U.S. patent application number 11/501473 was filed with the patent office on 2007-06-28 for compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets.
This patent application is currently assigned to AURORA BIOSCIENCES CORPORATION. Invention is credited to John D. Mendlein, Jimena Sierralta, Yumei Sun, Susan Tsunoda, Charles S. Zuker.
Application Number | 20070150970 11/501473 |
Document ID | / |
Family ID | 36758541 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070150970 |
Kind Code |
A1 |
Zuker; Charles S. ; et
al. |
June 28, 2007 |
Compositions and methods for identifying modulators of
transducisomes, a new class of therapeutic targets
Abstract
The invention provides cells and methods for identifying
modulators of signal transduction, based on transducisome proteins
that coordinate and assemble many types of signal transduction
proteins. A transducisome is a PDZ domain containing protein that
binds at least one signal transduction protein or a PDZ domain
containing protein with at least one signal transduction protein
bound. Examples of transducisome proteins include INAD, GRIP and
other recently identified multi-PDZ domain proteins. Examples of
signal transduction proteins include GPCRs, tyrosine kinase
receptors, tyrosine phosphatase receptors, ion channels,
phospholipases, adenylate cyclases, kinases and G-proteins. Also
provided are methods for identifying modulators of signal
transduction, proteins (and polynucleotides encoding the same)
corresponding to transducisomes, modified transducisomes or
defective transducisomes to use in assays of signal transduction,
and a screening assay system for detecting protein-protein
interactions.
Inventors: |
Zuker; Charles S.; (San
Diego, CA) ; Mendlein; John D.; (Encinitas, CA)
; Sun; Yumei; (Newton, MA) ; Tsunoda; Susan;
(San Diego, CA) ; Sierralta; Jimena; (Santiago,
CL) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.;DLA PIPER RUDNICK GRAY CARY US LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
AURORA BIOSCIENCES
CORPORATION
|
Family ID: |
36758541 |
Appl. No.: |
11/501473 |
Filed: |
August 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09462517 |
May 18, 2000 |
7087388 |
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PCT/US98/14667 |
Jul 15, 1998 |
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11501473 |
Aug 8, 2006 |
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60052588 |
Jul 15, 1997 |
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Current U.S.
Class: |
800/13 ; 435/194;
435/196; 435/348; 530/350; 536/23.2 |
Current CPC
Class: |
G01N 33/5041 20130101;
G01N 2333/726 20130101; G01N 2333/916 20130101; G01N 2333/912
20130101 |
Class at
Publication: |
800/013 ;
435/348; 536/023.2; 530/350; 435/194; 435/196 |
International
Class: |
A01K 67/033 20060101
A01K067/033; C07H 21/04 20060101 C07H021/04; C12N 5/06 20060101
C12N005/06; C07K 14/705 20060101 C07K014/705; C12N 9/12 20060101
C12N009/12; C12N 9/16 20060101 C12N009/16 |
Claims
1. A fly comprising an amino acid mutation in a transducisome
protein that prevents functional binding of a signal transduction
protein, wherein said amino acid mutation is not a naturally
occurring mutation of inaD.
2. The fly of claim 1, wherein said amino acid mutation results
from a mutation selected from the group consisting of inaD.sup.2
and inaD.sup.1.
3. An isolated cell comprising a polynucleotide encoding a
transducisome protein with an amino acid mutation that prevents
functional binding of a signal transduction protein, wherein said
amino acid mutation is a naturally occurring mutation of inaD.
4. The cell of claim 3, wherein said cell is a fly cell.
5. The cell of claim 4, wherein said amino acid mutation results
from a mutation selected from the group consisting of inaD.sup.2
and inaD.sup.1.
6. An isolated polynucleotide comprising a coding region for a
transducisome protein with an amino acid mutation in a PDZ domain
that prevents functional binding of a signal transduction protein,
wherein said amino acid mutation is not a naturally occurring
mutation inaD.sup.215.
7. The isolated polynucleotide of claim 6, wherein said
transducisome protein is INAD.
8. The isolated polynucleotide of claim 7, wherein said amino acid
mutation results from a mutation selected from the group consisting
of inaD.sup.2 and inaD.sup.1.
9. An isolated protein comprising a polypeptide of SEQ ID NO.:1
with an amino acid mutation in a PDZ domain that prevents
functional binding of a signal transduction protein, wherein said
amino acid mutation is not a naturally occurring mutation
inaD.sup.215.
10. The isolated protein of claim 9, wherein said amino acid
mutation results from a mutation selected from the group consisting
of inaD.sup.2 and inaD.sup.1.
11. A chimeric transducisome protein comprising at least one first
PDZ domain that binds a first signal transduction protein and at
least one second PDZ domain binds a second signal transduction
protein, wherein said chimeric transducisome protein is not a
naturally occurring protein.
12. The chimeric transducisome protein of claim 11, wherein said
first signal transduction protein is selected from the group of a
kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a
tyrosine phosphatase receptor, an ion channel, a G-protein, a
phospholipase and a calcium binding protein.
13. The chimeric transducisome protein of claim 11, wherein said
second signal transduction protein is selected from the groups PKC,
TRP, and PLC.beta..
14-29. (canceled)
30. An isolated, non-naturally occurring cell, comprising: a) a
heterologously expressed transducisome protein comprising one or
more PDZ domains and b) an expressed protein comprising a signal
transduction protein that binds to one or more said PDZ
domains.
31. An isolated, non-naturally occurring cell, comprising: a) a
cell capable of expressing: i) a non-naturally occurring
polynucleotide comprising a coding region for a transducisome
protein comprising one or more PDZ domains and ii) a non-naturally
occurring polynucleotide comprising a coding region for a
heterologous protein comprising a signal transduction protein.
32. The isolated, non-naturally occurring cell of claim 31, wherein
said signal transduction protein is selected from the group of a
kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a
tyrosine phosphatase receptor, an ion channel, a G-protein, a
phospholipase and a calcium binding protein.
33. The isolated, non-naturally occurring cell of claim 32, wherein
further comprising a test chemical.
34. The isolated, non-naturally occurring cell of claim 33, wherein
further comprising a signal transduction detection system.
35-40. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention relates to compositions and methods for
identifying modulators of signal transduction in cells,
particularly signal transduction related to cell surface receptors
and ion channels.
BACKGROUND
[0002] Many physiological signals (e.g., sensory, hormonal and
neurotransmitter signals) are transduced from extracellular to
intracellular environments by cell surface receptors. For example,
G-protein coupled receptors (GPCRs) (for a review, see Neer, 1995,
Cell 80:249-257), tyrosine kinase receptors and tyrosine
phosphatase receptors are involved in signal transduction.
[0003] As an example, GPCRs mediate signal transduction across a
cell membrane upon the binding of a ligand to an extracellular
portion of a GPCR. The intracellular portion of a GPCR interacts
with a G-protein to modulate signal transduction from outside to
inside a cell. A GPCR is therefore said to be "coupled" to a
G-protein. G-proteins are composed of three polypeptide subunits:
an .alpha. subunit, which binds and hydolyzes GTP, and a dimeric
.beta..gamma. subunit. In the basal, inactive state, the G-protein
exists as a heterotrimer of the .alpha. and .beta..gamma. subunits.
When the G-protein is inactive, guanosine diphosphate (GDP) is
associated with the .alpha. subunit of the G-protein. When a GPCR
is bound and activated by a ligand, the GPCR binds to the G-protein
heterotrimer and decreases the affinity of the G.alpha. subunit for
GDP. In its active state, the G submit exchanges GDP for guanine
triphosphate (GTP) and active G.alpha. subunit disassociates from
both the receptor and the dimeric .beta..gamma. subunit. The
disassociated, active G.alpha. subunit transduces signals to
effectors that are "downstream" in the G-protein signaling pathway
within the cell. Eventually, the G-protein's endogenous GTPase
activity returns active G subunit to its inactive state, in which
it is associated with GDP and the dimeric .beta..gamma.
subunit.
[0004] Currently available assays of signal transduction are often
hampered by low or non-specific signals. Receptor activation
pathways can cross talk, leading to a loss in signal specificity.
In addition, some receptors when heterologously expressed may not
function in the normal fashion due to the absence of protein(s)
integral to signal transduction function. Additionally, assay tools
for monitoring protein-protein interactions of signal transduction
are few and cumbersome, such as antibody labeling and not well
suited for high throughput screening.
[0005] Consequently, the inventors provide new methods and assays
components for biochemical and cell-based assays using a newly,
functionally identified class of therapeutic target, transducisome
proteins. Transducisome proteins as described further herein,
assembly and organize many types of signal transduction proteins
using PDZ domains to permit or enhance signal transduction.
SUMMARY OF THE INVENTION
[0006] Cells respond to a wide variety of external signals mediated
by cell surface receptors that transduce extracellular stimuli into
an intracellular response. Although receptors that recognize
different ligands are known to interact with the same intracellular
signaling molecules, the specificity of signaling, often essential
to a cell's physiological role, is maintained. How is this
specificity maintained, or rather, how is signal cross talk
avoided? One solution involves organizing different signaling
cascades into physically and functionally distinct signaling units.
Such assemblies can permit or enhance signal response time,
specificity and selectivity while minimizing cross talk. Until the
advent of present invention, know little was known about the
architectural organization of the corresponding signaling machinery
or how it can be used to discover useful modulators of signal
transduction.
[0007] The invention provides cells and methods for identifying
modulators of signal transduction based, in part, on transducisome
proteins that coordinate and assemble many types of signal
transduction proteins. Transducisomes can either permit or enhance
signal transduction. By including transducisome proteins in the
assays, as described herein, modulators of signal transduction can
be identified. "Transducisome" refers to a PDZ domain containing
protein that binds at least one signal transduction protein or a
PDZ domain containing protein with at least one signal transduction
protein bound to it (see FIG. 1A-C). Other types of transducisomes
are described herein.
[0008] The invention includes methods for identifying modulators of
signal transduction comprising contacting a transducisome in a
biochemical assay, cell assay or animal assay, with a test chemical
and detecting a change in signal transduction. As described herein
the invention includes animals (e.g., mice and flies), cells (e.g.,
mammalian and insect), with transducisomes, modified transducisomes
and defective transducisomes to use in assays of signal
transduction. The invention also includes proteins (as well as
polynucleotides encoding the same) corresponding to transducisomes,
modified transducisomes or defective transducisomes to use in
assays of signal transduction.
[0009] The invention also includes a screening assay system for
detecting protein-protein interactions. This screening assay system
comprises a recombinant protein comprising at least one PDZ domain;
a PDZ binding protein; and at least one test chemical. The
recombinant protein or the PDZ binding protein or both have a label
to facilitate the detection of specific binding. Preferably, the
recombinant protein has a donor and the PDZ binding protein has a
quencher, wherein the donor and quencher are energy transfer
partners, as described herein. The PDZ binding protein can be
selected from the group of a kinase, a phosphatase, a GPCR, a
tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion
channel, a G-protein, a phospholipase and calcium binding protein.
More preferably, the energy transfer partners are a modified GFP
FRET partner pair.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A shows an exemplary transducisome in the uncomplexed
and complexed state. A transducisome protein "T" has three
different PDZ domains that bind different signal transduction
proteins such as a phospholipase ("PL"), a phosphokinase ("PK") and
a cell surface receptor ("R"). The interlocking regions on the
transducisome protein illustrate PDZ domains that bind signal
transduction proteins. The interlocking region on each signal
transduction protein illustrates an amino acid region that binds a
PDZ domain on the transducisome protein.
[0011] FIG. 1B shows an exemplary transducisome in the uncomplexed
and complexed state. A transducisome protein "T" has three
different PDZ domains that bind signal transduction proteins such
as a phospholipase ("PL"), a phosphokinase ("PK") and two ion
channels of the same type ("IC"). The interlocking regions on the
transducisome protein illustrate PDZ domains that bind signal
transduction proteins. The interlocking region on each signal
transduction protein illustrates an amino acid region that binds a
PDZ domain on the transducisome protein.
[0012] FIG. 1C shows one embodiment of the invention related
monitoring protein-protein interactions in transducisomes in the
uncomplexed and complexed state. The hash-marked regions represent
a first energy transfer moiety for FRET. The dotted regions
represent a second energy transfer moiety for FRET. FRET is
substantially occurs in the complexed state between the first and
second energy transfer moieties.
[0013] FIG. 2A shows a diagram of INAD protein with the locations
of and size of each PDZ domain highlighted. Also shown are the
relative locations of the three inaD mutations related to
photoreceptor activation.
[0014] FIG. 2B shows an amino acid alignment of PDZ domains from
mammalian PSD-95.sup.9, nNOS.sup.23, Drosophila dlg.sup.8 and
inaD.sup.36. Black boxes indicate amino acid identities and gray
boxes show conservative substitutions. Stars above the sequence
indicate residues implicated in substrate binding.sup.27. The
circled residues refer to the site of point mutation in the three
Drosophila inaD alleles (see text for details).
[0015] FIG. 2C shows an immunoblot demonstrating the absence of
INAD protein in inaD.sup.1.
[0016] FIG. 3A shows that INAD antibodies co-immunoprecipitate TRP,
eye-PKC and PLC.sub..beta. from retinal extracts in an
immunoblot.
[0017] FIG. 3B shows the results obtained in vitro using a full
length GST-INAD fusion protein. Full length GST-INAD fusions
associate with TRP, PLC.beta. and PKC in cell extracts. FIG. 3B
also shows that the third PDZ domain of INAD is specific for
TRP.sup.37, while the fourth domain specifically interacts with
eye-PKC and the fifth domain specifically interacts with
PLC.sub..beta.. Overexpression of each of the individual PDZ
domains from INAD as GST-PDZ fusions produce highly preferred
interactions in biochemical assays.
[0018] FIG. 4 shows electrophysiology recordings with altered
kinetics from wildtype and mutant INAD protein in cells.
[0019] FIG. 5A-C show altered kinetics and quantal bumps in
photoreceptor cells.
DEFINITIONS
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein and the laboratory
procedures in spectroscopy, drug discovery, cell culture, and
molecular genetics, described below are those well known and
commonly employed in the art. Standard techniques are typically
used for preparation of signal detection, recombinant nucleic acid
methods, polynucleotide synthesis, and microbial culture and
transformation (e.g., electroporation, and lipofection). The
techniques and procedures are generally performed according to
conventional methods in the art and various general references (see
generally, Sambrook et al. Molecular Cloning: A Laboratory Manual,
2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., and Lakowicz, J. R. Principles of Fluorescence
Spectroscopy, New York: Plenum Press (1983) for fluorescence
techniques, which are incorporated herein by reference) which are
provided throughout this document. Standard techniques are used for
chemical syntheses, chemical analyses, and biological assays. As
employed throughout the disclosure, the following terms, unless
otherwise indicated, shall be understood to have the following
meanings:
[0021] "Fluorescent donor moiety" refers to the radical of a
fluorogenic compound, which can absorb energy and is capable of
transferring the energy to another fluorogenic molecule or part of
a compound. Suitable donor fluorogenic molecules include, but are
not limited to, coumarins and related dyes xanthene dyes such as
fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes,
bimanes, acridines, isoindoles, dansyl dyes, aminophthalic
hydrazides such as luminol and isoluminol derivatives,
aminophthalimides, aminonaphthalimides, aminobenzofurans,
aminoquinolines, dicyanohydroquinones, and europium and terbium
complexes and related compounds.
