length, preferably between about 2 and 30 amino
acid residues. A preferred linker moiety contains, or consists of,
the sequence Gly-Gly, Ser-Gly or Gly-Ser. Linker moieties and their
applications are well known in the art and described, for example,
in U.S. Pat. Nos. 5,998,204 and 5,981,200, and Newton et al.,
Biochemistry 35:545-553 (1996).
[0105] The invention provides isolated nucleic acid molecules,
which alone or in combination as components of a kit encode
membrane molecule indicator polypeptides, including each of the
exemplary indicators shown schematically in FIGS. 1, 9 and 10 and
described above.
[0106] As used herein, the term "nucleic acid molecule" refers to a
polynucleotide comprised of either DNA or RNA; which can be single-
or double-stranded; which can optionally contain one or more
non-natural nucleotides, such as nucleotides having modifications
to the base, the sugar, or the phosphate portion; and which can
optionally contain one or more non-natural linkages, such as
phosphothioate linkages.
[0107] As used herein, the term "kit" refers to two or more
component nucleic acid molecules packaged or sold for use together.
The kit components will be contained either in a single container
or separate containers. The kit can further optionally contain
written instructions for use of the components in the methods of
the invention, and/or buffers and components suitable for such
methods.
[0108] The invention nucleic acid molecules are preferably
operatively linked to a promoter of gene expression. As used
herein, the term "operatively linked" is intended to mean that the
nucleic acid molecule is positioned with respect to either the
endogenous promoter, or a heterologous promoter, in such a manner
that the promoter will direct the transcription of RNA using the
nucleic acid molecule as a template.
[0109] Methods for operatively linking a nucleic acid to a
heterologous promoter are well known in the art and include, for
example, cloning the nucleic acid into a vector containing the
desired promoter, or appending the promoter to a nucleic acid
sequence using PCR. A nucleic acid molecule operatively linked to a
promoter of RNA transcription can be used to express membrane
molecule indicator transcripts and polypeptides in a desired host
cell or in vitro transcription or transcription-translation
system.
[0110] The choice of promoter to operatively link to an invention
nucleic acid molecule will depend on the intended application, and
can be determined by those skilled in the art. For example, if the
encoded polypeptide may be detrimental to a particular host cell,
it may be desirable to link the invention nucleic acid molecule to
a regulated promoter, such that gene expression can be turned on or
off. Alternatively, it may be preferred to have expression driven
by either a weak or strong constitutive promoter. Exemplary
promoters suitable for mammalian cell systems include, for example,
the SV40 early promoter, the cytomegalovirus (CMV) promoter, the
mouse mammary tumor virus (MMTV) steroid-inducible promoter, and
the Moloney murine leukemia virus (MMLV) promoter. Promoters
suitable in yeast include, for example, ADH promoter (S.
cerevisiae) and the inducible Nmt promoter (S. pombe).
[0111] It will be appreciated that a nucleic acid molecule encoding
a polypeptide containing a MMID and a donor fluorescent domain, and
a nucleic acid molecule encoding a polypeptide containing a MMID
and an acceptor fluorescent domain (e.g. FIGS. 1B and 1D) can
optionally be present on the same vector or under the control of
the same promoter. Such constructs are advantageous, for example,
in simplifying introducing the nucleic acid molecules into a cell
and in ensuring 1:1 stoichiometry of the donor and acceptor in the
pair. Alternatively, the nucleotide sequences encoding the two
polypeptides can be present on separate vectors or under the
control of different promoters.
[0112] The invention further provides a vector containing an
isolated nucleic acid molecule encoding a membrane molecule
indicator polypeptide. Exemplary vectors include vectors derived
from a virus, such as a bacteriophage, a baculovirus or a
retrovirus, and vectors derived from bacteria or a combination of
bacterial sequences and sequences from other organisms, such as a
cosmid or a plasmid. The vectors of the invention will generally
contain elements such as an origin of replication compatible with
the intended host cells; one or more selectable markers compatible
with the intended host cells; and one or more multiple cloning
sites. The choice of particular elements to include in a vector
will depend on factors such as the intended host cells; the insert
size; whether regulated expression of the inserted sequence is
desired; the desired copy number of the vector; the desired
selection system, and the like. The factors involved in ensuring
compatibility between a host cell and a vector for different
applications are well known in the art.
[0113] For recombinant expression of the encoded polypeptide, the
isolated nucleic acid molecules will generally be operatively
linked to a promoter of gene expression, as described above, which
may be present in the vector or in the inserted nucleic acid
molecule.
[0114] Also provided are cells containing membrane molecule
indicators, including each of the exemplary indicators shown
schematically in FIGS. 1, 9 and 10 and described above, and cells
containing nucleic acid molecules encoding such indicators. The
cells of the invention can advantageously express the encoded
polypeptide(s) and thus be used in screens for agonists,
antagonists and inverse agonists of signaling pathways indicated by
properties of the membrane molecule; to functionally clone
modulatory components of the signal transduction pathway in which
the membrane molecule is involved; and to determine or confirm the
function of potential modulatory components of the signal
transduction pathway in which the membrane molecule is involved.
Such applications are described further below.
[0115] The isolated nucleic acid molecule(s) will generally be
contained within an expression vector, but optionally can be
expressible DNA or RNA not contained within a vector. The isolated
nucleic acid molecule(s) can be maintained episomally, or
incorporated into the host cell genome. The cells of the invention
can be prepared by introducing the nucleic acid molecules of the
invention by any suitable means, including, for example,
transfection, transduction, electroporation and microinjection, as
well as by transgenic technology.
[0116] The cells of the invention can be prepared from any
organism, including, for example, bacteria (e.g. E. coli), insects
(e.g. Drosophila), yeast (e.g. S. cerevisiae, S. pombe, or Pichia
pastoris), nematodes (e.g. C. elegans), amphibians (e.g. Xenopus
embryos and oocytes) and mammals (e.g. human, rodent or primate
primary cells and established cell lines, such as COS, CHO, 3T3,
N1E-115, HEK, etc., representing either a normal or diseased state
of the mammal).
[0117] The cells of the invention can further recombinantly
express, either stably or transiently, a known or candidate
modulator of the membrane molecule, such as a known or candidate
agonist, antagonist or reverse agonist peptide; a known or
candidate receptor; or a known or candidate effector molecule.
[0118] As used herein, the term "recombinant expression," with
respect to expression of a signaling polypeptide, refers to
transient or stable expression of a polypeptide from a recombinant
nucleic acid molecule. Recombinant expression is advantageous in
providing a higher level of expression of the polypeptide than is
found endogenously, and also allows expression in cells or systems
in which the polypeptide is not normally found.
[0119] The term "recombinant nucleic acid molecule" is intended to
refer to a nucleic acid molecule that has been constructed, at
least in part, by molecular biological methods, such as PCR,
restriction digestion or ligation. A recombinant nucleic acid
expression construct generally will contain a constitutive or
inducible promoter of RNA transcription appropriate for the host
cell or transcription-translation system, operatively linked to a
nucleotide sequence that encodes the polypeptide of interest. The
expression construct can be DNA or RNA, and optionally can be
contained in a vector, such as a plasmid or viral vector.
[0120] The construction of expression vectors and the expression of
genes in transfected cells involves the use of molecular cloning
techniques well known in the art and described, for example, in
Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) and
Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,
(Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (most recent
Supplement).
[0121] The nucleotide sequences of various receptors and effectors,
and methods of recombinantly expressing the encoded polypeptides in
a variety of cell types, are well known in the art.
[0122] The invention further provides membrane molecule indicator
polypeptides. The invention polypeptides include polypeptides
recombinantly expressed by the invention nucleic acid molecules, as
well as constructs produced by chemically coupling some or all of
the component domains, which themselves can be recombinantly
produced. The expressed polypeptides can optionally be isolated
from a transcription-translation system or cell, by biochemical and
immunological purification methods known in the art. To facilitate
isolation, the invention polypeptides can optionally be fused to a
tag sequence, such as an epitope tag, a GST polypeptide or a
6.times.His fusion.
[0123] The invention polypeptides can optionally be introduced into
a whole cell, such as by recombinant expression or microinjection,
and the cells used in the methods described herein. The invention
polypeptides can alternatively be introduced into a lipid bilayer,
such as a cellular membrane extract, or an artificial lipid bilayer
(e.g. a liposome vesicle). Methods of preparing lipid bilayers
containing desired amounts and types of molecules, including the
membrane molecules and MMID polypeptides described herein, are
known in the art.
[0124] The invention also provides a method of determining a
property of a membrane molecule. The method is practiced by
[0125] (a) providing a cell or lipid bilayer comprising a membrane
molecule indicator; and
[0126] (b) determining FRET between the donor fluorescent domain
and the acceptor fluorescent domain,
[0127] wherein FRET between the donor domain and the acceptor
domain is indicative of a property of the membrane molecule.
[0128] Also provided is a method of identifying a compound that
modulates a property of a membrane molecule. The method is
practiced by
[0129] (a) contacting a cell or lipid bilayer comprising a membrane
molecule indicator with one or more test compounds, wherein the
cell or bilayer further comprises the membrane molecule; and
[0130] (b) determining FRET between the donor fluorescent domain
and the acceptor fluorescent domain following contacting,
[0131] wherein increased or decreased FRET following contacting
indicates that the test compound is a compound that modulates a
property of the membrane molecule.
[0132] The lipid bilayer useful in such methods can be a whole
cell, which naturally or recombinantly expresses the membrane
molecule, or a cellular extract containing the plasma membrane or
vesicular membranes. Alternatively, the lipid bilayer can be a
lipid vesicle, which can include either natural or synthetic lipids
or both, into which a membrane molecule of interest is
incorporated. Lipid vesicles are advantageous in that the abundance
of the membrane molecule (and, optionally, of other signaling
molecules of interest) can be controlled.
[0133] The methods of the invention are useful in the practice of
essentially any application for which a readout of signal
transduction mediated through membrane molecules is useful. Such
applications are well known in the art.
[0134] Exemplary applications include 1) identifying test compounds
that act as agonists, antagonists, inverse agonists or natural
ligands of receptors (described further below); 2) expression
cloning of peptide agonists, antagonists and inverse agonists of
receptors; 3) expression cloning of novel modulators that affect
the abundance, localization, conformation or post-translational
modification state of the membrane molecule of interest (e.g.
enzymes, enzyme inhibitors, transcriptional regulators, and the
like), which themselves can be used as therapeutic drug targets; 4)
determining the function of variants of known or predicted
modulators of membrane molecules (e.g. determining the effect of
SNPs, disease-associated mutations and engineered variations in
receptors, effectors and the like); 5) establishing dose-response
curves of modulators of membrane molecules (e.g. for predicting
effective dose of a therapeutic); and 6) determining alterations in
membrane molecules and modulators that reflect disease state, which
can be applied to the development of diagnostic methods. Methods of
using the compositions and methods described herein for such
applications, and other applications relating to signal
transduction, will be readily apparent to the skilled person.
[0135] As an example, the methods of the invention can be used to
identify test compounds that are agonists, antagonists, inverse
agonists or natural ligands of receptors, including G-protein
coupled receptors (described further below), tyrosine kinase
receptors (e.g. PDGF, IGF, FGF and EGF receptors and the like) and
integrins. In the methods of the invention, the basal level of FRET
can be determined in an unstimulated lipid bilayer. The lipid
bilayers can then be contacted with a test compound, and FRET
compared with an unstimulated bilayer. FRET is advantageous over
fluorescent visualization methods in that both increases and
decreases, relative to the basal level, can be readily determined.
Increased or decreased FRET relative to the basal level is a
reflection of the activity of the test compound as an agonist,
antagonist or inverse agonist of the signaling pathway linked to
the membrane molecule.
[0136] As used herein, the term "agonist" refers to a molecule that
selectively activates or increases signal transduction. An agonist
can act by any mechanism, such as by binding a receptor at the
normal ligand binding site, thereby mimicking the natural ligand
and promoting receptor signaling. An agonist can also act, for
example, by potentiating the binding ability of the natural ligand,
or by favorably altering the conformation of the receptor. The
compositions and methods of the invention can advantageously be
used to identify agonists that acts through any agonistic
mechanism.
[0137] As used herein, the term "antagonist" refers to a compound
that selectively inhibits or decreases signal transduction. An
important subset of antagonist compounds that can advantageously be
identified by the methods described herein, are referred to as
"inverse agonists." Inverse agonists are antagonists that
selectively inhibit or decrease signal transduction below basal
levels.
[0138] An antagonist can act by any antagonistic mechanism, such as
by binding to a ligand or receptor, thereby inhibiting their
interaction. An antagonist can also act by modifying or altering
the native conformation of .alpha.-receptor. The methods of the
invention can advantageously be used to identify an antagonist that
acts through any antagonistic mechanism.
[0139] For therapeutic applications, an agonist preferably has an
EC.sub.50, and an antagonist or inverse agonist preferably has an
IC.sub.50, of less than about 10.sup.-7 M, such as less than
10.sup.-8 M, and more preferably less than 10.sup.-9 M. However,
depending on the stability, selectivity and toxicity of the
compound, an agonist with a higher EC.sub.50, or an antagonist with
a higher IC.sub.50, can also be useful therapeutically. EC.sub.50
and IC.sub.50 of such compounds can be established by dose-response
curves using the methods described herein.
[0140] As used herein, the term "test compound" refers to any
molecule that potentially acts as an agonist, antagonist, inverse
agonist or natural ligand of a signaling pathway reported by the
membrane molecule indicator compositions and methods of the
invention. A test compound can be a naturally occurring
macromolecule, such as a polypeptide, nucleic acid, carbohydrate,
lipid, or any combination thereof. A test compound also can be a
partially or completely synthetic derivative, analog or mimetic of
such a macromolecule, or a small organic molecule prepared by
combinatorial chemistry methods.
[0141] Methods for preparing large libraries of compounds,
including simple or complex organic molecules, metal-containing
compounds, carbohydrates, peptides, proteins, peptidomimetics,
glycoproteins, lipoproteins, nucleic acids, antibodies, and the
like, are well known in the art and are described, for example, in
Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem.
Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371
(1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med.
Res. Rev. 15:481-496 (1995); and the like. Libraries containing
large numbers of natural and synthetic compounds also can be
obtained from commercial sources.
[0142] The number of different test compounds to assay in the
methods of the invention will depend on the application of the
method. For example, one or a small number of test compounds can be
advantageous in manual screening procedures, or when it is desired
to compare efficacy among several predicted ligands, agonists or
antagonists. However, it is generally understood that the larger
the number of test compounds, the greater the likelihood of
identifying a compound having the desired activity in a screening
assay. Additionally, large numbers of compounds can be processed in
high-throughput automated screening assays. Therefore, "one or more
test compounds" can be, for example, 2 or more, such as 5, 10, 15,
20, 50 or 100 or more different compounds, such as greater than
about 10.sup.3, 10.sup.5 or 10.sup.7 different compounds.
[0143] A lipid bilayer can be contacted with a test compound by any
mode, such as by extracellular administration, by intracellular
uptake of the compound, or by recombinant expression of the
compound (in methods involving an intact cell). The contacting with
the test compound can optionally take place in the presence of a
known agonist or antagonist of the signaling pathway of interest,
and the effect of the compound on agonist or antagonist-mediated
signaling can be assessed.