[0022] "Quencher" refers to a chromophoric molecule or part of a
compound, which is capable of reducing the emission from a
fluorescent donor when attached to the donor. Quenching may occur
by any of several mechanisms including fluorescence resonance
energy transfer, photoinduced electron transfer, paramagnetic
enhancement of intersystem crossing, Dexter exchange coupling, and
excitation coupling such as the formation of dark complexes.
[0023] "Acceptor" refers to a quencher that operates via
fluorescence resonance energy transfer. Many acceptors can re-emit
the transferred as energy as fluorescence. Examples include
coumarins and related fluorophores, xanthenes such as fluoresceins,
rhodols, and rhodamines, resorufins, cyanines,
difluoroboradiazaindacenes, and phthalocyanines. Other chemical
classes of acceptors generally do not re-emit the transferred
energy. Examples include indigos, benzoquinones, anthraquinones,
azo compounds, nitro compounds, indoanilines, di- and
triphenylmethanes.
[0024] "Binding pair" refers to two moieties (e.g. chemical or
biochemical) that have an affinity for one another. Examples of
binding pairs include antigen/antibodies, lectin/avidin, target
polynucleotide/probe oligonucleotide, antibody/anti-antibody,
receptor/ligand, enzyme/ligand and the like. "One member of a
binding pair" refers to one moiety of the pair, such as an antigen
or ligand.
[0025] "Dye" refers to a molecule or part of a compound that
absorbs specific frequencies of light, including but not limited to
ultraviolet light. The terms "dye" and "chromophore" are
synonymous.
[0026] "Fluorophore" refers to a chromophore that fluoresces.
[0027] "Membrane-permeant derivative" refers a chemical derivative
of a compound that has enhanced membrane permeability compared to
an underivativized compound. Examples include ester, ether and
carbamate derivatives. These derivatives are made better able to
cross cell membranes, i.e. membrane permeant, because hydrophilic
groups are masked to provide more hydrophobic derivatives. Also,
masking groups are designed to be cleaved from a precursor (e.g.,
fluorogenic substrate precursor) within the cell to generate the
derived substrate intracellularly. Because the substrate is more
hydrophilic than the membrane permeant derivative it is now trapped
within the cells.
[0028] "Isolated polynucleotide" refers a polynucleotide of
genomic, cDNA, or synthetic origin or some combination there of,
which by virtue of its origin the "isolated polynucleotide" (1) is
not associated with the cell in which the "isolated polynucleotide"
is found in nature, or (2) is operably linked to a polynucleotide
which it is not linked to in nature.
[0029] "Isolated protein" refers a protein, usually of cDNA,
recombinant RNA, or synthetic origin or some combination thereof,
which by virtue of its origin the "isolated protein" (1) is not
associated with proteins that it is normally found with in nature,
(2) is isolated from the cell in which it normally occurs, (3) is
isolated free of other proteins from the same cellular source, e.g.
free of human proteins, (4) is expressed by a cell from a different
species, or (5) does not occur in nature. "Isolated naturally
occurring protein" refers to a protein which by virtue of its
origin the "isolated naturally occurring protein" (1) is not
associated with proteins that it is normally found with in nature,
or (2) is isolated from the cell in which it normally occurs or (3)
is isolated free of other proteins from the same cellular source,
e.g. free of human proteins.
[0030] "Polypeptide" as used herein as a generic term to refer to
native protein, fragments, or analogs of a polypeptide sequence.
Hence, native protein, fragments, and analogs are species of the
polypeptide genus. Preferred transducisome polypeptides, include
those with the polypeptide sequence represented in the SEQUENCE ID
LISTING (as well as human homologs hereof) and any other protein
having activity similar to such transducisome proteins as measured
by one or more of the assays described herein. SEQ. ID NO.: 1 is a
transducisome protein (fly) amino acid sequence and SEQ. ID NO. 2
is a transducisome protein (fly) nucleotide sequence. Transducisome
polypeptides or proteins can include any protein having sufficient
activity for detection in the assays described herein.
[0031] "Naturally-occurring" as used herein, as applied to an
object, refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0032] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences, such as when the appropriate
molecules (e.g., inducers and polymerases) are bound to the control
or regulatory sequence(s).
[0033] "Control sequence" refers to polynucleotide sequences which
are necessary to effect the expression of coding and non-coding
sequences to which they are ligated. The nature of such control
sequences differs depending upon the host organism; in prokaryotes,
such control sequences generally include promoter, ribosomal
binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, components whose presence can
influence expression, and can also include additional components
whose presence is advantageous, for example, leader sequences and
fusion partner sequences.
[0034] "Polynucleotide" refers to a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide.
The term includes single and double stranded forms of DNA.
[0035] Two amino acid sequences are homologous if there is a
partial or complete identity between their sequences. For example,
85% homology means that 85% of the amino acids are identical when
the two sequences are aligned for maximum matching. Gaps (in either
of the two sequences being matched) are allowed in maximizing
matching; gap lengths of 5 or less are preferred with 2 or less
being more preferred. Alternatively and preferably, two protein
sequences (or polypeptide sequences derived from them of at least
30 amino acids in length) are homologous, as this term is used
herein, if they have an alignment score of at more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O.,
in Atlas of Protein Sequence and Structure, 1972, Volume 5,
National Biomedical Research Foundation, pp. 101-110, and
Supplement 2 to this volume, pp. 1-10. The two sequences or parts
thereof are more preferably homologous if their amino acids are
greater than or equal to 30% identical when optimally aligned using
the ALIGN program.
[0036] "Corresponds to" refers to a sequence that is homologous
(i.e., is identical, not strictly evolutionarily related) to all or
a portion of a reference sequence.
[0037] The following terms are used to describe the sequence
relationships between two or more proteins: "reference sequence,"
"comparison window," "sequence identity," "percentage of sequence
identity," and "substantial identity." A "reference sequence" is a
defined sequence used as a basis for a sequence comparison; a
reference sequence may be a subset of a larger sequence, for
example, as a segment of a full-length protein given in a sequence
listing such as a SEQ. ID NO. 1, or may comprise a complete protein
sequence. Generally, a reference sequence is at least 400
nucleotides in length, frequently at least 600 nucleotides in
length, and often at least 800 nucleotides in length (or the
protein equivalent). Since two proteins may each (1) comprise a
sequence (i.e., a portion of the complete protein sequence) that is
similar between the two proteins, and (2) may further comprise a
sequence that is divergent between the two proteins, sequence
comparisons between two (or more) proteins are typically performed
by comparing sequences of the two proteins over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window," as used herein, refers to a
conceptual segment of at least 20 contiguous amino acid positions
wherein a protein sequence may be compared to a reference sequence
of at least 20 contiguous amino acids and wherein the portion of
the protein sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20 percent or less as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
(1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm
of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search
for similarity method of Pearson and Lipman (1988) Proc. Natl.
Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is
selected. The term "sequence identity" means that two protein
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over the window of comparison. The term "percentage of sequence
identity" is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical amino acids occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison (i.e., the window size), and multiplying the
result by 100 to yield the percentage of sequence identity.
[0038] As applied to proteins, the term "substantial identity"
means that two protein sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, typically
share at least 70 percent sequence identity, preferably at least 80
percent sequence identity, more preferably at least 90 percent
sequence identity, and most preferably at least 95 percent sequence
identity. Preferably, residue positions, which are not identical,
differ by conservative amino acid substitutions. Conservative amino
acid substitutions refer to the interchangeability of residues
having similar side chains. For example, a group of amino acids
having aliphatic side chains is glycine, alanine, valine, leucine,
and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, glutamic-aspartic, and
asparagine-glutamine.
[0039] "Transducisome protein" refers to a protein with the
transducisome protein activity of one of the transducisome proteins
of the SEQ. ID listing or another PDZ domain containing protein
that binds signal transduction proteins. Preferably, the
transducisome protein can functionally bind at least one signal
transduction protein. Examples of transducisome proteins include
INAD, GRIP and other recently identified multi-PDZ domain proteins.
Examples of signal transduction proteins include GPCRS, tyrosine
kinase receptors, tyrosine phosphatase receptors, ion channels,
phospholipases, adenylate cyclases, kinases and G-proteins.
Transducisome protein activity can be measured with endogenously or
heterologously expressed transducisome proteins and signal
transduction proteins using the assays described herein.
Preferably, a transducisome protein can functionally bind to at
least two different types of signal transduction proteins, more
preferably, a transducisome protein can functionally bind to at
least three different types of signal transduction proteins.
Transducisome proteins permit enhanced signal transduction by
signal transduction proteins compared to signal transduction by
signal transduction proteins not functionally bound to a
transducisome. Transducisome protein activity includes functional
binding of a signal transduction protein to a transducisome
protein, alteration of signal transduction protein activity when
the signal transduction protein is complexed to the transducisome
protein (e.g., a increase or decrease in activity or a change in
signal transduction protein specificity) and any other activity
related to signal transduction induced by a transducisome protein.
Mammalian transducisome proteins for use with mammalian signal
transduction proteins are preferred, particularly human
transducisome proteins.
[0040] "Modulation" refers to the capacity to either enhance or
inhibit a functional property of biological activity or process
(e.g., enzyme activity or receptor binding); such enhancement or
inhibition may be contingent on the occurrence of a specific event,
such as activation of a signal transduction pathway, and/or may be
manifest only in particular cell types.
[0041] The term "modulator" refers to a chemical (naturally
occurring or non-naturally occurring), such as a synthetic molecule
(e.g., nucleic acid, protein, non-peptide, or organic molecule), or
an extract made from biological materials such as bacteria, plants,
fungi, or animal (particularly mammalian) cells or tissues.
Modulators are evaluated for potential activity as inhibitors or
activators (directly or indirectly) of a biological process or
processes (e.g., agonist, partial antagonist, partial agonist,
inverse agonist, antagonist, antineoplastic agents, cytotoxic
agents, inhibitors of neoplastic transformation or cell
proliferation, cell proliferation-promoting agents, and the like)
by inclusion in screening assays described herein. The activity of
a modulator may be known, unknown or partially known.
[0042] "Sequence homology" refers to the proportion of base matches
between two nucleic acid sequences or the proportion amino acid
matches between two amino acid sequences. When sequence homology is
expressed as a percentage, e.g., 50%, the percentage denotes the
proportion of matches over the length of sequence from a desired
sequence (e.g., SEQ. ID NO. 1) that is compared to some other
sequence. Gaps (in either of the two sequences) are permitted to
maximize matching; gap lengths of 15 bases or less are usually
used, 6 bases or less are preferred with 2 bases or less more
preferred.
[0043] The term "test chemical" refers to a chemical to be tested
by one or more screening method(s) of the invention as a putative
modulator. A test chemical is usually not known to bind to the
target of interest. The term "control test chemical" refers to a
chemical known to bind to the target (e.g., a known agonist,
antagonist, partial agonist or inverse agonist). The term "test
chemical" does not include a chemical added as a control condition
that alters the function of the target to determine signal
specificity in an assay. Such control chemicals or conditions
include chemicals that 1) non-specifically or substantially disrupt
protein structure (e.g., denaturing agents (e.g., urea or
guandium), charotropic agents, sulfhydryl reagents (e.g.,
dithiotritol and .beta.-mercaptoethanol), and proteases), 2)
generally inhibit cell metabolism (e.g., mitochondrial uncouplers)
and 3) non-specifically disrupt electrostatic or hydrophobic
interactions of a protein (e.g., high salt concentrations, or
detergents at concentrations sufficient to non-specifically disrupt
hydrophobic interactions). The term "test chemical" also does not
include chemicals known to be unsuitable for a therapeutic use for
a particular indication due to toxicity of the subject. Usually,
various predetermined concentrations test chemicals are used for
screening such as 0.01 .mu.M, 0.1 .mu.M, 1.0 M, and 10.0 .mu.M.
[0044] The term "target" refers to a biochemical entity involved a
biological process. Targets are typically proteins that play a
useful role in the physiology or biology of an organism. A
therapeutic chemical binds to target to alter or modulate its
function. As used herein targets can include cell surface
receptors, G-proteins, kinases, ion channels, phopholipases and
other proteins mentioned herein.
[0045] The terms "label" or "labeled" refers to incorporation of a
detectable marker, e.g., by the incorporation of a radio labeled
amino acid or attachment to a polypeptide of biotinyl moieties that
can be detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical, or calorimetric methods). Various methods of labeling
polypeptides and glycoproteins are known in the art and may be
used. Examples of labels for polypeptides include, but are not
limited to, the following: radioisotopes (e.g., .sup.3H, .sup.14C,
.sup.35S, .sup.125I, .sup.131I), fluorescent labels (e.g., FITC,
rhodamine, and lanthanide phosphors), enzymatic labels or reporter
genes (e.g., horseradish peroxidase, .beta.-galactosidase,
.beta.-latamase, luciferase, alkaline phosphatase),
chemiluminescent groups, biotinyl groups, predetermined polypeptide
epitopes recognized by a secondary reporter (e.g., leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags). In some embodiments, labels are
attached by spacer arms of various lengths to reduce potential
steric hindrance.
[0046] "Fluorescent label" refers to incorporation of a detectable
fluorescent marker, e.g., by incorporation of a fluorescent moiety
to a chemical entity that binds to a target or attachment to a
polypeptide of secondary attachment molecules, such as biotinyl
moieties that can be detected by avidin (e.g., streptavidin
containing a fluorescent label or enzymatic activity that can be
detected by fluorescence detection methods). Examples of
fluorescent labels for polypeptides include, but are not limited to
dyes (e.g., FITC and rhodamine), intrinsically fluorescent
proteins, and lanthanide phosphors. In some embodiments, labels are
attached by spacer arms of various lengths to reduce potential
steric hindrance and in some embodiments to facilitate energy
transfer.
[0047] "Reporter gene" refers to a nucleotide sequence encoding a
protein that is readily detectable either by its presence or
activity, including, but not limited to, luciferase, fluorescent
protein (e.g., green fluorescent protein), chloramphenicol acetyl
transferase, .beta.-galactosidase, secreted placental alkaline
phosphatase, .beta.-lactamase, human growth hormone, and other
secreted enzyme reporters. Generally, reporter genes encode a
polypeptide not otherwise produced by the host cell, which is
detectable by analysis of the cell(s), e.g., by the direct
fluorometric, radioisotopic or spectrophotometric analysis of the
cell(s) and preferably without the need to kill the cells for
signal analysis. Preferably, the gene encodes an enzyme, which
produces a change in fluorometric properties of the host cell,
which is detectable by qualitative, quantitative or
semi-quantitative function of transcriptional activation. Exemplary
enzymes include esterases, .beta.-lactamase, phosphatases,
proteases (tissue plasminogen activator or urokinase) and other
enzymes whose function can be detected by appropriate chromogenic
or fluorogenic substrates known to those skilled in the art or
developed in the future. "Signal transduction" refers to the
coupling of an extracellular signal to an intracellular
response.
[0048] "Signal transduction detection system" refers to a system
for detecting signal transduction across a cell membrane, typically
a cell plasma membrane. Such systems typically detect at least one
activity or physical property directly or indirectly associated
with signal transduction. For example, an activity or physical
property directly associated with signal transduction is the
activity or physical property of either the receptor (e.g., GPCR),
or a coupling protein (e.g., a G.alpha. protein). Signal
transduction detection systems for monitoring an activity or
physical property directly associated with signal transduction,
include GTPase activity, ion channel activity and conformational
changes. An activity or physical property indirectly associated
with signal transduction is the activity or physical property
produced by a molecule other than by either the receptor (e.g.,
GPCRS, tyrosine kinase receptors, or tyrosine phoshphatase
receptors), ion channel or a coupling protein (e.g., a G.alpha.
protein) associated with a receptor (e.g., GPCRs, tyrosine kinase
receptors, or tyrosine phoshphatase receptors), or a coupling
protein (e.g., a G.alpha. protein). Such indirect activities and
properties include changes in intracellular levels of molecules
(e.g., ions (e.g., Ca, Na or K), second messenger levels (e.g.,
cAMP, cGMP and inositol phosphate)), kinase activities,
transcriptional activity, enzymatic activity, phospholipase
activities, ion channel activities and phosphatase activities.