[0144] In cases in which the test compound is a peptide, the
peptide can preferentially be targeted to the membrane location of
the membrane molecule of interest, such as the extracellular or
intracellular face of the plasma membrane, Golgi, ER or the like,
using known methods. For example, test compounds that are peptides
can be expressed on the extracellular membrane of a cell or the
invention, or on a second cell, by phage display methods known in
the art. Alternatively, test compounds that are peptides can be
secreted from a cell of the invention, or from a second cell, by
expressing the peptide with a secretory signal.
[0145] As an example, the methods of the invention can be used to
screen for G-protein coupled receptor (GPCR) agonists, antagonists
and inverse agonists, as well as to identify the natural ligands of
orphan GPCRs.
[0146] GPCRs are seven-transmembrane-domain polypeptides that
transduce G-protein coupled signals in response to ligands. The
natural agonists of different GPCRs range from peptide and
non-peptide neurotransmitters, hormones and growth factors, to
lipids, nucleoside-sugars, amino acids, light and odorants. GPCRs
are involved in a variety of critical biological functions,
including cell proliferation, differentiation and apoptosis. GPCRs
have proven to be important targets of pharmaceuticals that affect
a variety of diseases, including neurological and psychiatric
disorders, vascular diseases, endocrinological disorders, and
cancer. It is estimated that over 50% of current drugs are targeted
towards GPCRs, and represent about a quarter of the 100 top-selling
drugs worldwide.
[0147] The natural ligands of different GPCRs include peptides,
biogenic amines, glycoproteins, nucleotides, ions, lipids, amino
acids, light and odorants. Structurally, GPCRs can be divided into
three major subfamilies, each of which currently includes orphan
receptors as well as receptors whose ligands are characterized
(reviewed in Gether, Endocrine Reviews 21:90-113 (2000)). A
database containing links to the nucleotide and amino acid
sequences of numerous mammalian GPCRs, including orphan GPCRs, is
available at http://www.darmstadt.amd.de/.about.gpcrdb/.
[0148] As used herein, the term "G-protein" refers to a class of
heterotrimeric GTP binding proteins, with subunits designated
G.alpha., G.beta. and G.gamma., that couple to seven-transmembrane
cell surface receptors to couple extracellular stimuli to
intracellular messenger molecules. G-proteins are distinguished by
their G.alpha. subunits. The more than 20 different G.alpha.
subunits, encoded by 17 different genes, can be grouped into four
major families: G.alpha.s, G.alpha.i, G.alpha.q, and G.alpha.12.
Signaling through GPCRs that couple to G.alpha.q-containing G
proteins activates PLC enzymes to hydrolyze PIP2 in the plasma
membrane to DAG and IP3.
[0149] The specificity of G.alpha. subunits for GPCRs is determined
by the C-terminal five amino acids of the G.alpha.. Thus, a variety
of signal transduction pathways can be assayed to determine
signaling through a GPCR, by co-expressing a chimeric G.alpha.
containing the five C-terminal residues of a G.alpha. known or
predicted to couple to the receptor of interest (such as G.alpha.i,
G.alpha.s or the promiscuous G.alpha.16), with the remainder of the
protein corresponding to a G.alpha. coupled to the GPCR that
signals through a membrane molecule of interest (see Conklin et
al., Nature 363:274-276 (1993), and Komatsuzaki et al., FEBS
Letters 406:165-170 (1995)).
[0150] For example, in instances in which the membrane molecule
indicator polypeptides are designed to indicate abundance of PIP2,
cells (or other lipid bilayers) can contain a GPCR of interest, and
optionally a G.alpha.q or G.alpha.16 (or chimeric or variant
G.alpha. which functions as a G.alpha.q). The basal level of FRET
between acceptor and donor fluorescent domains linked to a MMID (or
two MMIDs) that associate with PIP2 can be determined. In response
to agonist-induced signal transduction through the GPCR, PIP2 is
hydrolyzed and FRET is decreased, as exemplified in the cells
described in the Example, below. Likewise, antagonistic or inverse
agonistic effects can be determined by an increase in
agonist-induced, or basal, levels of FRET.
[0151] In the cells described in the Example, below, in which the
MMID associates with PIP2 in the plasma membrane, FRET is high in
unstimulated cells. In the presence of a test compound that
activates PLC (e.g. bradykinin), FRET is significantly lower than
in unstimulated cells, as PIP2 in the membrane is hydrolyzed, and
the donor and acceptor fluorescent domains are no longer in close
proximity. Thus, the compositions and methods described in the
Example, below, can be used to identify and compare test compounds
that stimulate the activation of PLC, that decrease the basal level
of PLC activation, or that antagonize agonist-induced PLC
activation.
[0152] PIP2 hydrolysis leads to the production of the second
messengers DAG and IP3. IP3 mediates the release of Ca.sup.2+ from
intracellular stores. Ca.sup.2+ release has been used as a
signaling assay in variety of research, diagnostic and screening
applications. The methods described herein, which detect PIP2
hydrolysis, can be used in most applications in which determination
of Ca.sup.2+ release has proven useful.
[0153] As disclosed herein, determining PIP2 hydrolysis has certain
advantages over determining Ca.sup.2+ release, in that PIP2
hydrolysis is more proximal to receptor activation, and is thus
less dependent on intermediate signaling steps that may introduce
variability. As shown herein, signals that yield similar Ca.sup.2+
responses have different PLC activation kinetics, suggesting that
PIP2 hydrolysis follows receptor activation more faithfully than
Ca.sup.2+ responses.
[0154] Optionally, PIP2 hydrolysis, as determined by the FRET
methods described herein, and Ca.sup.2+ release, as determined
using Ca.sup.2+ indicator dyes known in the art, can both be
assayed. Because Ca.sup.2+ release is downstream of PIP2
hydrolysis, Ca.sup.2+ release can be assayed simultaneously with
FRET to confirm that the observed FRET reflects PIP2
hydrolysis.
[0155] Methods of determining and quantitating FRET at the single
cell level, or in cell populations, are well known in the art or
can be determined by the skilled person. For example, FRET can be
measured using dual emission fluorescence microscopy, as described
in the Example, below. Alternatively, FRET can be measured using
fluorescent microscopy imaging methodology, which allows for
simultaneous recordings from multiple cells.
[0156] As a further example, FRET can be determined with
fluorescent lifetime. Briefly, upon excitation with an ultrashort
pulse of light (e.g. about 0.01 ns), fluorophores have a
characteristic decay in emission that is single exponential, and
may last 0.1-10 ns, dependent on the fluorophore and conditions. It
has been shown that the presence of a FRET acceptor dramatically
shortens the decay time of the donor, which can be detected either
using direct monitoring of the decay time (time domain monitoring),
or using sine-modulated light, in the frequency domain (see, for
example, Verveer et al., Biophys. J., 78:2127-37 (2000)).
[0157] For high-throughput screening applications, FRET can be
measured using fluorescence activated cell sorting (FACS), such as
with a HeCd laser or frequency-double diode laser. FACS is
advantageous in permitting the analysis of around 50,000 cells per
second, which is orders of magnitude faster than visual detection
methods. FACS also allows the isolation of cells for further
growth, manipulation and identification of nucleic acid molecules
encoding compounds that modulate association between membrane
molecules and the MMIDs of the invention compositions.
[0158] Therefore, the compositions and methods of the invention are
amenable to high-throughput screening for potential
therapeutics.
[0159] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Preparation and Use of Membrane Molecule Indicator Compositions
[0160] This example shows the preparation of two pairs of nucleic
acid molecules of the invention. In the first pair, the first
nucleic acid molecule encodes a polypeptide containing a membrane
molecule indicator domain (PH domain) and a donor fluorescent
domain (CFP), and the second nucleic acid molecule encodes a
polypeptide containing a membrane molecule indicator domain (PH
domain) and an acceptor fluorescent domain (YFP). In the second
pair, the first nucleic acid molecule encodes a polypeptide
containing a membrane molecule indicator domain (PH domain) and a
donor fluorescent domain (CFP), and the second nucleic acid
molecule encodes a polypeptide containing a membrane anchoring
domain (CaaX) and an acceptor fluorescent domain (YFP).