Signal transduction assays are further described in commonly owned
U.S. applications by Negulescu et al, 60/020,234, filed Jun. 21,
1996 and serial number not yet available Jun. 19, 1997. Signal
transduction detection systems for monitoring an activity or
physical property indirectly associated with signal transduction,
include transcriptional-based assays, enzymatic assays,
intracellular ion assays and second messenger assays.
[0049] "Transducisome" refers to a PDZ domain containing protein
that binds at least one signal transduction protein or a PDZ domain
containing protein with at least one signal transduction protein
bound to it. For example, a transducisome may be an "uncomplexed
transducisome" comprising only the PDZ domain containing protein.
Alternatively, a transducisome may be a "complexed transducisome"
comprising the PDZ domain containing protein and a signal
transduction protein. For instance, a complexed transducisome may
contain at least two different PDZ domains bound to at least two
different signal transduction protein partners that separately
recognize their respective PDZ domains. Typically, a complexed
transducisome can enhance signal transduction activity compared 1)
signal transduction proteins that are not functionally bound to a
transducisome or 2) an uncomplexed transducisome. Transducisome
protein only refers to the uncomplexed transducisome.
[0050] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill,
San Francisco, incorporated herein by reference).
A DETAILED DESCRIPTION OF THE INVENTION
[0051] Cells respond to a wide variety of external signals mediated
by cell surface receptors that transduce extracellular stimuli into
an intracellular response. Receptors vary in type based on their
properties, including structural similarities, signaling properties
and intracellular pathways (e.g., G protein coupled receptors,
tyrosine kinase receptors, and tyrosine phophatase receptors).
Although receptors that recognize different ligands are known to
interact with the same intracellular signaling molecules, the
specificity of signaling, often essential to a cell's physiological
role is maintained. How is this specificity maintained, or rather,
how is signal cross talk avoided? One solution involves organizing
different signaling cascades into physically and functionally
distinct signaling units. Such assemblies can permit or enhance
signal response time, specificity and selectivity while minimizing
cross talk. Until the advent of present invention, little was known
about the architectural organization of the corresponding signaling
machinery or how it can be used to discover useful modulators of
signal transduction.
[0052] The invention provides cells and methods for identifying
modulators of signal transduction based, in part, on tmmsducisome
proteins that coordinate and assemble many types of signal
transduction proteins. Transducisomes can either permit or enhance
signal transduction. By including transducisome proteins in the
assays, as described herein, modulators of signal transduction can
be identified. "Transducisome" refers to a PDZ domain containing
protein that binds at least one signal transduction protein or a
PDZ domain containing protein with at least one signal transduction
protein bound to it.
[0053] The invention includes methods for identifying modulators of
signal transduction comprising contacting a transducisome in a
biochemical assay, cell assay or animal assay, with a test chemical
and detecting a change in signal transduction. As described herein
the invention includes animals (e.g., mice and flies), cells (e.g.,
mammalian and insect), with transducisomes, modified transducisomes
and defective transducisomes to use in assays of signal
transduction. The invention also includes proteins (as well as
polynucleotides encoding the same) corresponding to transducisomes,
modified transducisomes or defective transducisomes to use in
assays of signal transduction.
[0054] The Drosophila phototransduction is described herein as an
example of signal transduction, as it was used by the inventors to
demonstrate for the first time the function and structure of a
transducisome. The phototransduction cascade is a G
protein-coupled, PLC-signaling pathway that shares many features
with other signaling cascades.sup.30. Drosophila photoreceptor
neurons also show a high degree of architectural organization, with
most of the molecules involved in phototransduction localized to
the rhabdomeres, a specialized subcellular compartment consisting
of approximately 60,000 tightly packed microvilli, 1-2 im in length
and 50 nm wide.sup.31. In rhabdomeres, light activation of
rhodopsin activates a Gqa, which in turn activates a
PLC.sub.a.sup.32. PLC.sub..beta. catalyzes the breakdown of
phosphatidylinositol bisphosphate (PIP.sub.2) into the two
intracellular messengers inositol triphosphate (IP.sub.3) and
diacylglycerol (DAG), which leads to the eventual opening (and
modulation) of the TRP and TRPL light-activated channels.sup.33.
Following termination of the stimulus, calcium-dependent regulatory
processes, including activation of eye-PKC, mediate deactivation of
the light response.sup.34. InaD is one of the many loci that have
been identified in genetic screens designed to dissect this
signaling cascade. A single mutant allele, InaD.sup.215, was
isolated and shown to have a dominant negative phenotype on
photoreceptor deactivation.sup.35,36. The INAD protein was shown to
contain at least two PDZ domains.sup.36, and to interact with the
TRP ion channel.sup.37. More recently, several groups reported that
INAD can be found associated with multiple components of the
phototransduction cascade, including PLCa, eye-PKC, rhodopsin and
calmodulin.sup.37-39. These results were used to argue that INAD is
a regulatory component involved in feedback regulation of the light
response.sup.39. In surprising contrast to such previous work, the
present invention shows that the INAD protein is composed of five
distinct PDZ domains, and functions as the organizing scaffold for
photoreceptor signaling complexes in vivo. The genetic,
physiological and cell biological studies of the inventors
demonstrate the presence of a multivalent adapter protein that
links multiple signaling components within the same cascade, and
provides a fundamental role for PDZ domains in the assembly of
transduction complexes in vivo.
[0055] The advantages of spatially and temporarily restricting
signal transduction proteins with transducisomes are significant. A
cell can optimize, and tune, its responses to different pathways by
controlling the recruitment of different signaling molecules into
the different transduction complexes, thus enhancing specificity
and speed, while minimizing cross talk. An example is the yeast
mating response, in which the product of the sterile 5 gene
functions as a scaffold protein, coordinating the recruitment of
several kinases within the same signaling pathway.sup.45-47.
[0056] The inventors used phototransduction in Drosophila as a
model to study the organization of G protein-coupled transduction
complexes in vivo and in vitro in the Examples. In this signaling
pathway, photoreceptor neurons report activity with exquisite
sensitivity and specificity. In addition, photoreceptors achieve
superb temporal resolution by ensuring that the transduction
machinery is reset quickly after generating a response.
Phototransduction in Drosophila is the fastest known G
protein-coupled cascade, taking just a few tens of milliseconds to
go from light activation of rhodopsin to the generation of a
receptor potential, and less than 100 ms to shut-off following
termination of the stimulus.sup.30. The transducisome protein,
INAD, coordinates the recruitment of components involved both in
activation (PLC.sub..beta. and TRP), as well as in deactivation
(eye-PKC).sup.34. An important strategy used by photoreceptors to
attain high response speed is to assemble signaling molecules into
organized transducisomes. In this setting, response time would not
be limited by the diffusion of different signaling components
within a microvillus. This model of signal transduction, as well as
other examples of drosophila biology, applies to signal
transduction in humans.
[0057] A photoreceptor neuron attains high sensitivity by having an
exceptionally large number of receptor molecules in its surface
(.about.100.times.10.sup.6 rhodopsins/cell).sup.48,49. To rapidly
and efficiently couple this large number of receptor molecules to
the downstream transduction complexes, a diffusable coupling
molecule would be required. The G protein is an ideal candidate for
this function. In this model, each G protein would need only to
sample a small number of receptors in the membrane (i.e. an
ultra-microdomain of signaling) and report their activity to the
downstream transduction complexes. The Examples show that neither
rhodopsin, nor Ga are included in the INAD complexes
(transducisomes) while PKC, TRP ion channel and PLCP are part of
the transducisome.
[0058] Transducisomes also contribute to the nature of unitary
(i.e. single photon) responses in the eye. A quantum bump
represents the coordinated activation of a few hundred
light-activated ion channels in response to the activation of a
single rhodopsin molecule.sup.30. The organization of INAD
complexes into a supramolecular complex, either via PDZ-PDZ domain
interactions or PDZ-cytoskeletal interactions within a microvillus,
represents the structural basis of a quantum bump, insuring both
reliability and coordinated signaling.
[0059] Transducisomes can be identified and selected for screens
and assays described herein, by analyzing the structural basis of,
and the interaction between, PDZ domains and their targets. For
instance, the X-ray crystallographic structure of the third PDZ
domain from the synaptic protein PSD-95, alone or in complex with
its peptide ligand has been determined and can be used as a
model.sup.5,27,28. In addition, peptide libraries can be generated
to display different target sites that bind PDZ domains and show
preferences for distinct PDZ domains..sup.40 Using such in vitro
assays, PDZ domains that share structural elements and represent
different PDZ domains can be easily separated to show specificity
for different target proteins. The inventors have also showed this
to be the case in vivo. The finding that INAD is composed primarily
of PDZ domains indicates the importance of this protein motif in
the organization of signaling pathways. Recently, a new protein,
GRIP (glutamate receptor interacting protein), composed solely of
seven PDZ domains has been identified in mammalian cells.sup.50 and
can be involved in functioning as a signaling scaffold, organizing
specific transduction complexes at the synapse. Current models of
PDZ-target interaction involve a binding site composed of S/TXV
residues at the absolute C-terminal end of the target protein,
which is a preferred region to leave unaltered in many embodiments
of the invention..sup.5,27,29,40 In other embodiments of the
invention, PDZ binding regions of targets can be located at other
sites, such as located within 120 amino acid of the n-terminus of
the target. For example, all three INAD targets lack such a motif
(position -4 from the C-terminus in TRP, positions -122 and -507 in
eye-PKC and several sites PLC.sub.a, the closest at residue -26).
In addition, transducisome proteins can function as modular
proteins by maintaining separate binding functions in different
regions. For example, INAD functions as a modular protein and is
demonstrated by the fact that eliminating one target does not
prevent INAD's ability to interact with the others. By using the
methods described herein, it is possible to custom design
transduction complexes by manipulating the number, orientation and
distribution of PDZ domains in of transducisome proteins. The
availability of mutant transducisomes, such as null inaD mutant,
makes assays based on such compositions now possible.
[0060] Many signal transduction proteins can be recruited and
assembled into larger complexes, transducisomes, by protein-protein
interaction domains. Proteins interacting with, or containing, PDZ
domains.sup.8-12, are often localized at the plasma
membrane.sup.13-15. Thus, PDZ domains provide a framework for
recruiting target molecules into membrane-bound macromolecular
complexes. Recently, the PDZ-domain protein PSD-95 has been shown
to mediate the clustering of both NMDA receptors.sup.12,16-18 and
K.sup.+ channels.sup.19-22. In addition, members of the PSD-95/93
family form synaptic complexes.sup.16,23-25, and a PDZ domain in
FAP1 binds to the FAS membrane receptor.sup.26. In other
embodiments, PDZ-PDZ interactions can mediate of protein-protein
interactions.sup.26. To date, PDZ domains have been found in more
than 50 proteins, including many involved in cell signaling and can
be used as a source of signal transduction proteins..sup.27-29
[0061] One aspect of the invention includes a method of identifying
modulators of signal transduction. The method comprises contacting
a first cell with a test chemical, wherein the first cell comprises
at least one signal transduction protein and a polynucleotide
encoding a transducisome protein. The transducisome protein
functionally binds to the signal transduction protein to permit or
enhance signal transduction. The method can include activating
signal transduction in the first cell either with the test chemical
or with another chemical. The method includes the step of detecting
signal transduction from the first cell with a signal transduction
detection system. Different types of cells are described herein and
mammalian cells, particularly human cells, are preferred. Signals
from the first cell can be compared with signals from a second cell
assayed under different conditions, for instance under control
conditions.
[0062] For example, the method includes contacting a second cell
with the test chemical, wherein the second cell comprises the
signal transduction protein and a polynucleotide encoding a
defective transducisome protein. The defective transducisome
protein fails to functionally bind at least one signal transduction
protein to permit or enhance signal transduction. Alternatively,
second cell may be used that fails to express the transducisome
protein to permit the transducisome protein to functionally bind to
at least one signal transduction protein to permit or enhance
signal transduction. The method can include the step of activating
signal transduction in the second cell with a test chemical or
another chemical and detecting signal transduction from the second
cell with a signal transduction detection system. The method
includes the step of comparing signal transduction from the first
cell with signal transduction from the second cell. Often the
second cell is the same type of cell as the first cell. The second
cell can also comprise an amino acid mutation in a PDZ domain of
the defective transducisome protein that prevents functional
binding of a signal transduction protein, as described herein.
[0063] Another aspect of the invention includes a method of
identifying modulators of signal transduction by overexpressing the
transducisome protein to enhance or permit signal transduction. The
method comprises contacting a cell with a test chemical, wherein
the first cell comprises at least one signal transduction protein
and a polynucleotide encoding a transducisome protein, the
polynucleotide permits increased expression of the transducisome
protein and the transducisome protein functionally binds to the
signal transduction protein to permit or enhance signal
transduction compared to the absence of increased expression of the
transducisome protein. The method includes optionally activating
the signal transduction with a signal that increases or activates
the signal transduction in the cell, and detecting signal
transduction from the first cell with a signal transduction
detection system.
[0064] The method can also include contacting a second cell with
the test chemical. Wherein the second cell comprises the signal
transduction protein and a polynucleotide encoding a defective
transducisome protein. The defective transducisome protein fails to
functionally bind the signal transduction protein to permit or
enhance signal transduction or the second cells fails to express
the transducisome protein to permit the transducisome protein to
functionally bind to the signal transduction protein to permit or
enhance signal transduction. Signal transduction from the second
cell is detected with a signal transduction detection system, and
compared to signal transduction from the first cell. Alternatively,
the method can include contacting a second cell with the test
chemical, wherein the second cell lacks the signal transduction
protein to permit the transducisome protein to functionally bind to
the signal transduction protein to permit or enhance signal
transduction.
[0065] The signal for signal transduction can be any signal
compatible with the assay being used. The signal may be a test
chemical itself or a known activator of signal transduction. The
activating step can include activating signal transduction with a
signal selected from the group consisting of a chemical signal
found in blood, a chemical signal found in a synaptic cleft, a
chemical signal found in interstitial fluid, light or a chemical
signal found in air or other signals known the art or developed in
the future.
[0066] In preferred embodiments described herein, the signal
transduction protein is heterologously expressed and is selected
from the group consisting of a kinase, a phosphatase, a GPCR, a
tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion
channel, a G-protein, a phospholipase and a calcium binding
protein.
[0067] Another aspect of the invention, is a method of identifying
modulators of a cell surface receptor using transducisome proteins.
The method comprises contacting a cell with a test chemical,
wherein the cell comprises at least one cell surface receptor and a
polynucleotide encoding a transducisome protein. The polynucleotide
permits increased expression of the transducisome protein and the
transducisome protein functionally binds to the cell surface
receptor to permit or enhance signal transduction compared to the
absence of increased expression of the transducisome protein.
Signals can be detected associated with the cell surface receptor
in the presence and absence of the test chemical or known ligand of
the receptor. Various cells and receptors described herein or known
in the art can be used.