[0161] This example also shows the use of the pairs of nucleic acid
molecules to determine the abundance of a membrane molecule (PIP2),
by determining FRET between the donor and acceptor fluorescent
domains. High FRET results from high PIP2 abundance at the plasma
membrane, which indicates the resting state of the cell; decreased
FRET results from PIP2 hydrolysis, which indicates signaling
through a G-protein coupled receptor linked to PLC activation.
[0162] Experimental Procedures
[0163] Materials
[0164] 1-oleoyl LPA, histamine, bradykinin (BK), phenyl-arsine
oxide and quercetin were from Sigma Chemical Co. (St. Louis, Mo.);
neurokinin A, caged IP3 (cat.# 407135) and ionomycin were from
Calbiochem-Novabiochem Corp. (La Jolla, Calif.);
Myo-[.sup.3H]inositol (60 Ci/mmol) was from Amersham-Pharmacia
Biotech. Fura-red (K salt) was from Molecular Probes Inc. (Eugene,
Oreg.). All other chemicals were of analytical grade.
[0165] Constructs
[0166] The pleckstrin homology-domain of human phospholipase C
.delta.1 was obtained from the Superhiro-PLC.delta.1 PH construct
(AA 1-174, obtained from T. Meyer) and cloned into the eukaryotic
expression vector pECFP-C1 (Clontech, Calif.). Two primers
(PLC.delta.PH1; 5' CCTGC GGCCG CGGTA CCGAT ATCAG ATGTT GAGCT CCTTC
AC 3' (SEQ ID NO:3) and PLC.delta.PH2; 5' CCGAA TTCCC GGGTC TCAGC
CATGG ACTCG GGCCG GGACT TC 3' (SEQ ID NO:4)) were designed to
generate the PH-domain in frame behind the CFP followed by a stop
codon. The PCR-product was cloned into the pECFP plasmid with the
restriction sites EcoRI and EcoRV on EcoRI and SmaI, leading to
pECFP-PH.
[0167] YFP was obtained from yellow Cameleon 2.0 (obtained from A.
Miyawaki and R. Tsien) and subcloned into cloning vector PGEM3z
(Promega), via SacI and EcoRI, and subsequently into pcDNA3
(Invitrogen) via BamHI and EcoRI. PCR on YFP-pcDNA-3 with primers
T7 (Promega) and GFP3; 5' GGCTG AGACC CGGGA ATTCG GCTTG TACAG CTCGT
CCATG 3' (SEQ ID NO:5) was done to remove the stop codon. The PH
domain PCR-product, taken between primers PLC.delta.PH1 and
PLC.delta.PH2, was cloned in frame behind YFP with EcoRI and NotI,
leading to pcDNA3YFPPH. To obtain pcDNA3eGFPPH, YFP was swapped
with EGFP, using primers T7 and GFP3 on pcDNA3eGFP and restriction
enzymes BamHI and EcoRI.
[0168] For YFP-CAAX and GFP-CAAX, the membrane localization
sequence of K-Ras (KMSKD GKKKK KKSKT KCVIM; SEQ ID NO:6) was
obtained by PCR from Bp180-CAAX (GenBank accession number M54968
and M38506), using primers CAAX3 5'CCGAA TTCCC GGGTC AAGAT GAGCA
AAGAT GGTAA AAAG 3' (SEQ ID NO:7), containing an EcoRI site, and
CAAX2; 5' CCTGC GGCCG CGGTA CCGAG ATCTT TACAT AATTA CACAC TT 3'
(SEQ ID NO:8), that contained a NotI-site behind the stop codon.
The final constructs were made by exchanging the PH domain from
YFP-PH and GFP-PH for the CAAX domain using EcoRI and NotI. All
clones were verified by sequence analysis. YFP-CAAX contained a
point mutation (V instead of G in the CAAX domain), but this did
not influence the membrane localization.
[0169] Constitutively active mutants of G.alpha..sub.q and
G.alpha..sub.12 subunits in pcDNA3 vectors were obtained from Dr.
O. Kranenburg (Kranenburg et al., Mol. Biol. Cell 10:1851-1857
(1999)).
[0170] Cell Culture and Transfections
[0171] N1E-115 neuroblastoma cells were seeded in 6-well plates at
about 25,000 cells per well on 25 mm glass coverslips, and cultured
in 3 ml Dulbecco's Modified Eagle's medium (DMEM) supplemented with
10% FCS and antibiotics. Unless otherwise indicated, constructs
were transfected for 6-12 hours using calcium phosphate
precipitate, at 0.8 .mu.g DNA/well. Following transfection, cells
were incubated in serum-free DMEM for 12-48 hours. For fluorescence
detections, coverslips with cells were transferred to a culture
chamber and mounted on an inverted microscope. All experiments were
performed in bicarbonate-buffered saline (containing, in mM, 140
NaCl, 5 KCl, 1 MgCl.sub.2, 1 CaCl.sub.2, 10 glucose, with 10 mM
HEPES added), pH 7.2, kept under 5% CO.sub.2, at 37.degree. C.
[0172] Inositol Phosphate Determinations
[0173] Preparation, culture and labeling of bovine adrenal
glomerulosa cells have been described in Balla et al., J. Biol.
Chem. 269:16101-16107 (1994). Cells labeled with
myo-[.sup.3H]inositol for 24-48 hrs were stimulated by angiotensin
II (30 nM) for the indicated times in a medium containing either
Sr.sup.2+ or Ca.sup.2+. Reactions were terminated with perchloric
acid and inositol phosphates were separated by HPLC essentially as
described in Balla et al., supra (1994).
[0174] Confocal Microscopy and Image Analysis
[0175] For confocal imaging, a Leica DM-IRBE inverted microscope
fitted with TCS-SP scanhead was used. Excitation of EGFP was with
the 488 nm Argon ion laserline, and emission was collected at
500-565 nm. For translocation studies, a series of confocal images
were taken at 2-10 second intervals and stored on disk.
Determination of the ratio of membrane to cytosolic fluorescence by
directly assigning regions of interest (ROIs) for membrane and
cytosol was hampered by the shape changes of cells during
experiments. Using Qwin software (Leica) this ratio was therefore
calculated by post-acquisition automated ROI assignment and
analysis. In brief, a binary mask of the transfected cell was lined
out using a thresholding step on a smoothed image. From this mask,
the area corresponding to the membrane was eroded by a selectable
amount to delineate the membrane. Further erosion was then applied
to reliably separate membrane from cytosol area, and the remaining
area was taken to represent cytosol. This mask was updated for each
image in a series, and translocation was expressed as ratio of the
fluorescence values for membrane and cytosol area, to correct for
bleaching. This approach corrects fully for cell movements and
shape changes, and was able to reliably detect very minor
translocations (using e.g. diluted agonists).
[0176] Fluorescence Determinations
[0177] For FRET experiments, cells were transferred to an inverted
Zeiss Axiovert 135 microscope equipped with dry Achroplan 63.times.
(NA 0.75) objective. Excitation of CFP was at 425.+-.5 nm, and
emission was collected with a 460 nm dichroic mirror. Emission of
CFP and YFP was split using an additional 505 dichroic and filtered
with 475DF30 and 540DF40 bandpass filters, respectively. Detection
was with PTI model 612 analog photomultipliers, and for data
acquisition, the FELIX software (PTI Inc.) was used. FRET was
expressed as the ratio of CFP to YFP signals, the value of which
was set as 1.0 at the onset of the experiment. Changes are
expressed as percent deviation from this initial value of 1.0. For
detection of intracellular Ca.sup.2+, Yellow Cameleon 2.1 was used
at the same wavelengths (Miyawaki et al., Nature 388: 882-887
(1997)).
[0178] For sustained stimulation, agonists and inhibitors were
added to the medium from concentrated stocks. Stimulation with
short pulses of NKA was performed by placing a glass micropipette
(tip diamenter about 2 .mu.m) at about 25 .mu.m from the cell using
an Eppendorf microinjection system and applying pulses of pressure
for 10 seconds. It was verified using Lucifer Yellow in the pipette
that following termination of the pressure pulse the concentration
at the cell rapidly dropped towards zero.
[0179] Loading and flash Photolysis of Caged IP3
[0180] Before electroporation, adherent cells grown on coverslips
were washed twice in intracellular buffer (containing in mM: 70
KCl, 70 Kglutamate, 2 MgCl.sub.2, 0 CaCl.sub.21 5 phosphate buffer,
pH 7.1) and then 70 .mu.l of this intracellular buffer was added to
the cells with 20 .mu.M of Fura-red tetrapotassium salt and either
1, 10 or 100 .mu.M of caged IP3. Electroporation was achieved by a
series of 15 high-frequency square wave pulses, (1-second spaced,
amplitude 150V, frequency 80 kHz, lasting 0.5 ms each) using 2
platina electrodes of 8.times.3 mm with 2.5 mm spacing. The
efficiency of this method was assessed by control permeabilizations
that were performed on the stage of a confocal microscope. This
protocol caused complete permeabilization (based on equilibration
of intracellular calcein concentrations with the extracellular
buffer) of the cells in the area between the electrodes.
[0181] For photorelease of caged IP3, a single cell was illuminated
with a short pulse of UV light (340-410 nm) from a 100W HBO lamp
using a shutter. The shutter open time was adjusted to give full
release of caged IP3, that is no response being observed with a
subsequent illumination. For partial photolysis, the flash
intensity was adjusted by using neutral density filters placed in
the illumination pathway.
[0182] Quantitation of Expression Levels
[0183] For quantitation of expression levels of CFP-PH and YFP-PH,
cellular fluorescence was compared to the fluorescence of a
solution of known concentration of purified, bacterially expressed
CFP-PH or YFP-PH, following the method of Miyawaki et al., "Calcium
signaling: a practical approach," Oxford University Press (in
press). In short, CFP-PH and YFP-PH were expressed as GST-fusion
proteins, and purified on glutathione sepharose beads. Protein
concentration was measured by the BCA* Protein Assay (Pierce, Ill.,
USA). The solution (4.8 .mu.M) was then introduced in a linear
wedge-shaped chamber (0-170 .mu.M thickness) that was placed on the
microscope (using NA 0.7 objective), and the position of the
chamber was adjusted to give a fluorescence readout that matched
that of a single, CFP or YFP expressing cell. The estimate of the
fluorescent protein concentration in the cell was obtained by
comparing the local thickness of the wedge to that of an average
cell (17 .mu.M). Relative amounts of CFP-PH and YFP-PH expression
in cells were always determined under conditions of full cytosolic
localization of the constructs.
[0184] At the onset of each experiment, photomultiplier gains (high
voltage) were adjusted to give a standard 6V output for the resting
cell. Noting that over an extended range of light input, every
2-fold change in intensity corresponds to a 35V change in cathode
voltage, cell intensities were measured. By comparing these to the
values obtained with the GFP-wedge, estimates of expression levels
were obtained.
[0185] Fluorescence Recovery After Photobleaching (FRAP)
[0186] For FRAP experiments, cells were imaged using a Leica TCS-SP
confocal microscope equipped with 63.times. (NA 1.3) oil immersion
objective. The beam from an external ArKr laser (25 mW) was coupled
into the backfocal plane of the objective via the epifluorescence
excitation port, using a 30/70 beamsplitter, thus allowing
simultaneous imaging and spot bleaching. Spots of about 1.3 .mu.m
(full width half maximum) were bleached (>95%) in the basal
membrane using a single 30 ms pulse from the ArKr laser during data
collection in linescan mode at 1000, 500 or 125 Hz. Data were
corrected for slight (<7%) background bleaching and fitted with
single exponents using Clampfit software (Axon Instruments,
CA).
[0187] Results
[0188] Fluorescence Resonance Energy Transfer Between Plasma
Membrane-Localized PLC.delta.1PH-CFP and PLC.delta.1PH-YFP
[0189] PH-CFP and PH-YFP chimera were transiently transfected into
N1E-115 mouse neuroblastoma cells at a 1:1 molar ratio. After 1-2
days, cells were transferred to an inverted epifluorescence
microscope and assayed for FRET by simultaneously monitoring the
emission of CFP (475.+-.15 nm) and of YFP (530.+-.20 nm), while
exciting CFP at 425.+-.5 nm. In resting cells, PH-CFP and PH-YFP
reside at the plasma membrane bound to PI(4,5)P2, and the two
fluoriphores remain within resonance distance. Upon activation of
PLC by the addition of bradykinin (BK), PI(4,5)P2 is rapidly
hydrolyzed and consequently PH domains can no longer bind to the
plasma membrane. Depending on cell type (surface to volume ratio),
it is estimated that the distance(s) between fluoriphores increase
about 200-1000 fold), and therefore FRET does not occur (FIG. 2).
As a result, the donor (CFP) emission intensity increases, while
the acceptor (YFP) emission decreases. By taking the ratio of CFP
to YFP emission, the FRET signal becomes essentially independent on
excitation intensity fluctuations and photobleaching.
[0190] The kinetics of BK-induced PLC activation in N1E-115 cells
as detected by FRET is characterized by a rapid onset, with
translocation peaking at 20-30 s after addition of the agonist. The
decaying phase is somewhat slower, usually returning to baseline
within 1 to 4 minutes. This time course is very similar to that
deduced from confocal detection of PLC.delta.1PH-GFP translocation
recorded under identical conditions (FIG. 2C). In this latter case,
the data were extracted from a time series using post-acquisition
automated image analysis (see Experimental Procedures). Similar
translocation responses can be obtained by FRET in other cell
types, including A431 epidermoid carcinoma cells, HEK293 embryonal
kidney cells, and COS monkey kidney cells stimulated with a variety
of ligands to Gq-coupled receptors.
[0191] The above described kinetics with a fast and rather complete
translocation induced by BK, suggest that PIP2 depletion after
stimulation is quite extensive. While most reports of
agonist-induced PIP2 hydrolysis, as detected biochemically from
[.sup.3H]-inositol-labele- d cells, show slower and less pronounced
decreases in phosphoinositide levels, considerable agonist- and
cell type-dependent variations exist, e.g. (Tilly et al., Biochem.
J. 252:857-863(1988); van der Bend et al., Biochem. J 285 (Pt
I):235-240 (1992); Zhang et al., Mol. Pharmacol. 50:864-869
(1996)). Where early time points were also studied, rapid decreases
in PIP2 levels have been detected (Wijelath et al., Biochem.
Biophys. Res. Commun. 152:392-397 (1988); Divecha et al., EMBO J.
10:3207-3214 (1991); Stephens et al., Biochem. J. 296 (Pt
2):481-488 (1993)). For example, significant bradykinin-induced
PIP2 decreases were reported to occur within 10 seconds in bovine
aortic endothelial cells (Myers et al., Cell Signal. 1:335-343
(1989)), and at 1 minute in bombesin-stimulated 3T3 cells (Divecha
et al., supra (1991)). Rapid recovery towards basal levels has also
been found. Wijelath et al. reported as much as 85% hydrolysis of
PIP2 at 5 seconds after stimulation of macrophages with
interleukin, while PIP2 levels had recovered to 50% at 60 seconds
(Wijelath et al., supra (1988)). Similar fast recovery was also
seen in other cell types (Divecha et al., supra (1991); Stephens et
al., supra (1993)). Since biochemical analyses have to rely upon
measurements on cell populations, where not all cells give
synchronized and identical responses (and many cells may not
respond at all), it is not surprising to find differences between
the results of measurements with these two alternative
approaches.