[0068] In regard to receptors, the invention also provides a method
of identifying modulators of a GPCR. The method includes contacting
a cell with a test chemical, wherein the cell comprises a
polynucleotide encoding a GPCR comprising one or more regions that
bind a PDZ domain(s) and a polynucleotide encoding a heterologous
protein comprising a PDZ domain that binds the GPCR. The method
includes detecting a signal associated with the activity the GPCR.
Various methods may be employed with the present invention
concerning the screening of GPCRS, including the use of response
elements and promiscuous G proteins, as described in commonly owned
U.S. applications by Negulescu et al, 60/020,234, filed Jun. 21,
1996 and serial number not yet available, filed Jun. 19, 1997,
herein incorporated by reference.
[0069] Such embodiments, as well as other embodiments described
herein, are particularly well suited for identifying modulators to
prevent or reduce the association of specific signal transduction
proteins with a transducisome. Thus, such modulators alter signal
transduction by reducing the amount of complexed transducisomes.
Such modulations can often reduce signal transduction without
completely eliminating signal transduction, which can enable to the
cell to perform a basal level of activity. This can be an advantage
compared to a antagonist that completely inactivates the
receptor.
[0070] The invention also includes a method of identifying
modulators of an ion channel using transducisome proteins. The
method comprises contacting a cell with a test chemical, wherein
the cell comprises at least one ion channel and a polynucleotide
encoding a transducisome protein, the polynucleotide permits
increased expression of the transducisome protein. The
transducisome protein functionally binds to the ion channel to
permit or enhance signal transduction compared to the absence of
increased expression of the transducisome protein. The method
includes detecting a signal associated with the activity the ion
channel.
[0071] Alternatively, the invention provides another a method of
identifying modulators of an ion channel. The method comprises
contacting a cell with a test chemical, wherein the cell comprises
a polynucleotide encoding an ion channel comprising one or more
regions that bind PDZ domains and a polynucleotide encoding a
heterologous protein comprising a PDZ domain that binds the ion
channel. The method includes detecting a signal associated with the
activity the ion channel.
[0072] Another aspect of the invention provides for a screening
assay system for identifying modulators of transducisomes. The
screening assay system comprises an isolated, non-naturally
occurring cell comprising at least one signal transduction protein
and a polynucleotide encoding a transducisome protein. The
polynucleotide permits increased expression (including inducible or
constitutive expression) of the transducisome protein. The
transducisome protein functionally binds to the signal transduction
protein to permit or enhance signal transduction compared to the
absence of increased expression of the transducisome protein. The
screening assay system can include a signal transduction detection
system for signal transduction in the isolated, non-naturally
occurring cell. The assay system can further include at least one
test chemical. Often such chemicals will be tested in arrays
enabling high throughput screening of at least 10,000 chemicals per
day.
[0073] The invention also includes a screening assay system for
detecting protein-protein interactions. This screening assay system
comprises a recombinant protein comprising at least one PDZ domain,
a PDZ binding protein, and at least one test chemical. The
recombinant protein can be either in solution or membrane
associated. The PDZ binding protein can also be either in solution
or membrane associated. The test chemical is in solution. For
example, the recombinant protein or the PDZ binding protein are
membrane bound and the test chemical is in solution. The
recombinant protein or the PDZ binding protein or both have a label
to facilitate detecting specific binding. Preferably, the
recombinant protein has a donor and the PDZ binding protein has a
quencher, wherein the donor and quencher are energy transfer
partners, as described herein. The PDZ binding protein can be
selected from the group of a kinase, a phosphatase, a GPCR, a
tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion
channel, a G-protein, a phospholipase and calcium binding protein.
More preferably the energy transfer partners are a GFP or modified
GFP FRET partner pair.
[0074] As described further herein, the invention provides for
isolated, non-naturally occurring cells. Such cells can be used
with the methods described herein and comprise 1) a heterologously
expressed transducisome protein comprising one or more PDZ domains
and 2) an expressed protein comprising a signal transduction
protein that binds to one or more the PDZ domains.
[0075] Alternatively, the invention provides for isolated,
non-naturally occurring cells, comprising: a cell capable of
expressing: a) a non-naturally occurring polynucleotide comprising
a coding region for a transducisome protein comprising one or more
PDZ domains and b) a non-naturally occurring polynucleotide
comprising a coding region for a heterologous protein comprising a
signal transduction protein. The signal transduction protein can be
selected from the group of a kinase, a phosphatase, a GPCR, a
tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion
channel, a G-protein, a phospholipase and a calcium binding
protein. For cell-based assays such cells will typically be
contacted with a test chemical. The cell may also optionally
include a signal transduction detection system.
[0076] The invention also provides for chimeric transducisome
proteins. Such chimerics typically comprise at least two PDZ
binding domains not found in naturally occurring proteins. Such
proteins can be used to generate signal transduction systems with
chimeric properties. Such system can be useful for screening for
modulators of signal transduction proteins. Often such systems will
have enhanced signal transduction. For example, a chimeric can
include a PDZ domain that binds a phospholipase and a GPCR, where
neither the phospholipase nor the GPCR normally bind to a signal
PDZ containing protein. By binding to the chimeric, coupling of
signal transduction will be increased between the phospholipase and
the GPCR. Chimerics can comprise at least one first PDZ domain that
binds a first signal transduction protein and at least one second
PDZ domain binds a second signal transduction protein, wherein said
chimeric transducisome protein is not a naturally occurring
protein. Preferably, The chimeric transducisome protein can bind a
first signal transduction protein(s) selected from the group of a
kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a
tyrosine phosphatase receptor, an ion channel, a G-protein, a
phospholipase and a calcium binding protein. More preferably, The
chimeric transducisome protein can bind second signal transduction
protein is selected from the group consisting of PKC, TRP, and
PLC.beta..
[0077] Another aspect of the invention concerns mutant
transducisome proteins that can be used in assays of transducisome
function and signal transduction. Transducisome proteins with
altered function can be used to determine the specificity of
signals. For example, a transducisome protein that normally binds
four different signal transduction proteins can be mutated to only
bind signal transduction proteins A, B and C but not D. Such a
transducisome protein can be used as a control for binding of
signal transduction protein D. Phenotypes related to transducisomes
can also be ascertained using mutant transducisome proteins.
[0078] Another aspect the invention provides for animals, such as
mammals (particularly mice) or insects with mutant transducisome
proteins. For example, the invention includes a fly comprising an
amino acid mutation in a transducisome protein that prevents
functional binding of a signal transduction protein, wherein the
amino acid mutation is not a naturally occurring mutation of inaD.
Preferably, the amino acid mutation results from a mutation
selected from the group consisting of inaD.sup.2 and
inaD.sup.1.
[0079] Alternatively, the mutation may be in a human homolog of
inaD.
[0080] In another aspect, the invention provides for an isolated
cell comprising a polynucleotide encoding a transducisome protein
with an amino acid mutation that prevents functional binding of a
signal transduction protein, wherein the amino acid mutation is a
naturally occurring mutation of inaD. Preferably, the cell is a
insect cell or a mammalian cell, particularly a human cell.
Preferably, the amino acid mutation results from a mutation
selected from the group consisting of inaD.sup.2 and
inaD.sup.1.
[0081] The invention provides for an isolated polynucleotide
comprising a coding region for a transducisome protein with an
amino acid mutation in a PDZ domain that prevents functional
binding of a signal transduction protein. Typically, the amino acid
mutation is not a naturally occurring mutation inaD.sup.215.
Preferably, the transducisome protein is INAD or a human homolog
thereof. Preferably, the amino acid mutation results from a
mutation selected from the group consisting of inaD.sup.2 and
inaD.sup.1. The invention also provides for an isolated protein
comprising a polypeptide of SEQ ID NO.: 1 with an amino acid
mutation in a PDZ domain that prevents functional binding of a
signal transduction protein.
[0082] The invention also includes chemicals identified by the
methods described herein. For example, the invention includes
chemicals identified by preventing the binding of a transducisome
protein with a signal transduction protein. As a further example,
the invention includes chemicals identified by the modulation of
signal transduction in a either: 1) a cell comprising: a) a
heterologously expressed transducisome protein comprising one or
more PDZ domains and b) an expressed protein comprising a signal
transduction protein that binds to one or more the PDZ domains, or
2) a cell capable of expressing: a) a non-naturally occurring
polynucleotide comprising a coding region for a transducisome
protein comprising one or more PDZ domains and b) a non-naturally
occurring polynucleotide comprising 1 coding region for a
heterologous protein comprising a signal transduction protein.
[0083] The invention also includes a method of treating a
transducisome related disease. The method comprises administering a
therapeutically effective amount of a chemical to modulate the
association of a transducisome and at least one PDZ binding
protein. The invention also includes a method of modulating a
signal transduction in a cell, comprising contacting a cell with a
chemical to modulate the association of a transducisome and at
least one PDZ binding protein. The signal transduction is typically
selected from the group consisting of G-protein coupled, ion
channels, kinases and phospholipases. Such methods can use a
therapeutic compound for treating a transducisome related disease,
comprising a chemical to modulate the association of a
transducisome and at least one PDZ binding protein.
Energy Transfer Compositions and Methods
[0084] In one aspect of the invention, transducisome protein
interactions with signal transduction proteins can be monitored and
used for identifying modulators of such interactions. The
transducisome protein, a signal transduction protein, or both
include a label that permits an assay for specific binding of the
transducisome protein to the signal transduction protein. For
example, a PDZ domain containing fragment of the transducisome
protein, a fragment of a signal transduction protein that
specifically binds a PDZ domain of the transducisome protein or
both include a label that permits an assay for specific binding of
the transducisome protein fragment to the signal transduction
protein fragment. Specific binding is determined by established
methods for binding assays and can include comparison to binding in
the presence of control chemicals, amino acid mutations that
prevent binding and other conditions as known in the art or
developed in the future or described herein.
[0085] In the preferred embodiment, transducisome protein
interactions with signal transduction proteins are monitored using
energy transfer moieties and methods. For example, a transducisome
protein can include a first energy transfer moiety (e.g., donor)
and a signal transduction protein can include a second energy
transfer moiety (e.g., quencher). The transducisome protein has at
least one, preferably two, specific PDZ domains that separately and
functionally bind at least one, preferably two, signal transduction
proteins. Often the transducisome protein will recognize more than
signal transduction protein, in such cases the first and second
energy transfer moieties may be on signal transduction proteins to
permit energy transfer between signal transduction proteins. The
first energy transfer moiety and the second energy transfer moiety
are suitably matched to permit to energy transfer when one is a
donor and the other is an acceptor or quencher. Preferably, the
energy transfer moieties are FRET (fluorescence resonance energy
transfer) pairs and more preferably fluorescent proteins that are
FRET pairs or partners. Although, other types of donor or acceptors
can be used, as described herein and in PCT Application WO
96/30540(Tsien et al) and PCT Application WO 96/41166 (Tsien et
al), particularly with labeling techniques.
[0086] Fluorescent protein FRET pairs are chosen such that the
excitation spectrum of one of the moieties (the acceptor
fluorescent protein moiety) overlaps with the emission spectrum of
the excited protein moiety (the donor fluorescent protein moiety).
The donor and acceptor fluorescent protein moieties are attached to
either a transducisome protein or signal transduction protein
(e.g., fusion proteins). Binding of the signal transduction protein
to the transducisome protein creates a change in relative position
(e.g., distance) and orientation of the donor and acceptor
fluorescent protein moieties compared to the respective proteins in
the unbound state (e.g., free in solution). Binding of the signal
transduction protein to the transducisome protein alters the
relative amounts of fluorescence, due to energy transfer from the
two fluorescent protein moieties when the donor is excited by
irradiation. In particular, binding of the signal transduction
protein to the transducisome protein changes the ratio of the
amount of light emitted by the donor and acceptor fluorescent
protein moieties at a particular excitation wavelength. The ratio
between the two emission wavelengths provides a measure of the
binding of the signal transduction protein to the transducisome
protein in the sample. Affinities related to the binding of the
signal transduction protein to the transducisome protein can be
readily measured, as well as reduced or increased by binding in the
presence of test chemicals.
[0087] FIG. 1C shows, the donor fluorescent protein moiety is
covalently linked to a first region (e.g., the amino terminus) of
the transducisome protein, and the acceptor fluorescent protein
moiety is covalently linked a first region of to the signal
transduction protein (e.g., the carboxy terminus). Functional
binding of the signal transduction protein to the transducisome
protein decreases the distance between the donor and acceptor
moieties. Both the signal transduction protein and the
transducisome protein can be free in solution, membrane bound or
both depending on the signal transduction system. Alternatively,
the donor and acceptor moieties can move farther apart upon
disassociation. A linker moiety can be added between the
fluorescent protein and the transducisome protein or the signal
transduction protein to enhance energy transfer. Typically, the
linker moiety is flexible enough to permit binding of the signal
transduction protein to the transducisome protein and allow closer
association of the fluorescent moieties to facilitate a form of
pseudodimerization. The fluorescent moieties themselves can be
selected from dimerizing proteins to enhance energy transfer.
Linking moieties are described, for example, in Huston, J. S., et
al., PNAS 85:5879-5883 (1988), Whitlow, M., et al., Protein
Engineering 6:989-995 (1993), and Newton, D. L., et al.,
Biochemistry 35:545-553 (1996).
[0088] The donor moiety is excited by light of appropriate
intensity within the excitation spectrum of the donor moiety
(.lamda..sub.excitation). The donor moiety emits the absorbed
energy as fluorescent light (.lamda..sub.emission 1). When the
acceptor fluorescent protein moiety is positioned to quench the
donor moiety in the excited state, the fluorescence energy is
transferred to the acceptor moiety that can emit fluorescent light
(.lamda..sub.emission 2). FRET can be manifested as a reduction in
the intensity of the fluorescent signal from the donor moiety
(.lamda..sub.emission 1), reduction in the lifetime of the excited
state of the donor moiety, or emission of fluorescent light at the
longer wavelengths (lower energies) characteristic of the acceptor
moiety (.lamda..sub.emission 2). When binding of the signal
transduction protein to the transducisome protein occurs, the
fluorescent protein moieties come closer, and FRET is
increased.
[0089] The efficiency of FRET depends on the separation distance
and the orientation of the donor and acceptor fluorescent protein
moieties. For example, the Forster equation describes the
efficiency of excited state energy transfer, based in part on the
fluorescence quantum yield of the donor moiety and the energetic
overlap with the acceptor moiety.
[0090] The Forster equation is:
E=(F.sub.0-F)/F.sub.0=R.sub.0.sup.6/(R.sup.6+R.sub.0.sup.6) where E
is the efficiency of FRET, F and F.sub.0 are the fluorescence
intensities of the donor moiety in the presence and absence of the
acceptor, respectively, and R is the distance between the donor
moiety and the acceptor moiety.
[0091] The characteristic distance R.sub.0 at which FRET is 50%
efficient depends on the quantum yield of the donor moiety (i.e.,
the shorter-wavelength fluorophore), the extinction coefficient of
the acceptor moiety (i.e., the longer-wavelength fluorophore), and
the overlap between the emission spectrum of the donor moiety and
the excitation spectrum of the acceptor moiety. R.sub.0 is given
(in A) by: R.sub.0=9.79.times.10.sup.3(K.sup.2QJn.sup.-4).sup.1/6
where K.sup.2 is an orientation factor having an average value
close to 0.67 for freely mobile donors and acceptors, Q is the
quantum yield of the unquenched donor moiety, n is the refractive
index of the medium separating the donor moiety and the acceptor
moiety, and J is the overlap integral. J can be quantitatively
expressed as the degree of spectral overlap between the donor
moiety and the acceptor moiety according to the equation:
J?.sup.8.sub.0.sup.o.sub.eF.sub.ee.sup.4de/?.sup.8.sub.0F.sub.ede
where .orgate.a.sub.e (M.sup.-1cm.sup.-1) is the molar absorptivity
of the acceptor and F.sub..lamda.2 is the donor moiety fluorescence
intensity at wavelength e. See, for example, Forster, T. Ann.
Physik 2:55-75 (1948). Tables of spectral overlap integrals are
readily available to those working in the field (for example,
Berlman, I. B. Energy transfer parameters of aromatic compounds,
Academic Press, New York and London (1973)). FRET is a
nondestructive spectroscopic method that can monitor proximity and
relative angular orientation of fluorophores in living cells. See,
for example, Adams, S. R., et al., Nature 349:694-697 (1991), and
Gonzalez, J. & Tsien, R. Y. Biophy. J 69:1272-1280 (1995).
[0092] These factors need to be balanced to optimize the efficiency
and detectability of FRET from assay systems described herein. The
emission spectrum of the donor fluorescent protein moiety should
overlap as much as possible with the excitation spectrum of the
acceptor fluorescent protein moiety to maximize the overlap
integral J. Also, the quantum yield of the donor fluorescent
protein moiety and the extinction coefficient of the acceptor
fluorescent protein moiety should be as large as possible to
maximize R.sub.0. In addition, the excitation spectra of the donor
and acceptor moieties should overlap as little as possible so that
a wavelength region can be found at which the donor moiety can be
excited selectively and efficiently without directly exciting the
acceptor moiety. Direct excitation of the acceptor moiety should be
avoided since it can be difficult to distinguish direct emission
from fluorescence arising from FRET. Similarly, the emission
spectra of the donor and acceptor moieties should have minimal
overlap so that the two emissions can be distinguished. High
fluorescence quantum yield of the acceptor moiety is desirable if
the emission from the acceptor moiety is to be monitored to
determine the amount of its labeled protein in a sample. In a
preferred embodiment, the donor fluorescent protein moiety is
excited by ultraviolet (<400 nm) and emits blue light (<500
nm), and the acceptor fluorescent protein moiety is efficiently
excited by blue but not by ultraviolet light and emits green light
(>500 nm), for example, P4-3 and S65T, respectively.
[0093] In another preferred embodiment, the donor fluorescent
moiety is excited by violet (400-430 nm) and emits blue-green
(450-500 nm) and the acceptor fluorescent moiety is efficiently
excited by blue-green (450-500 nm) and emits yellow-green light
(?520 nm), for example WIB and 10 C respectively.
[0094] The amount of a labeled protein in a sample can be
determined by determining the degree of FRET in the sample. Labeled
protein concentration can be determined by monitoring FRET at
different concentrations to establish a calibration curve.
[0095] The degree of FRET can be determined by any spectral or
fluorescence lifetime characteristic of the excited donor moiety.
For example, intensity of the fluorescent signal from the donor,
the intensity of fluorescent signal from the acceptor, the ratio of
the fluorescence amplitudes near the acceptor's emission maxima to
the fluorescence amplitudes near the donor's emission maximum, or
the excited state lifetime of the donor can be monitored.
[0096] Preferably, changes in the degree of FRET are determined as
a function of the change in the ratio of the amount of fluorescence
from the donor and acceptor moieties; a process referred to as
"ratioing." Changes in the absolute amount of indicator, excitation
intensity, and turbidity or other background absorbances in the
sample at the excitation wavelength affect the intensities of
fluorescence from both the donor and acceptor approximately in
parallel. Therefore the ratio of the two emission intensities is a
more robust and preferred measure of cleavage than either intensity
alone.
[0097] Fluorescence in a sample is measured using a fluorometer. In
general, excitation radiation, from an excitation source having a
first wavelength, passes through excitation optics. The excitation
optics cause the excitation radiation to excite the sample. In
response, fluorescent proteins in the sample emit radiation which
has a wavelength that is different from the excitation wavelength.
Collection optics then collect the emission from the sample. The
device can include a temperature controller to maintain the sample
at a specific temperature while it is being scanned. According to
one embodiment, a multi-axis translation stage moves a microtiter
plate holding a plurality of samples in order to position different
wells to be exposed. The multi-axis translation stage, temperature
controller, auto-focusing feature, and electronics associated with
imaging and data collection can be managed by an appropriately
programmed digital computer. The computer also can transform the
data collected during the assay into another format for
presentation.
[0098] Methods of performing assays on fluorescent materials are
well known in the art and are described in, e.g., Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, New York: Plenum Press
(1983); Herman, B., Resonance energy transfer microscopy, in:
Fluorescence Microscopy of Living Cells in Culture, Part B, Methods
in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San
Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modern
Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing
Col, Inc. (1978), pp. 296-361.
[0099] The excited state lifetime of the donor moiety is, likewise,
independent of the absolute amount of substrate, excitation
intensity, or turbidity or other background absorbances. Its
measurement requires equipment with nanosecond time resolution.
[0100] Quantum yields of wild-type GFP, S65T, and P4-1 mutants can
be estimated by comparison with fluorescein in 0.1 N NaOH as a
standard of quantum yield 0.91. J. N. Miller, ed., Standards in
Fluorescence Spectrometry, New York: Chapman and Hall (1981).
Mutants P4 and P4-3 were likewise compared to 9-aminoacridine in
water (quantum yield 0.98).
[0101] Any fluorescent protein can be used in the invention,
including proteins that fluoresce due intramolecular rearrangements
or the addition of cofactors that promote fluorescence. For
example, green fluorescent proteins of cnidarians, which act as
their energy-transfer acceptors in bioluminescence, are suitable
fluorescent proteins for use in the fluorescent indicators. A green
fluorescent protein ("GFP") is a protein that emits green light,
and a blue fluorescent protein ("BFP") is a protein that emits blue
light. GFPs have been isolated from the Pacific Northwest
jellyfish, Aequorea Victoria, the sea pansy, Renilla reniformis,
and Phialidium gregarium. See, Ward, W. W., et al., Photochem.
Photobiol., 35:803-808 (1982); and Levine, L. D., et al., Comp.
Biochem. Physiol., 72B:77-85 (1982).
[0102] A variety of Aequorea-related GFPs having useful excitation
and emission spectra have been engineered by modifying the amino
acid sequence of a naturally occurring GFP from Aequorea victoria.
See, Prasher, D. C., et al., Gene, 111:229-233 (1992); Heim, R., et
al., Proc. Natl. Acad. Sci., USA, 91:12501-04 (1994); U.S. Ser. No.
08/337,915, filed Nov. 10, 1994; International application
PCT/US95/14692, filed Nov. 10, 1995; and U.S. Ser. No. 08/706,408,
filed Aug. 30, 1996. The cDNA of GFP can be concatenated with those
encoding many other proteins; the resulting fusions often are
fluorescent and retain the biochemical features of the partner
proteins. See, Cubitt, A. B., et al., Trends Biochem. Sci.
20:448-455 (1995). Mutagenesis studies have produced GFP mutants
with shifted wavelengths of excitation or emission. See, Heim, R.
& Tsien, R. Y. Current Biol. 6:178-182 (1996). Suitable pairs,
for example a blue-shifted GFP mutant P4-3 (Y66H/Y145F) and an
improved green mutant S65T can respectively serve as a donor and an
acceptor for fluorescence resonance energy transfer (FRET). See,
Tsien, R. Y., et al., Trends Cell Biol. 3:242-245 (1993). A
fluorescent protein is an Aequorea-related fluorescent protein if
any contiguous sequence of 150 amino acids of the fluorescent
protein has at least 85% sequence identity with an amino acid
sequence, either contiguous or non-contiguous, from the wild type
Aequorea green fluorescent protein. More preferably, a fluorescent
protein is an Aequorea-related fluorescent protein if any
contiguous sequence of 200 amino acids of the fluorescent protein
has at least 95% sequence identity with an amino acid sequence,
either contiguous or non-contiguous, from the wild type Aequorea
green fluorescent protein. Similarly, the fluorescent protein can
be related to Renilla or Phialidium wild-type fluorescent proteins
using the same standards.
[0103] Some Aequorea-related engineered versions described in Table
I. Other variants or mutants are within the scope of the invention
as described in the art or developed in the future. TABLE-US-00001
TABLE I Extinct Coef- Quan- Excitation Emission ficient tum Clone
Mutation(s) max (nm) max (nm) (M.sup.-1cm.sup.-1) yield Wild type
none 395 508 21,000 0.77 (475) (7,150) P4 Y66H 383 447 13,500 0.21
P4-3 Y66H; Y145F 381 445 14,000 0.38 W7 Y66W; N146I 433 475 18,000
0.67 (453) (501) (17,100) M153T V163A N212K W2 Y66W; I123V 432 480
10,000 0.72 (453) (9,600) Y145H H148R M153T V163A N212K S65T S65T
489 511 39,200 0.68 P4-I S65T; M153A 504 514 14,500 0.53 (396)
(8,600) K238E S65A S65A 471 504 S65C S65C 479 507 S65L S65L 484 510
Y66F Y66F 360 442 Y66W Y66W 458 480 10c S65G; V68L 513 527 S72A;
T203Y W1B F64L, S65T 432 476 (453) (503) Y66W; N146I M153T V163A
N212K Emerald S65T; S72A 487 508 N149K M153T I167T Sapphire S72A;
Y145F 395 511 T203I
[0104] Other fluorescent proteins can be used in the proteins of
the invention, such as, for example, yellow fluorescent protein
from Vibrio fischeri strain Y-1, Peridinin-chlorophyll a binding
protein from the dinoflagellate Symbiodinium sp. phycobili proteins
from marine cyanobacteria such as Synechococcus, e.g.,
phycoerythrin and phycocyanin, or oat phytochromes from oat
reconstructed with phycoerythrobilin. These fluorescent proteins
have been described in Baldwin, T. O., et al., Biochemistry
29:5509-5515 (1990), Morris, B. J., et al., Plant Molecular
Biology, 24:673-677 (1994), and Wilbanks, S. M., et al., J. Biol.
Chem. 268:1226-1235 (1993), and Li et al, Biochemistry 34:7923-7930
(1995).
[0105] A localization sequence may be used to enhance targeting the
transducisome or signal transduction protein to a predetermined
cellular location. Localization sequences can be targeting
sequences which are described, for example, in "Protein Targeting",
chapter 35 of Stryer, L., Biochemistry (4th ed.). W.H. Freeman,
1995. The localization sequence can also be a localized protein.
Some important localization sequences include those targeting the
nucleus (KKKRK), mitochondrion (amino terminal
MLRTSSLFTRRVQPSLFRNILRLQST-), endoplasmic reticulum (KDEL at
C-terminus, assuming a signal sequence present at N-terminus),
peroxisome (SKF at C-terminus), prenylation or insertion into
plasma membrane (CaaX, CC, CXC, or CCXX at C-terminus), cytoplasmic
side of plasma membrane (fusion to SNAP-25), or the Golgi apparatus
(fusion to furin).
Production of Assay Systems and Proteins of the Invention Using
Cells
[0106] Many of the assays systems and proteins described herein can
be produced as fusion proteins, heterologously expressed proteins
and endogenous expressed proteins by recombinant DNA technology,
molecular biology and cell biology or a combination thereof.
Recombinant and cellular production of fluorescent proteins,
transducisomes and signal transduction involves expressing nucleic
acids having sequences that encode the proteins. Nucleic acids
encoding transducisome proteins can be obtained by methods known in
the art. For example, a nucleic acid encoding other transducisome
proteins can be isolated by polymerase chain reaction of cDNA from
various tissues and organism using primers based on the DNA
sequences of proteins containing PDZ domains or using cDNA probes
from the same. PCR methods are described in, for example, U.S. Pat.
No. 4,683,195; Mullis, et al. Cold Spring Harbor Symp. Quant. Biol.
51:263 (1987), and Erlich, ed., PCR Technology, (Stockton Press,
NY, 1989). In addition to such cloning approaches to find new or
orphan transducisomes, amino acid sequences in existing databases
such as GENBANK or EMBL, can be scanned for proteins having PDZ
domains and the protein sequences analyzed as described herein.
Putative or orphan transducisomes can be readily screened with
known signal transduction proteins to find their corresponding
transducisome protein. Many of the assays described herein are
particularly suitable for such screening.
[0107] Mutant versions of transducisomes can be made by
site-specific mutagenesis of nucleic acids encoding transducisome
proteins (particularly PDZ domains), or by random mutagenesis
caused by increasing the error rate of PCR of the original
polynucleotide with 0.1 mM MnCl.sub.2 and unbalanced nucleotide
concentrations. Other mutant screening methods as known in the art,
developed in the future or described herein can be used. Such
mutants can be readily tested for their ability to decrease binding
to signal transduction proteins. Such defective transducisome
proteins can be used as controls in the assays described herein,
both as controls in biochemical assays and in cell-based assays
(e.g., cell lines with defective transducisomes as described
herein). New fluorescent proteins can also be made by
mutagensis.
[0108] The construction of expression vectors and the expression of
genes in transfected cells involve the use of molecular cloning
techniques also well known in the art. 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).
[0109] Nucleic acids used to transfect cells with sequences coding
for expression of the polypeptide of interest generally will be in
the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for
expression of the polypeptide. As used, the term "nucleotide
sequence coding for expression of" a polypeptide refers to a
sequence that, upon transcription and translation of mRNA, produces
the polypeptide. This can include sequences containing, e.g.,
introns. As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are operatively linked to a nucleic acid sequence
when the expression control sequences control and regulate the
transcription and, as appropriate, translation of the nucleic acid
sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
stop codons.
[0110] Methods which are well known to those skilled in the art can
be used to construct expression vectors comprising a transducisome
protein or signal transduction protein coding sequence, or both,
and appropriate transcriptional/translational control signals.
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. (See, for example, the techniques described in
Maniatis, et al., Molecular Cloning A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y., 1989).
[0111] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0112] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransfected with DNA
sequences encoding the fusion polypeptide of the invention, and a
second foreign DNA molecule encoding a selectable phenotype, such
as the herpes simplex thymidine kinase gene. Another method is to
use a eukaryotic viral vector, such as simian virus 40 (SV40) or
bovine papilloma virus, to transiently infect or transform
eukaryotic cells and express the protein. (Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
Preferably, a eukaryotic host is utilized as the host cell as
described herein.
[0113] Techniques for the isolation and purification of either
microbially or eukaryotically expressed polypeptides of the
invention may be by any conventional means such as, for example,
preparative chromatographic separations and immunological
separations such as those involving the use of monoclonal or
polyclonal antibodies or antigen.
[0114] A variety of host-expression vector systems may be utilized
to express transducisome protein or signal transduction protein
coding sequence or both. These include but are not limited to
microorganisms such as bacteria transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing a protein of the invention coding sequence; yeast
transformed with recombinant yeast expression vectors containing
the a protein of the invention coding sequence; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing a protein of the invention coding sequence;
insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus) containing a transducisome protein or
signal transduction protein coding sequence, or both; or animal
cell systems infected with recombinant virus expression vectors
(e.g., retroviruses, adenovirus, vaccinia virus) containing a
transducisome protein or signal transduction protein coding
sequence, or both, or transformed animal cell systems engineered
for stable expression.