[0192] Characterization of Fluorescence Signals
[0193] During agonist-induced translocation, several factors may
affect the fluorescent properties of these PH domain chimeras as
well as the transfer of fluorescent energy between them (Tsien,
Annu. Rev. Biochem. 61:509-544 (1998)). For example, the move away
from a compartment adjacent to the lipophilic membrane could alter
fluorescent characteristics, and is also likely to alter FRET by
increasing the degree of rotational freedom. While the relative
influence of increased rotational freedom on the
translocation-induced decrease in FRET is difficult to assess in
this model sytem, fluorescence changes were analyzed in some
further detail.
[0194] Cells were transfected with only one of the
PLC.delta.1PH-CFP or PLC.delta.1PH-YFP constructs. After
stimulation, a small but consistent transient fluorescence decrease
was observed with either the CFP or the YFP-tagged PH domains (FIG.
3). The original green construct (PLC.delta.1PH-GFP) displayed
similar behavior (not shown). This transient decrease is likely
caused by fluoriphore displacement from the membrane, since it is
not observed in cells that express a more stably membrane-anchored
GFP-CAAX, nor is it seen in cells that express a mutated
PLC.delta.1PH-GFP (R40L) (Varnai et al., J. Cell Biol. 143:501-510
(1998)) that can not bind PI(4,5)P2 and, therefore, is cytosolic
throughout the experiment. The precise mechanism that causes this
decrease of emission upon cytosolic translocation is unknown;
however, influence of the local microenvironment (e.g.
hydrophobicity, charged groups, changing ion concentrations etc.)
on the spectral properties of GFP seems likely (Tsien, supra
(1998)). The "displacement" effect may explain why the
translocation-induced decrease in YFP signal usually is somewhat
larger than the increase in CFP fluorescence. However, expressing
FRET as an emission ratio largely eliminates this effect.
[0195] FRET could also be measured in cells that coexpress
PLC.delta.1PH-CFP with YFP-CAAX (not shown); however, using this
pair, ratioing did not cancel the above mentioned displacement
effect.
[0196] To assess the effects of construct concentrations on FRET,
cells expressing various levels of the chimeric proteins were
compared. Intracellular fluorescent protein concentrations were
estimated by comparing the emission intensities of individual cells
to those of a solution of bacterially expressed, purified protein
of known concentration (Miyawaki et al., supra (in press); see
Experimental Procedures). Based on these estimates, resonance could
be observed in cells with expression levels between about 2-200
.mu.M, over a 100-fold concentration range. However, FRET was not
observed in cells expressing less than about 1 .mu.M of each of the
constructs. Very high expression levels, on the other hand,
appeared to be detrimental to the cells (as judged from the
appearance of membrane blebs and detachment of cells 2-3 days after
transfection). Such cells were excluded from analysis. These data
also revealed that PLC.delta.1PH-CFP expression levels (detected in
fully translocated cells) did not differ more than about 2-fold
from those of PLC.delta.1PH-YFP in most cells.
[0197] It was of interest to determine whether estimates of CFP and
YFP concentrations can be used to calculate lipid concentrations
and molecular proximity in the cells studied. Assuming a typical
attached N1E-115 cell to be a pyramid having a 20.times.20 .mu.m
base and 10 .mu.m height (having 1.3 pl volume and 1100 .mu.m.sup.2
surface), and assuming that (I) the concentration of both chimera
is 20 .mu.M; (II) 50% of fluoriphores are located at the membrane
(complete translocation roughly doubles the fluorescence in the
cytosol); (III) the distribution of fluoriphores is homogenous
along the membrane; and (IV) fluoriphores are insensitive to the
local environment, then the calculated mean distance between
fluoriphores is 7-8 nm, which is close to the reported Forster
radius (50 Angstrom) for FRET between this pair of fluoriphores
(Tsien, supra (1998)). However, it should be emphasized that these
assumptions are valid only as first approximations. For example, we
and others (Tall et al., Curr. Biol. 10:743-746 (2000)) noted that
GFP-PH is not homogeneously localized along the plasma membrane.
Also, as discussed above, the spectral properties of the
fluorescent proteins are sensitive to the microenvironment.
Nevertheless, these data set a lower limit for the density of PIP2
molecules available for PH binding at the inner surface of the
plasma membrane.
[0198] GFP-PH Rapidly Shuttles Between Membrane and Cytosol
[0199] Another important characteristic to address was membrane
association and dissociation rates of the PH chimera. These rates
directly influence reliability of FRET in reporting rapid changes
in PLC activity, and are also relevant to the ability of PLC to
hydrolyze PI(4,5)P2 in cells that express high levels of the
PLC.delta.1PH-GFP protein. Accordingly, fluorescence recovery after
photobleaching (FRAP) experiments were performed to estimate the
binding and dissociation kinetics of PLCSIPH-GFP in the membrane.
FIG. 4 shows representative results from such FRAP experiments in
N1E-115 cells. In panels A and B, the recovery rates are depicted
for GFP-CAAX and PLC.delta.1PH(R40L)-GFP, constructs that are
delimited to the plasma membrane and the cytosol, respectively. The
former presents the extreme of slow, purely membrane-delimited
diffusion (2.81.+-.0.31 s, n=15), and the latter of fast cytosolic
diffusion (0.201.+-.0.022 s, n=15). Since FRAP of
membrane-localized PLC.delta.1PH-GFP is significantly faster than
that of the membrane-delimited GFP-CAAX (1.22.+-.0.23 s, n=40;
p<0.0005; compare panel A and C), its recovery has to be
partially through the cytoplasm. Thus, PI(4,5)P2-PH binding is a
dynamic process, with on-off rates in the order of seconds. In
support of this notion, FRAP times further decreased during
agonist-induced partial translocation, when association rates are
increased due to the raised cytosolic GFP-PH levels (panel D). The
rapid shuttling between membrane and cytosol of individual
PLC.delta.1PH-GFP molecules could explain why PI(4,5)P2 is still
available for PLC-mediated hydrolysis or for binding of other
proteins in cells expressing these chimeras.
[0200] Widefield FRET Detection Allows Prolonged Monitoring
Independent of Cell Shape Changes.
[0201] Rapid confocal scanning of cells transfected with
PLC.delta.1PH-GFP leads to considerable photobleaching (within 100
frames) and often causes severe phototoxic damage, manifested as
membrane blebbing and loss of membrane integrity within minutes.
Using wide-field optical detection and integrating emission from an
entire cell (or even clusters of cells) allowed excitation
intensity to be dimmed by as much as 100 to >1000 fold, while
still retaining acceptable signal-to-noise ratio. Thus, FRET can be
followed in single cells for extended periods of time without
detectable cell damage. This permits recording of complex
stimulation protocols, as shown in FIG. 5A. As shown therein, a
single N1E-115 cell that is repeatedly stimulated with short pulses
of neurokinin A (NKA) from a puffer pipette showed repeated PLC
activation. The response to NKA displays incremental partial
homologous desensitization of PLC activation, while the response to
subsequently added BK is unaltered. Optimizing for low excitation
intensity, recordings of several hours can be obtained with
sub-second resolution.