[0115] Depending on the host/vector system utilized, any of a
number of suitable transcription and translation elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, etc. may be used in
the expression vector (see, e.g., Bitter, et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
?, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be
used. When cloning in mammalian cell systems, promoters derived
from the genome of mammalian cells (e.g., metallothionein promoter)
or from mammalian viruses (e.g., the retrovirus long terminal
repeat; the adenovirus late promoter; the vaccinia virus 7.5K
promoter) may be used. Promoters produced by recombinant DNA or
synthetic techniques may also be used to provide for transcription
of the inserted transducisome protein or signal transduction
protein coding sequence, or both.
[0116] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
transducisome protein or signal transduction protein coding
sequence expressed. For example, when large quantities of the
transducisome protein or signal transduction protein are to be
produced, vectors which direct the expression of high levels of
fusion protein products that are readily purified may be desirable.
Those which are engineered to contain a cleavage site to aid in
recovering transducisome protein or signal transduction protein
coding sequence are preferred.
[0117] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Grant, et
al., Expression and Secretion Vectors for Yeast, in Methods in
Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol.
153, pp. 516-544, 1987; Glover, DNA Cloning, Vol. II, IRL Press,
Washington D.C., Ch. 3, 1986; and Bitter, Heterologous Gene
Expression in Yeast, Methods in Enzymology, Eds. Berger &
Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684, 1987; and The
Molecular Biology of the Yeast Saccharomyces, Eds. Strathem et al.,
Cold Spring Harbor Press, Vols. I and II, 1982. A constitutive
yeast promoter such as ADH or LEU2 or an inducible promoter such as
GAL may be used (Cloning in Yeast, Ch. 3, R. Rotistein In: DNA
Cloning Vol. II, A Practical Approach, Ed. DM Glover, IRL Press,
Washington D.C., 1986). Alternatively, vectors may be used which
promote integration of foreign DNA sequences into the yeast
chromosome.
[0118] In cases where plant expression vectors are used, the
expression of a transducisome protein or signal transduction
protein coding sequence may be driven by any of a number of
promoters. For example, viral promoters such as the 35S RNA and 19S
RNA promoters of CaMV (Brisson, et al., Nature 310:511-514, 1984),
or the coat protein promoter to TMV (Takamatsu, et al., EMBO J.
6:307-311, 1987) may be used; alternatively, plant promoters such
as the small subunit of RUBISCO (Coruzzi, et al., 1984, EMBO J.
3:1671-1680; Broglie, et al., Science 224:838-843, 1984); or heat
shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley, et
al., Mol. Cell. Biol. 6:559-565, 1986) may be used. These
constructs can be introduced into plant cells using Ti plasmids, Ri
plasmids, plant virus vectors, direct DNA transformation,
microinjection, electroporation, etc. For reviews of such
techniques see, for example, Weissbach & Weissbach, Methods for
Plant Molecular Biology, Academic Press, NY, Section VIII, pp.
421-463, 1988; and Grierson & Corey, Plant Molecular Biology,
2d Ed., Blackie, London, Ch. 7-9, 1988.
[0119] An alternative expression system which could be used to
express a transducisome protein or signal transduction protein
coding sequence is an insect system. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes. The virus grows in Spodoptera frugiperda
cells. The transducisome protein or signal transduction protein
coding sequences may be cloned into non-essential regions (for
example, the polyhedrin gene) of the virus and placed under control
of an AcNPV promoter (for example the polyhedrin promoter).
Successful insertion of the transducisome protein or signal
transduction protein coding sequence coding sequence will result in
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat coded
for by the polyhedrin gene). These recombinant viruses are then
used to infect Spodoptera frugiperda cells in which the inserted
gene is expressed, see Smith, et al., J. Viol. 46:584, 1983; Smith,
U.S. Pat. No. 4,215,051.
[0120] Eukaryotic systems, and preferably mammalian expression
systems, allow for proper post-translational modifications of
expressed mammalian proteins to occur. Eukaryotic cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, phosphorylation, and, advantageously
secretion of the gene product should be used as host cells for the
expression of proteins of the invention. Such host cell lines may
include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,
Jurkat, HEK-293, and WI38.
[0121] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, the transducisome protein
or signal transduction protein coding sequence may be ligated to an
adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene
may then be inserted in the adenovirus genome by in vitro or in
vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the proteins of the
invention in infected hosts (e.g., see Logan & Shenk, Proc.
Natl. Acad. Sci. USA, 81: 3655-3659, 1984). Alternatively, the
vaccinia virus 7.5K promoter may be used. (e.g., see, Mackett, et
al., Proc. Natl. Acad. Sci. USA, 79: 7415-7419, 1982; Mackett, et
al., J. Virol. 49: 857-864, 1984; Panicali, et al., Proc. Natl.
Acad. Sci. USA 79: 4927-4931, 1982). Of particular interest are
vectors based on bovine papiloma virus which have the ability to
replicate as extrachromosomal elements (Sarver, et al., Mol. Cell.
Biol. 1: 486, 1981). Shortly after entry of this DNA into mouse
cells, the plasmid replicates to about 100 to 200 copies per cell.
Transcription of the inserted cDNA does not require integration of
the plasmid into the host's chromosome, thereby yielding a high
level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such as
the neo gene. Alternatively, the retroviral genome can be modified
for use as a vector capable of introducing and directing the
expression of the fluorescent indicator gene in host cells (Cone
& Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353, 1984).
High level expression may also be achieved using inducible
promoters, including, but not limited to, the metallothionine IIA
promoter and heat shock promoters.
[0122] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with the transducisome protein or signal
transduction protein cDNA controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci
which in turn can be cloned and expanded into cell lines. For
example, following the introduction of foreign DNA, engineered
cells may be allowed to grow for 1-2 days in an enriched media, and
then are switched to a selective media. A number of selection
systems may be used, including but not limited to the herpes
simplex virus thymidine kinase (Wigler, et al., Cell, 11: 223,
1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska
& Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and
adenine phosphoribosyltransferase (Lowy, et al., Cell, 22: 817,
1980) genes can be employed in tk.sup.-, hgprt or aprt cells
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA, 77: 3567,
1980; O'Hare, et al., Proc. Natl. Acad. Sci. USA, 8: 1527, 1981);
gpt, which confers resistance to mycophenolic acid (Mulligan &
Berg, Proc. Natl. Acad. Sci. USA, 78: 2072, 1981; neo, which
confers resistance to the aminoglycoside G-418 (Colberre-Garapin,
et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers
resistance to hygromycin (Santerre, et al., Gene, 30: 147, 1984)
genes. Recently, additional selectable genes have been described,
namely trpB, which allows cells to utilize indole in place of
tryptophan; hisD, which allows cells to utilize histinol in place
of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA,
85:8047, 1988); and ODC (ornithine decarboxylase) which confers
resistance to the ornithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., In: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory,
ed., 1987).
[0123] DNA sequences encoding the transducisome protein or signal
transduction protein coding sequence polypeptide of the invention
can be expressed in vitro by DNA transfer into a suitable host
cell. "Host cells" are cells in which a vector can be propagated
and its DNA expressed. The term also includes any progeny of the
subject host cell. It is understood that all progeny may not be
identical to the parental cell since there may be mutations that
occur during replication. However, such progeny are included when
the term "host cell" is used. Methods of stable transfer, in other
words when the foreign DNA is continuously maintained in the host,
are known in the art.
[0124] Recombinant transducisome protein or signal transduction
protein coding sequence can be produced by expression of nucleic
acid encoding the protein in prokaryotes, such as E. coli or in
eukaryotes, such as yeast cells or mammalian cells.
[0125] The construct can also contain a tag to simplify isolation
of the transducisome protein or signal transduction protein coding
sequence. For example, a polyhistidine tag of, e.g., six histidine
residues, can be incorporated at the amino terminal end of the
fluorescent protein. The polyhistidine tag allows convenient
isolation of the protein in a single step by nickel-chelate
chromatography.
[0126] In a preferred embodiment, the transducisome protein or
signal transduction protein coding sequence is a fusion protein
with a fluorescent protein produced by recombinant DNA technology
in which a single polypeptide includes a donor moiety, a peptide
linker moiety or an acceptor moiety and a peptide linker moiety.
The donor moiety can be positioned at the amino-terminus relative
to the acceptor moiety in the polypeptide. Such a fusion protein
has the generalized structure: (amino terminus) donor or acceptor
fluorescent protein moiety--peptide linker moiety--transducisome
protein or signal transduction protein (carboxy terminus).
Alternatively, the donor moiety can be positioned at the
carboxy-terminus of the transducisome protein or signal
transduction protein. To optimize the location of the donor and
acceptor for a particular binding pair of transducisome protein and
signal transduction protein all combinations (such as
N-donor-C--N-transducisome protein-C/N-acceptor-C--N-signal
transduction protein-C, N-transducisome
protein-C--N-donor-C/N-acceptor-C--N-signal transduction protein-C,
N-donor-C--N-transducisome protein-C/N-signal transduction
protein-C--N-acceptor-C, N-transducisome
protein-C--N-donor-C/N-signal transduction protein-C--N-acceptor-C,
N-acceptor-C--N-transducisome-C/N-donor-C--N-signal transduction
protein-C, and N-transducisome-C--N-acceptor-C/N-donor-C--N-signal
transduction protein-C) can be readily made and tested within a
short period of time or person days. Tranducisome protein fragments
and signal transduction fragments can be made this way as well. The
invention also envisions fusion proteins that contain extra amino
acid sequences at the amino and/or carboxy termini, for example,
polyhistidine tags. Such fluorescent transducisome and signal
transduction proteins or fragments thereof can also be used as
non-FRET proteins in fluorescent binding assays for screening
compounds.
[0127] Thus, FRET binding pair of transducisome protein and signal
transduction proteins can be encoded by a recombinant nucleic acid
sequences. The elements are selected so that upon expression into
fusion proteins, the donor and acceptor moieties exhibit FRET when
the donor moiety is excited and binding occurs. The recombinant
nucleic acid can be incorporated into an expression vector
comprising expression control sequences operatively linked to the
recombinant nucleic acid. The expression vector can be adapted for
function in prokaryotes or eukaryotes by inclusion of appropriate
promoters, replication sequences, markers, etc.
[0128] The expression vector can be transfected into a host cell
for expression of the recombinant nucleic acid. Host cells can be
selected for high levels of expression in order to purify the
fluorescent binding pair of transducisome protein and signal
transduction proteins. E. coli is useful for this purpose,
especially if larger quantities of full-length proteins or
functionally fragments are desired for biochemical assays.
Alternatively, the host cell can be a prokaryotic or eukaryotic
cell selected to study the activity of an enzyme produced by the
cell. In this case, the linker peptide is selected to include an
amino acid sequence recognized by the protease. The cell can be,
e.g., a cultured cell or a cell in vivo.
[0129] A primary advantage of proteins of the invention is that
they are prepared by normal protein biosynthesis, thus completely
avoiding organic synthesis and the requirement for customized
unnatural amino acid analogs. The constructs can be expressed in E.
coli in large scale for in vitro assays. Purification from bacteria
is simplified when the sequences include polyhistidine tags for
one-step purification by nickel-chelate chromatography.
Alternatively, the substrates can be expressed directly in a
desired host cell for assays in situ, as described herein for
screening assays.
Cells and Targets
[0130] Any cell expressing a protein target in sufficient quantity
for measurement in cellular assays can be used with the invention.
Cells endogenously expressing proteins of the invention are
suitable for many embodiments, as well as proteins expressed from
heterologous nucleic acids. For example, cells may be transfected
with a suitable vector encoding one or more targets that are known
to those of skill in the art or may be identified by those of skill
in the art. Although essentially any cell which expresses
endogenous ion channel or receptor activity may be used, when using
receptors or channels as targets it is preferred to use cells
transformed or transfected with heterologous DNAs encoding such ion
channels and/or receptors so as to express predominantly a single
type of ion channel or receptor. Many cells that may be genetically
engineered to express a heterologous cell surface protein are
known. Exemplary membrane proteins include, but are not limited to,
surface receptors and ion channels.
[0131] One method of the present invention uses targets for
identifying chemicals that are useful in modulating the activity of
a target in the presence of a transducisome protein. The target can
be any biological entity, such as a protein, sugar, nucleic acid or
lipid. Typically, targets will be proteins such as cell surface
proteins or enzymes. Targets can be assayed in either biochemical
assays (targets free of cells), or cell based assays (targets
associated with a cell).
[0132] For example, cells expressing transducisome proteins may be
loaded with ion or voltage sensitive dyes to report receptor or ion
channel activity, such as calcium channels or N-methyl-D-aspartate
(NMDA) receptors, GABA receptors, kainate/AMPA receptors, nicotinic
acetylcholine receptors, sodium channels, calcium channels,
potassium channels excitatory amino acid (EAA) receptors, and
nicotinic acetylcholine receptors. Assays for determining activity
of such receptors can also use agonists and antagonists to use as
negative or positive controls to assess activity of tested
chemicals. In preferred embodiments of automated assays for
identifying chemicals that have the capacity to modulate the
function of receptors or ion channels (e.g., agonists,
antagonists), changes in the level of ions in the cytoplasm or
membrane voltage will be monitored using an ion-sensitive or
membrane voltage fluorescent indicator, respectively. Among the
ion-sensitive indicators and voltage probes that may be employed,
are those disclosed in the Molecular Probes 1997 Catalog, herein
incorporated by reference.
[0133] Other methods of the present invention concern determining
the activity of receptors in the presence of transducisome
proteins. Receptor activation can sometimes initiate subsequent
intracellular events that release intracellular stores of calcium
ions for use as a second messenger. Activation of some
G-protein-coupled receptors stimulates the formation of inositol
triphosphate (IP3 a G-protein coupled receptor second messenger)
through phospholipase C-mediated hydrolysis of
phosphatidylinositol, Berridge and Irvine (1984), Nature 312:
315-21. IP3 in turn stimulates the release of intracellular calcium
ion stores. Thus, a change in cytoplasmic calcium ion levels caused
by release of calcium ions from intracellular stores can be used to
reliably determine G-protein-coupled receptor function. Among
G-protein-coupled receptors are muscarinic acetylcholine receptors
(mAChR), adrenergic receptors, serotonin receptors, dopamine
receptors, angiotensin receptors, adenosine receptors, bradykinin
receptors, metabotropic excitatory amino acid receptors and the
like. Cells expressing such G-protein-coupled receptors may exhibit
increased cytoplasmic calcium levels as a result of contribution
from both intracellular stores and via activation of ion channels,
in which case it may be desirable although not necessary to conduct
such assays in calcium-free buffer, optionally supplemented with a
chelating agent such as EGTA, to distinguish fluorescence response
resulting from calcium release from internal stores.
[0134] Other assays can involve determining the activity of
receptors in the presence of transducisome proteins which, when
activated, result in a change in the level of intracellular cyclic
nucleotides, e.g., cAMP, cGMP. For example, activation of some
dopamine, serotonin, metabotropic glutamate receptors and
muscarinic acetylcholine receptors results in a decrease in the
cAMP or cGMP levels of the cytoplasm. Furthermore, there are cyclic
nucleotide-gated ion channels, e.g., rod photoreceptor cell
channels and olfactory neuron channels (see, Altenhofen, W. et al.
(1991) Proc. Natl. Acad. Sci. U.S. 88: 9868-9872 and Dhallan et al.
(1990) Nature 347: 184-187) that are permeable to cations upon
activation by binding of cAMP or cGMP. In cases where activation of
the receptor results in a decrease in cyclic nucleotide levels, it
may be preferable to expose the cells to agents that increase
intracellular cyclic nucleotide levels, e.g., forskolin, prior to
adding a receptor-activating compound to the cells in the assay.
Cells for this type of assay can be made by co-transfection of a
host cell with DNA encoding a cyclic nucleotide-gated ion channel,
transducisome protein and DNA encoding a receptor (e.g., certain
metabotropic glutamate receptors, muscarinic acetylcholine
receptors, dopamine receptors, serotonin receptors, and the like),
which, when activated, causes a change in cyclic nucleotide levels
in the cytoplasm.
[0135] Ion channels can also be used with the invention and
include, but are not limited to, calcium channels comprised of the
human calcium channel .alpha..sub.2.beta. and/or .gamma.-subunits
(see also, WO89/09834; human neuronal .alpha..sub.2 subunit);
rabbit skeletal muscle al subunit (Tanabe, et al. (1987) Nature
328, pp. 313-E318); rabbit skeletal muscle .alpha..sub.2 subunit
(Ellis, et al. (1988) Science 241, pp. 1661-1664); rabbit skeletal
muscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118);
rabbit skeletal muscle .gamma. subunit (Jay, et al. (1990) Science
248, pp. 490-49.sup.2); and the like; potassium ion channels, e.g.,
rat brain (BK2) (McKinnon, D. (1989) J. Biol. Chem. 264, pp.
9230-8236); mouse brain (BK1) (Tempel, et al. (1988) Nature 332,
pp. 837-839); and the like; sodium ion channels, e.g., rat brain I
and II (Noda, et al. (1986) Nature 320, pp. 188-192); rat brain III
(Kayano, et al. (1988) FEBS Lett. 228, pp. 187-1.94); human II
(ATCC No. 59742, 59743 and Genomics 5: 204-208 (1989); chloride ion
channels (Thiemann, et al. (1992), Nature 356, pp. 57-60 and
Paulmichl, et al. (1992) Nature 356, pp. 238-241), and others known
or developed in the art.
[0136] GPCRs that can be used with the invention include, but are
not limited to, muscarinic receptors, e.g., human M2 (GenBank
accession #M16404); rat M3 (GenBank accession #M16407); human M4
(GenBank accession #M16405); human M5 (Bonner, et al., (1988)
Neuron 1, pp. 403-410); and the like; neuronal nicotinic
acetylcholine receptors, e.g., the human .alpha..sub.2,
.alpha..sub.3, and .beta..sub.2, subtypes. The human .alpha..sub.5
subtype (Chini, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:
1572-1576), the rat .alpha..sub.2 subunit (Wada, et al. (1988)
Science 240, pp. 330-334); the rat .alpha..sub.3 subunit (Boulter,
et al. (1986) Nature 319, pp. 368-374); the rat .alpha..sub.4
subunit (Goldman, et al. (1987) Cell 48, pp. 965-973); the rat
.alpha..sub.5 subunit (Boulter, et al. (1990) I. Biol. Chem. 265,
pp. 4472-4482); the chicken .alpha..sub.7 subunit (Couturier et al.
(1990) Neuron 5: 847-856); the rat .beta..sub.2 subunit Deneris, et
al. (1988) Neuron 1, pp. 45-54) the rat .beta..sub.3 subunit
(Deneris, et al. (1989) J. Biol. Chem. 264, pp. 6268-6272); the rat
.beta..sub.4 subunit (Duvoisin, et al. (1989) Neuron 3, pp.
487-496); combinations of the rat .alpha. subunits, and s.beta.
subunits and a and p subunits; GABA receptors, e.g., the bovine x,
and .beta..sub.1, subunits (Schofield, et al. (1987) Nature 328,
pp. 221-227); the bovine X.sub.2, and X.sub.3, subunits (Levitan,
et al. (1988) Nature 335, pp. 76-79); the .gamma.-subunit
(Pritchett, et al. (1989) Nature 338, pp. 582-585); the
.beta..sub.2, and .beta..sub.3, subunits (Ymer, et al. (1989) EMBO
J. 8, pp. 1665-1670); the 8 subunit (Shivers, B. D. (1989) Neuron
3, pp. 327-337); and the like; glutamate receptors, e.g., rat GluR1
receptor (Hollman, et al. (1989) Nature 342, pp. 643-648); rat
GluR2 and GluR3 receptors (Boulter et al. (1990) Science
249:1033-1037; rat GluR4 receptor (Keinanen et al. (1990) Science
249: 556-560); rat GluR5 receptor (Bettler et al. (1990) Neuron 5:
583-595); rat GluR6 receptor (Egebjerg et al. (1991) Nature 351:
745-748); rat GluR7 receptor (Bettler et al. (1992) neuron
8:257-265); rat NMDAR1 receptor (Moriyoshi et al. (1991) Nature
354:31-37 and Sugihara et al. (1992) Biochem. Biophys. Res. Comm.
185:826-832); mouse NMDA el receptor (Meguro et al. (1992) Nature
357: 70-74); rat NMDAR2A, NMDAR2B and NMDAR2C receptors (Monyer et
al. (1992) Science 256: 1217-1221); rat metabotropic mGluR1
receptor (Houamed et al. (1991) Science 252: 1318-1321); rat
metabotropic mGluR2, mGluR3 and mGluR4 receptors (Tanabe et al.
(1992) Neuron 8:169-179); rat metabotropic mGluR5 receptor (Abe et
al. (1992) I. Biol. Chem. 267: 13361-13368); and the like;
adrenergic receptors, e.g., human 31 (Frielle, et al. (1987) Proc.
Natl. Acad. Sci. 84, pp. 7920-7924); human .alpha..sub.2 (Kobilka,
et al. (1987) Science 238, pp. 650-656); hamster2p (Dixon, et al.
(1986) Nature 321, pp. 75-79); and the like; dopamine receptors,
e.g., human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6);
mammalian dopamine D2 receptor (U.S. Pat. No. 5,128,254); rat
(Bunzow, et al. (1988) Nature 336, pp. 783-787); and the like; and
the like; serotonin receptors, e.g., human 5HT1a (Kobilka, et al.
(1987) Nature 329, pp. 75-79); serotonin 5HT1C receptor (U.S. Pat.
No. 4,985,352); human 5HT1D (U.S. Pat. No. 5,155,218); rat 5HT2
(Julius, et al. (1990) PNAS 87, pp. 928-932); rat 5HTIc (Julius, et
al. (1988) Science 241, pp. 558-564), and the like.
[0137] Various methods of identifying activity of chemical with
respect to a target in the presence of a transducisome protein can
be applied, including: ion channels (PCT publication WO 93/13423),
cell surface receptors (U.S. Pat. Nos. 5,401,629, and 5,436,128 and
PCT Application WO 93/13423 (Akong et al) and intracellular
receptors (PCT publication WO 96/41013,U.S. Pat. No. 5,548,063,
U.S. Pat. No. 5,171,671, U.S. Pat. No. 5,274,077, U.S. Pat. No.
4,981,784, EP 0 540 065 A1, U.S. Pat. No. 5,071,773, and U.S. Pat.
No. 5,298,429). All of the foregoing references are herein
incorporated by reference in their entirety.
[0138] If desired (e.g., for commercial purposes), a cell(s) of the
invention can packaged into a container that is packaged within a
kit. Such a kit may also contain any of the various isolated
nucleic acids, antibodies, proteins, signal transduction detection
systems, substrates, and/or drugs described herein, known in the
art or developed in the future. A typical kit also includes a set
of instructions for any or all of the methods described herein.
EXAMPLES
[0139] The following examples are intended to illustrate but not
limit the invention. While examples are typical of those methods
compositions that might be used to practice the invention, other
procedures known to those skilled in the are may alternatively be
used as appropriate.
Example 1--Materials and Methods Used in the Examples
Mutant Screens and Western blots
[0140] Males of cn bw genotype were aged for 5 days, treated with
EMS, and crossed en masse to flies carrying the dominant
temperature sensitive DTS91 allele. Single F1 males were collected
and crossed in single vials to CyO/DTS91 virgin females. The vials
were then shifted to 29.degree. C. for 72 hrs to eliminate any eggs
or larvae carrying the DTS allele. The parents were then removed
and the vials were incubated at 29.degree. C. for an additional 48
hrs before returning to 25.degree. C. The progeny from this cross
were transferred to fresh food, and their homozygous white-eyed
offspring (cn bw) were subjected to a protein immunoblot screen for
the loss of the INAD antigen.sup.33.
Antibodies
[0141] To generate antibodies specific to INAD, we generated a
T7-fusion protein consisting of the last 300 residues of the
protein. All antibodies were checked for specificity and affinity
using wild type, mutant, and transgenic controls. For
immunostaining, the INAD antibody was diluted 1:500 in phosphate
buffered saline, 1% BSA, 0.1% saponin (PBS-S); the TRP antibody was
first preabsorbed with a homogenate of trp mutant heads to reduce
background staining and used at a final dilution of 1:100.
Rhodopsin (1:300), eye-PKC (1:50), PLC (1:1000), TRPL (1:100), and
DGq (1:200) were detected using polyclonal antibodies as previously
described.sup.33,34,44.
Immunoprecipitation
[0142] Frozen heads (500-1,000) were homogenized in 2 to 3 mL of
buffer A (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA and
protease inhibitors) using a glass-glass homogenizer. The
homogenate was centrifuged at 4,000.times.g for one minute to
remove chitinous-material. Membranes were isolated by
centrifugation at 100,000.times.g for 30 minutes at 4.degree. C.,
and resuspended in 0.8 to 1 ml of buffer A to determine protein
concentration. Samples were re-centrifuged, resuspended in buffer B
(150 mM NaCl, 1% triton, 50 mM Tris-HCl, pH 8.0 and protease
inhibitors), mixed (100 .mu.g of protein) with anti-INAD antibodies
and incubated for 1 hour at 4.degree. C. At this time, 30 .mu.l of
protein A-agarose beads (Pierce) were added and incubated for 2
additional hours. Samples were washed in buffer B, resuspended in
SDS buffer and fractionated by SDS PAGE. The entire
immunoprecipitate was loaded on the gels. Studies using GST-INAD
protein fusions used similar incubation conditions but also
contained affinity-purified GST-fusion proteins. GST-fusions
containing individual PDZ domains (PDZ1 to PDZ5) were constructed
according to the boundaries shown in FIG. 2. All GST-fusions were
overproduced and purified by affinity chromatography on
glutathione-agarose beads as described.sup.19,23,50.
Electrophysiological Recordings
[0143] Photoreceptors were isolated from adult flies (<6 hr
after eclosion) and whole-cell, patch-clamp recordings were
performed as previously described.sup.33. Photoreceptors were
stimulated by a 75 W Xenon source connected to the epifluorescence
port of an inverted Fluovert FS (Leitz) microscope; light was
bandpass-filtered (.lamda.=580.+-.10 nm) and focused onto the
photoreceptor cells with a 0.5 numerical aperture, 40.times.
objective. Signals were recorded with an Axopatch 200A patch-clamp
amplifier (Axon Instruments, Foster City, Calif.) and data were
analyzed using pClamp6.02 (Axon) and Origin (Microcal) software.
The membrane of the photoreceptors was voltage-clamped at a holding
potential of 40 mV. Traces were low-pass filtered at 2 kHz (Bessel
filter) and digitized at 2 kHz, unless stated otherwise. Measured
series resistance, 16 M.OMEGA. on average, was 80% compensated. The
bath solution contained (in mM): 124 NaCl, 4 KCL, 10 HEPES, 5
proline, 25 sucrose, 1.5 CaCl.sub.2, 1 MgCl.sub.2, pH 7.15. Pipette
solution contained 95 KGluconate, 40 KCl, 10 mM HEPES, 2
MgCl.sub.2, 2 EGTA, pH 7.15.
[0144] For quantum-bump analysis, photoreceptors were clamped at
-70 mV, and stimulated with a dim light flash to generate quantum
bumps around 50% of the time. Signals were lowpass-filtered at 1
kHz and digitized at 2 kHz.
Example 2--Exemplary Transducisome Structure
[0145] To investigate a putative transducisome and its structure,
the inventors analyzed the protein structure of INAD (SEQ. ID NO.:
1). INAD was selected as a candidate transducisome because mutants
in the gene, InaD, produce a dominant negative phenotype for
photoreceptor activation. The primary structure of INAD was
analyzed using BLAST. INAD is a modular protein composed of five
closely linked PDZ domains (see FIG. 2). Each of these domains
contains the structural hallmarks of a prototypical PDZ motif,
including the conserved amino acid region target binding. Each PDZ
domains, however, is different and displays sufficient differences
in amino acid sequence to permit binding of different signal
transduction proteins and to allow distinct protein-protein
interactions. This finding was quite surprising, as previous
studies had shown that INAD contains two PDZ domains instead of
five.
[0146] FIG. 2A shows a diagram of INAD protein with the locations
of and size of each PDZ domain highlighted. Also shown is the
relative location of the three inaD mutations related to
photoreceptor activation.
[0147] FIG. 2B shows an amino acid alignment of PDZ domains from
mammalian PSD-95.sup.9, nNOS.sup.23, Drosophila dlg.sup.8 and
inaD.sup.36. Sequences were aligned to maximize similarities and
the full-length sequences of the references are herein incorporated
by reference. Black boxes indicate amino acid identities and gray
boxes show conservative substitutions. Stars above the sequence
indicate residues implicated in substrate binding.sup.27. The
circled residues refer to the site of point mutation in the three
Drosophila inaD alleles.
[0148] FIG. 2C shows an immunoblot demonstrating the absence of
INAD protein in inaD.sup.1. Another photoreceptor protein NinaA was
used as a control in the blots. Note normal levels of NinaA in both
lanes.
Example 3--Transducisomes Organize Signal Transduction in a
Membranes
[0149] To investigate the ability of transducisomes to organize
signal transduction proteins in membranes, the inventors measured
binding of signal transduction proteins to INAD. The inventors
assayed for binding of different phototransduction cascade proteins
to INAD. Immunoprecipitations and GST-INAD protein fusions were
used to identify INAD binding entities or targets.
[0150] FIG. 3A shows that INAD antibodies co-immunoprecipitate TRP,
eye-PKC and PLC.sub..beta. from retinal extracts.sup.38. Rhodopsin
and Gq.sub.a failed to immunoprecipate despite the fact that both
proteins are extremely abundant in photoreceptor cells. Membranes
prepared from the heads of wt flies (IP) or trp, norpA and inaC
mutants were immunoprecipitated (100 ug) with anti-INAD antibody as
described in Example 1. The immunoprecipitated proteins were
separated by SDS-PAGE, transferred to nitrocellulose and separately
probed with antibodies specific for TRP, PLC, eye-PKC, G alpha
subunit (G.alpha.) and rhodopsin (Rh1). Heads refers to membranes
before immunoprecipitation. As a negative control for antibody and
immunoprecipation specificity, immunoprecipitations from inaD nulls
did not precipitate TRP, PKC or PLC (data not shown).
[0151] FIG. 3B shows similar results obtained in vitro using a full
length GST-INAD fusion protein. Full length GST-INAD (N-GST-INAD-C)
fusions associate with TRP, PLC.beta. and PKC in cell extracts.
[0152] To define the site on INAD that interacts with TRP, eye-PKC
and PLC.sub.?, the inventors dissected INAD. The inventors produced
individual PDZ domains of INAD as GST-fusion proteins and assayed
each domain for interaction with each of target proteins from whole
retinal extracts.