[0202] In N1E-115 and other cells, addition of certain agonists
causes rapid and significant shape changes. For instance, LPA
causes neurites to retract and the cell soma to round up within 60
seconds (Jalink et al., Cell Growth Differ. 4:247-255 (1993)). In
contrast, addition of BK has opposite effects, promoting a
differentiated phenotype (van Leeuwen et al., Nat. Cell Biol.
1:242-248 (1999)). During confocal imaging, such shape changes (as
well as the slight drift in focal plane that inevitably occurs over
prolonged times) seriously complicate the quantification of GFP-PH
translocation. Since FRET analysis uses the total integrated
emission from a cell, shape changes and focal drift do not present
problems.
[0203] In very flat and small cell structures such as neurites and
lamellipodia (below approximately 2 .mu.m in thickness), confocal
imaging cannot detect translocation due to its inherent limit in
z-axis resolution. However, in such cases changes in FRET can still
be reliably detected as shown by the agonist-induced PLC activation
recorded over a single neurite (FIG. 5B). FRET can also be recorded
from cell populations (FIG. 5C) providing with an average response
that would need analysis of hundreds of single cell recordings.
Thus, detecting resonance between fluorescent protein-labeled PH
domains overcomes a number of the limitations that are associated
with confocal detection.
[0204] Determination of Whether FRET Reports Changes in Membrane
PI(4,5)P2 or Increases in Cytosolic IP3
[0205] While PLC.delta.1PH-GFP has been introduced as an indicator
of membrane PI(4,5)P2 (Stauffer et al., Curr. Biol. 8:343-346
(1998); Varnai et al., supra (1998)), it also displays high
affinity to IP3 (Hirose et al., Science 284:1527-1530 (1999)) which
may exceed its affinity to PI(4,5)P2, although it is difficult to
accurately measure the latter as it is displayed in vivo. Based on
such relative affinity estimates, Hirose and coworkers recently
suggested that PLC.delta.1PH-GFP actually monitors IP3 increases
rather than the changes in lipid levels in MDCK cells (Hirose et
al., supra (1999)). They reported that microinjection of IP3 in
MDCK cells was sufficient to cause displacement of
PLC.delta.1PH-GFP from the membrane to the cytosol through
competition for binding of the fluorescent construct to membrane
PI(4,5)P2. They also showed that expression of an IP3-5-phosphatase
completely blocked the agonist-induced translocation of the
fluorescent protein, and concluded that PI(4,5)P2 changes do not
make a significant contribution to the translocation response
during stimulation.
[0206] While FRET analysis effectively monitors the result of PLC
activation regardless of whether it is the lipid decrease or the
IP3 increase that is more important for the translocation response,
this question deserved a more detailed analysis. First it was
determined whether intracellular applications of IP3 that generate
a Ca.sup.2+ signal comparable to that evoked by an agonist would
cause translocation of the PLC.delta.1PH that is similar to what is
caused by agonist stimulation. N1E-115 cells were loaded with 20 pM
of the calcium indicator Fura red and 100 pM caged IP3 by in situ
high frequency electroporation. Unlike microinjection, this
technique allows setting of the final concentration of caged IP3 in
the cytosol with high precision (see Experimental Procedures), as
confirmed by the observation that upon electroporation,
intracellular and extracellular fluorescence levels were equal. As
shown in FIG. 6A, UV flash photolysis of 1 PM of caged IP3 rapidly
mobilized Ca.sup.2+ from internal stores, with no visible
translocation of PLC.delta.1PH-GFP to the cytosol. Subsequent
release of 10 .mu.M of caged IP3 caused a higher Ca.sup.2+ response
and a small translocation. Only high IP3 concentrations that evoked
a large and prolonged Ca.sup.2+ increase were able to displace
PLC.delta.1PH-GFP from the plasma membrane. In contrast, BK
stimulation caused a larger translocation response than the highest
amounts of IP3 with a Ca.sup.2+ signal that was comparable to that
induced by the smallest amount of IP3 (FIG. 6A). In cells
electroporated with no caged IP3 in the electroporation buffer,
intense UV flashes did not influence intracellular Ca.sup.2+
levels, membrane localization of the chimera, or any of the
BK-induced changes herein (not shown).
[0207] Next, the effects of interfering with PI(4,5)P2 resynthesis
on the kinetics of translocation in N1E-115 cells was studied.
PI(4,5)P2 resynthesis was inhibited by low concentrations (5 .mu.M)
of phenyl arsine oxide (PAO) (FIG. 6B) or quercetin (Wiedemann et
al., EMBO J. 15:2094-2101 (1996)), or by depletion of free inositol
using prolonged incubation in inositol-free medium (not shown). In
PAO-treated cells, BK induced a sustained translocation of
PLC.delta.1PH-GFP to the cytosol, while IP3 increases in such cells
are only transient (Hunyady et al., J. Biol. Chem. 266:2783-2788
(1991)). In control experiments, these pretreatments did not
influence signaling events such as BK-induced Ca.sup.2+ signaling
(peak Ca.sup.2+ values of 870.+-.130 nM in control cells, and
845.+-.114 nM, in PAO pretreated cells, n=6, mean.+-.SEM) or the
thrombin- and lysophosphatidate-induced actinomyosin contraction
(Jalink et al., supra (1993); Jalink et al., J. Cell Biol.
118:411-419 (1992)). Similar observations were made in HEK293 cells
(not shown), suggesting that the translocation of PH domains under
these conditions reports the depleted PI(4,5)P2 pool rather than
the transient IP3 increase.
[0208] Moreover, when adrenal glomerulosa cells were stimulated
with angiotensin II in the presence of Sr.sup.2+, a condition under
which IP3 metabolism via Ins(1,3,4,5)P4 is greatly reduced (Balla
et al., supra (1994)), hence yielding significantly higher
Ins(1,4,5)P3- and diminished Ins(1,3,4)P3 increases (FIGS. 6C,D),
the translocation of PLC.delta.1PH-GFP was not significantly
different (FIG. 6E) from that observed in the presence of
Ca.sup.2+. Translocation responses of N1E-115 cells in response to
BK were also similar in the presence of Ca.sup.2+ or Sr.sup.2+
(FIG. 6F).
[0209] Taken together, these results indicate that, at least for
the cells and agonists described above, PLC.delta.1PH-GFP
translocation primarily reports changes in membrane PI(4,5)P2
content and not IP3 increases. The reason for the apparently
stronger binding of PLC.delta.1PH to membranes observed in live
cells compared to the reported low in vitro affinity (Hirose et
al., supra (1999)) to PIP2 containing lipid vesicles or BiaCore
surface (Lemmon et al., Proc. Natl. Acad. Sci. U.S.A 92:10472-10476
(1995)) is unclear at present, but may indicate a more complex
interaction of the PLC.delta.1PH domain with the native membranes
that is not mimicked by the in vitro experiments. However, the
finding reported in Hirose et al., supra (1999) that high IP3
levels can make significant contributions to the translocation
response was confirmed. Whether such high levels or IP3 occur under
the experimental conditions used with intact cells remains to be
elucidated. Nevertheless, possible interference from large IP3
increases should be kept in mind during interpretations of the
results of such translocation experiments.
[0210] FRET Reveals Response Heterogeneity to Different GPCR
Agonists that is not Reflected in Ca.sup.2+ Mobilization.
[0211] Having characterized the use of FRET between CFP and
YFP-tagged PH domains of PLC.delta.1 to record PLC activation, the
kinetics of responses to a set of calcium-mobilizing GPCR agonists
were compared. Included in this panel were the peptide agonists BK
and NKA, as well as the bioactive lipid lysophosphatidate (LPA),
the protease thrombin, and the bioactive amine, histamine. Thrombin
and LPA, in addition to inducing Ca.sup.2+ mobilizations from
internal stores, are also strong inducers of Rho-dependent
remodeling of the actin cytoskeleton in these cells (Jalink et al.,
supra (1993); Jalink et al., supra (1992)). Histamine, on the other
hand, does not induce Rho-dependent actin remodeling, but is known
to induce Ca.sup.2+ oscillations in several cell types (e.g.
Paltauf-Doburzynska et al., J. Physiol.(Lond) 524 Pt.3:701-713
(2000); Zhu et al., J. Biol. Chem. 275:6063-6066 (2000))
[0212] These agonists evoke very similar Ca.sup.2+ mobilizations in
N1E-115 cells, characterized by a fast onset and rapid termination
well within 2 minutes (FIG. 7). Estimated peak Ca.sup.2+ levels
ranged from 0.6-2 .mu.M, and, again, showed no consistent
differences between agonists. When PLC activation patterns were
recorded by FRET analysis, using the same agonists under identical
conditions, several distinct profiles of PLC activation kinetics
were obtained (FIG. 7). First, both NKA and BK caused fast and
near-complete translocation of the probe. This response was
transient, returning to baseline within 2-5 minutes. Stimulation
with thrombin or LPA evoked a different type of response: these
translocations had slower onset and smaller amplitude, averaging
25% of BK response control values (n=22). They also returned to
baseline at a slower rate. The response to histamine was much
slower and of small amplitude (40% of BK-induced peak values,
n=15), but it was long-lasting (at least for 15 minutes, but often
much longer).
[0213] Differences in degree of PIP2 hydrolysis induced by
activation of different Gq-coupled receptors have also been
reported (van der Bend et al., supra (1992), Tilly et al., Biochem.
J. 266:235-243 (1990)) in biochemical studies. However, so far only
cytosolic Ca.sup.2+ responses could be used to analyze receptor
activation patterns at the single cell level. On the other hand,
the shape of the Ca.sup.2+ response is determined by several other
factors: it can be triggered at relatively low levels of IP3 and
its shape is also determined by the Ca.sup.2+-induced Ca.sup.2+
release and inactivation properties of the IP3-receptor-channels,
as well as by the activities of the various Ca.sup.2+ sequestration
mechanisms. The present approach provides an opportunity to study a
more upstream receptor-mediated event, namely PLC activation, and
its regulation in detail at the single cell level.
[0214] PH Domain Translocation Kinetics Mirror Receptor
Activation.
[0215] These results thus suggest that PLC activation as assessed
by FRET is a more faithful index of receptor activity than the more
distal Ca.sup.2+ transients. However, inactivation could occur at
various steps in the signal cascade, including at the levels of
receptor, G protein and PLC and, conceivably, also by modulation
(upregulation) of PI(4,5)P2 resynthesis. To test whether there is
desensitization at the level of PLC, G proteins were directly
activated using AlF.sup.4- (FIG. 8A). While onset of AlF.sup.4-
induced PLC activation was slow, no desensitization was observed in
any of these experiments. Similarly, cells expressing a
constitutively active G.alpha.q mutant showed mostly cytosolic
localization of PLC.beta.1 PH-GFP domains for at least 2 days (FIG.
8B). Control transfection with activated G.alpha.12 had no effect.
At lower expression levels, the activating mutant G.alpha.q induced
sustained partial translocation that also persisted for several
days. These experiments suggested that no significant
desensitization occurs downstream of Gq and PLC. In line with this
notion, significant heterologous desensitization between
sequentially added agonists was not observed (compare e.g. FIGS. 5
and 7, last panel), whereas prolonged exposure of cells to each
individual agonist induced complete (homologous)
desensitization.
[0216] To further determine whether such monitoring of PLC activity
truly follows receptor activity (in other words coupling and
uncoupling between receptors and G proteins), the FRET responses of
N1E-115 cells expressing either the wild-type NK2 receptors or a
C-terminally truncated form, which is greatly impaired in its
ability to desensitize (Alblas et al., J. Biol. Chem. 270:
8944-8951 (1995)), were compared. After stimulation of the
wild-type human NK2 receptors the translocation response decays
towards baseline within minutes (average 50%) recovery time
83.+-.38 s, n=25; compare FIGS. 7 and 8C). Application of short
pulses of agonist using a puffer pipette resulted in incomplete
desensitization, and decayed significantly faster (45.+-.7 s, n=60,
FIG. 8C) between applications of stimuli due to the rapid
dissociation of the ligand from the receptor (Vollmer et al., J.
Biol. Chem. 274:37915-37922 (1999)). Conversely, stimulation of a
C-terminally truncated mutant human NK2 receptor, that was reported
to be transforming in Rat-1 fibroblasts, and which has been found
to display prolonged coupling to PLC (Alblas et al., supra (1995);
Alblas et al., EMBO J. 15:3351-3360 (1996); Alblas et al., J. Biol.
Chem. 268:22235-22238 (1993)) induced a much prolonged cytosolic
translocation as assessed in FRET analysis (FIG. 8C). However, in
the majority of cells, the FRET signal eventually slowly returned
to baseline (FIG. 8D; note the different time scale), with an
average 50% recovery time of 1365.+-.599 s (n=19) in the truncated
receptor. This result indicates the existence of an alternative and
much slower desensitization mechanism that functions even in NK2
receptors lacking the C-terminus. The kinetics of this slow
desensitization closely paralleled those of receptor
internalization (not shown), suggesting that one of the main
determinants for termination of NKA-induced PLC signaling could be
receptor internalization. Analysis of receptor activity by
monitoring PLC activity by FRET will greatly aid further studies
addressing these questions in more detail.
[0217] In summary, described above is a fluorescence
resonance-based detection scheme of membrane localization of tagged
PLC.delta.1 PH domains for analysis of activation-inactivation
kinetics of PLC in single cells with high temporal resolution. This
method has a number of significant advantages over confocal
detection of membrane localization, including: (i) a significant
decrease in excitation intensity allowing prolonged experiments or
very fast sampling with little photobleaching and phototoxicity;
(ii), suitability for very flat cells such as fibroblasts and
motile cells; (iii), extendibility to record from cell populations
as well as from small subregions such as neurites; and (iv) a
simpler detection hardware. FRET detection of PLC activation is a
fairly robust response that can be routinely obtained in a variety
of cell types.
[0218] Analysis of the translocation responses suggests that
localization of PLC.delta.1PH-GFP largely reports PI(4,5)P2
dynamics, although at high concentrations IP3 can also contribute
to translocation of the PH domains to the cytosol. Comparison of
the Ca.sup.2+ and FRET-recorded responses of several agonists of
GPCRs suggest that PLC activation detected by FRET is a more
faithful reflection of receptor activity than the Ca.sup.2+ signal
and that little if any "desensitization" or "uncoupling" occurs
beyond the levels of G proteins.
[0219] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0220] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention.
Sequence CWU 1
1
8 1 9 PRT Artificial Sequence synthetic construct 1 Arg Xaa His His
Cys Arg Xaa Cys Gly 1 5 2 4 PRT Homo sapiens 2 Lys Asp Glu Leu 1 3
42 DNA Artificial Sequence primer 3 cctgcggccg cggtaccgat
atcagatgtt gagctccttc ac 42 4 42 DNA Artificial Sequence primer 4
ccgaattccc gggtctcagc catggactcg ggccgggact tc 42 5 40 DNA
Artificial Sequence primer 5 ggctgagacc cgggaattcg gcttgtacag
ctcgtccatg 40 6 20 PRT Homo sapiens 6 Lys Met Ser Lys Asp Gly Lys
Lys Lys Lys Lys Lys Ser Lys Thr Lys 1 5 10 15 Cys Val Ile Met 20 7
39 DNA Artificial Sequence primer 7 ccgaattccc gggtcaagat
gagcaaagat ggtaaaaag 39 8 42 DNA Artificial Sequence primer 8
cctgcggccg cggtaccgag atctttacat aattacacac tt 42
* * * * *
References