[0153] FIG. 3B also shows that the third PDZ domain of INAD is
specific for TRP.sup.37, while the fourth domain specifically
interacts with eye-PKC and the fifth domain specifically interacts
with PLC.sub.?. Overexpression of each of the individual PDZ
domains from INAD as GST-PDZ fusions (N-GST-PDZ 1, 2, 3, 4 or 5-C)
produce highly preferred interactions in biochemical assays. PDZ 1,
2, 3, 4 or 5 are shown in FIG. 2.
[0154] These results demonstrate that loss of one PDZ domain and
its corresponding signal transduction protein from a transducisome
does not prevent binding of the other signal transduction proteins
to other PDZ domains or a transducisome protein.
[0155] Immunoprecipitation of INAD from trp mutants, PLC.sub..beta.
nulls (norpA), or PKC nulls (inaC) still co-precipitates the
remaining two targets. These results demonstrate some important
aspects of transducisome and PDZ domain function. First, different
PDZ domains can have different and highly specific targets. Second,
INAD functions as a modular multivalent PDZ protein interacting
with different components of the same pathway. Third, the
transducisome complex, with signal transduction proteins bound to
it, does not require binding interactions between the different
signal transduction proteins.
[0156] These results are surprising because previous work only
showed that INAD can associate with individual components of the
phototransduction cascade.sup.37-39, rather than the formation of a
complex that organizes signal transduction proteins of a membrane.
This is the first demonstration that transducisomes exist as PDZ
domain containing proteins to mediate protein-protein interactions
and the assembly of transduction complexes with multiple types of
signal transduction proteins in the bound in the same complex.
Example 4--Transducisomes Permit or Enhance Signal Transduction and
Transducisome Assembly In Vivo
[0157] To investigate the function of transducisomes in signal
transduction in vivo, the inventors isolated new inaD mutant
alleles responsible for the dominant negative phenotype of
photoreceptor deactivation and tested their corresponding proteins
for the ability to assemble transducisomes and perform signal
transduction in vivo.
[0158] If INAD functions in vivo as a scaffold to localize or
assemble multiple components of the phototransduction cascade into
transducisomes, then a null inaD mutant should display a complete
loss of signaling complexes and a redistribution of individual
signaling molecules. Unfortunately, only a single inaD mutant
allele had been isolated and it behaved genetically and
physiologically as a partially dominant negative
mutation.sup.35,36. The inventors isolated new inaD alleles.
[0159] Using flies, the screening strategy was based on the loss of
INAD antigen on immunoblots rather than on a hypothetical
physiological or behavioral phenotype. Fly stocks containing
individual homozygous mutagenized second chromosomes (inaD maps to
the second chromosome at position 59B1-2) were generated and each
stock was then subjected to immunoblot analysis for the loss of
anti-INAD immunoreactivity.sup.33. Analysis of 2847 lines yielded
two alleles, inaD.sup.1 and inaD.sup.2. inaD.sup.1 has a complete
loss of the protein, while inaD.sup.2 expresses reduced levels of
protein.
[0160] Using the polymerase chain reaction (PCR), the inventors
isolated the inaD.sup.1 gene from each mutant allele and determined
their entire nucleotide sequence. inaD.sup.1 has an amber non-sense
mutation at position 811, leading to premature termination of the
polypeptide chain at amino acid residue 270 (see FIG. 2 and
SEQUENCE ID LISTING). This represents a complete null allele.
InaD.sup.2 has a A->G change at nucleotide 1814, leading to the
substitution of a conserved glycine for glutamic acid in the fifth
PDZ domain (see FIG. 2). Nucleotide positions are based on the
published nucleotide sequence of INAD, Shieh and Niemeyer Neuron
14, 201-210 (1995), herein incorporated by reference.
[0161] The inventors then used immunofluorescent staining of frozen
tissue sections to test for the subcellular localization of
signaling molecules in the inaD.sup.1 null mutant cells. The data
demonstrate that TRP, PLC.sub..beta., and eye-PKC are completely
mislocalized in the inaD.sup.1 mutant. These signal transduction
proteins no longer localize to the rhabdomeres, but instead are
found randomly distributed either throughout the plasma membrane
(TRP) or the cytoplasm (PLC.sub..beta. and eye-PKC). In contrast,
rhodopsin, Gq.sub.a and TRPL distribute normally in inaD.sup.1.
Immunofluorescent staining was performed on one micron thick
cross-sections of wild type (showing localization), and inaD.sup.1
mutant (no localization of signal transduction proteins as
described herein) photoreceptors for different transduction
proteins. No INAD-immunoreactive material was found in inaD.sup.1
mutants.
[0162] To investigate the ability of transducisomes to permit or
enhance signal transduction of photoreceptors, the inventors
performed electrophysiological recordings on photoreceptor cells
from wildtype, inaD.sup.1, and inaD.sup.2 flies. Wildtype cells,
with a functional transducisome, showed enhanced signal
transduction in response to light compared to inaD.sup.1 mutants.
InaD.sup.1 mutants, compared to wildtype, are much less sensitive
to light, responding only at the highest light intensities and with
profoundly altered kinetics (see FIG. 4). InaD.sup.2 mutants,
compared to wildtype, are less sensitive to light, but more
sensitive to light compared to inaD.sup.1 mutants, and have
intermediate kinetics (see FIG. 4). The electroretinograms (ERG)
recordings are from wild type (wt), inaD.sup.1 and inaD.sup.2
mutant flies at <1 day after eclosion. Stimulus was a 10 second
pulse of orange light (570 nm longpass filter). Right traces show
responses to 10.times. the amount of light in the left traces
(log[I]=-2 log[I]=-1, respectively). Arrows indicate the onset of
the stimulus. Both mutants have an increase in loss of
responsiveness as a function of age.
[0163] These results demonstrate that when transducisomes are not
formed in inaD.sup.1 mutants, which have severe truncation of the
transducisome protein, extraordinary defects in phototransduction
occur. Conversely, when the transducisome is functional, as in the
wild type normal signal transduction is permitted. Thus, the
transducisomes permit normal signal transduction to occur and
enhance signal transduction compared to signal transduction by
signal transduction proteins that are not complexed. InaD.sup.2
mutants, which have point mutation of a conserved amino acid in a
PDZ domain of a transducisome protein, displayed intermediate
defects in phototransduction, consistent with the role of
transducisomes enhancing and organizing signal transduction.
[0164] To investigate the affect of the loss transducisome
scaffolding or localization on signal transduction proteins, the
inventors examined the instability of the signal transduction
proteins. The inventors assayed the steady-state levels of signal
transduction proteins by immunoblot analysis at different stages
post-eclosion. The levels of TRP, PLC.sub..beta. and eye-PKC are
all markedly reduced in the inaD.sup.1 mutants, and by 10 days
post-eclosion are less than 10% of wild type levels. In contrast,
the levels of rhodopsin, Ga and TRPL are unaffected. Immunoblot
analysis of transduction proteins were performed in wild type and
inaD mutants. Protein levels were measured at approximately 24 hrs
or sooner, after eclosion (0d) and 10 days (10d). Levels of all
signal transduction proteins in wild type flies remained constant
with age, while TRP, eye-PKC, and PLC declined drastically in
inaD.sup.1 mutants. Only TRP declines in inaD.sup.215 (PDZ3), and
only PLC declines in inaD.sup.2 (PDZ5). The equivalent of one fly
head per lane was run for wild type and inaD.sup.215, and two fly
heads for inaD.sup.1 and inaD.sup.2.
[0165] These results demonstrate a role for transducisomes for
organizing signal transduction complexes, enhancing signal
transduction, permitting normal signal transduction, and improving
the intracellular stability of the signal transduction
proteins.
Example 5--Mutations in Transducisomes Lead to Signal Transduction
Destabilization and Decreased Signal Transduction
[0166] To further investigate the role of transducisomes in signal
transduction, the inventors assessed the affect of mutations of the
INAD protein on signal transduction and signal transduction protein
stability. As described herein, INAD interacts with multiple
components of the phototransduction cascade and is essential for
the assembly of signaling complexes, i.e. transducisomes. The
inventors therefore postulated that mutation of the PDZ domain for
a particular target should prevent the recruitment of that protein
into the transduction complexes. This should generate in vivo
phenotypes that resemble mutations in the target proteins.
[0167] The original inaD allele, inaD.sup.215, has a missense
mutation in the third PDZ domain.sup.36. Since this domain is
involved in the interaction of INAD with TRP, as described herein,
and this mutation abolishes the interaction of TRP with
INAD.sup.37,39 the stability of TRP, its subcellular localization
and its function might be disrupted in inaD.sup.215 mutants. The
inventors examined TRP protein levels by immunoblot analysis, TRP
subcellular localization by immunofluorescent staining of tissue
sections and its function by performing whole-cell voltage-clamp
recordings and electroretinograms (ERG).
[0168] TRP protein levels decline with age in inaD.sup.215 mutants.
In young inaD.sup.215 flies (less than 24 hours old), TRP levels
are indistinguishable from control flies. However, by 10 days the
protein is barely detectable in immunoblots. In contrast,
PLC.beta., eye-PKC, and other transduction protein levels remain
constant in either mutant. TRP channels are also completely
mislocalized in the inaD.sup.215 mutant, and are found randomly
distributed throughout the plasma membrane (newly assayed eclosed
flies to prevent degradation of TRP), while PLC is mislocalized in
inaD.sup.2 mutants. Immunofluorescent staining for TRP, eye-PKC,
and PLC was conducted in one micron thick cross-section of
inaD.sup.215 and inaD.sup.2 mutant photoreceptors.
[0169] To examine the mislocalization of TRP in more detail,
ImmunoEM staining was conducted using gold-conjugated secondary
antibodies. These studies confirmed and extended the
immunofluorescence observations: the TRP channel randomly localizes
to the plasma membrane in such mutants. The inventors found no
evidence for mislocalization of any other phototransduction
protein, including PLC.sub..beta. and eye-PKC in inaD.sup.215
mutants. In wild type photoreceptors, TRP is present at
significantly higher levels and exclusively in the microvillar
membranes of the rhabdomeres. In inaD.sup.215, TRP levels are
significantly reduced in the rhabdomeres (newly enclosed flies) and
TRP prominently distributed throughout the plasma membrane.
[0170] These results are surprising, as in contrast to Chevesich et
al..sup.39, TRP was never found in the extracellular matrix, nor
were significant levels of cytoplasmic labeling observed. The
results also demonstrate that transducisomes are important for
organizing signal transduction in the correct membrane and in
specific regions of the cell, e.g., a region specialized for signal
transduction.
[0171] To characterize the physiology of inaD.sup.215 mutants
immunoblot and ERG studies were performed. InaD.sup.215 mutants
display an ERG phenotype that approaches that of trp mutants, and
does so on a time course similar to that of the decay of TRP
protein seen in immunoblots. Whole-cell voltage-clamp recordings of
macroscopic currents and quantum bumps were also recorded.
inaD.sup.215 mutants were originally characterized as displaying
slow deactivation kinetics in response to a flash of light.sup.36
(see FIG. 5A). ERG recordings were from wildtype, trp.sup.301, and
inaD.sup.215 mutant eyes at 10 days after eclosion. Light stimulus
was a 30 sec. pulse of orange light (570 nm longpass filter). Note
the transient response of trp mutants (trp=transient receptor
potential), and older inaD.sup.215 flies.
[0172] To determine the basis for the slow deactivation component
in inaD.sup.215 photoreceptors, quantal responses were
characterized. In wild-type photoreceptors, single-photons give
rise to unitary events known as quantum bumps.sup.41,42. Quantum
bumps are the result of the activation of a single rhodopsin
molecule and reflect the amplification of the entire signaling
pathway, leading to the opening of the light-activated
channels.sup.43. Surprisingly, the quantum bumps from inaD.sup.215
flies display normal termination kinetics (wild type
t.sub.90%=13.6.+-.0.58 ms, inaD.sup.215 t.sub.90%=13.8.+-.0.60 ms;
FIG. 5B, right panel), indicating that the macroscopic defect of
inaD.sup.215 mutants cannot be due to an underlying defect in
deactivation. FIG. 5B shows whole-cell recordings (left traces) and
quantum bumps (right traces) from wild type (wt) and inaD.sup.215
mutant photoreceptors.
[0173] For macroscopic responses, cells were stimulated (arrow)
with a 10 ms flash of 580 nm light of log[I]=-1. The deactivation
time course of inaD.sup.215 is well fit by the sum of two
exponentials (time constants of 14.7.+-.2.5 and 143.1.+-.12.1 ms;
N=7), while the time course of decay of wild type responses is
fitted by a single exponential with a time constant of 14.5.+-.2.2
ms (N=6). For quantum bumps.sup.44, cells were stimulated with a 10
ms flash of 580 nm light of log[I]=-6.5 (arrow). This stimulus
produced a probability of not seeing a bump of 0.40 in wild type
and 0.65 in inaD.sup.215. Note the normal termination kinetics of
inaD.sup.215 bumps. InaD.sup.215 quantum bumps have defective
latency. Latency to first bump from wild type (open bars) and
inaD.sup.215 mutant (solid bars) were 47.6.+-.1.3 ms (N=210 bumps
from 7 cells), and 67.0.+-.3.1 ms (N=160 bumps from 7 cells),
respectively. Instead of defective termination kinetics, the mean
latency times between stimulus and quantum bump generation were
significantly altered in inaD.sup.215 mutants (47.6.+-.1.3 ms in
wild type vs. 67.0.+-.3.2 ms in inaD.sup.215; FIG. 5C). Thus,
inaD.sup.215 photoreceptors do not have a defect in
termination.sup.36, nor do they display problems with feedback
regulation.sup.37,39. The phenotype is consistent with the
mislocalization of TRP channels that leads to longer latencies and
a corresponding macroscopic defect in deactivation kinetics. Again
these results are surprising, as previous work postulated that
deactivation related INAD might be due to an affect TRP channel
activity.sup.37-39, rather than affecting TRP localization or
stability while maintaining channel activity.
[0174] To further define the physiological importance of the
interaction between INAD and its individual targets, the
interaction between INAD and PLC.sub..beta. was also studied.
PLC.sub.? is randomly distributed in the cytoplasm of inaD.sup.2
photoreceptor cells, which has mutation in the fifth PDZ domain
(see FIG. 2) of INAD. InaD.sup.2 mutation failed to affect the
distribution of other signal transduction proteins like eye-PKC,
TRP, Rhl and DGq. These results from fly eyes are consistent with
the specific redistribution of PLC.sub..beta. in vivo due to
inaD.sup.2 mutant photoreceptors and a failure of PLC.sub..beta. to
be recruited into mutant transducisomes, which leads to
PLC.sub..beta. instability and a decay of PLC.sub..beta. over
time.
[0175] The loss of PLC.sub..beta. from transduction complexes,
leads to significant defects in phototransduction. ERG recordings
from inaD.sup.2 mutant photoreceptors exhibit major defects in
response kinetics: latency, activation, and deactivation, which are
all significantly slower in the mutant cells. Because these
recording were carried out in newly enclosed flies, a time at which
there are near normal levels of PLC as described herein, these
findings clearly illustrate that it is not the mere presence of a
transducisome, but rather its location that promotes effective
signaling. Taken together data validate the existence of a highly
organized signaling unit, a transducisome, demonstrate that it is
possible to experimentally manipulate the composition of signaling
complexes, and substantiate the essential role of PDZ domains in
the assembly and function of signal transduction complexes in
vivo.
Example 5--A GPCR Based Screen for Modulators of Signal
Transduction Function
[0176] A screen for identifying modulators of signal transduction
was designed using cells that can express a GPCR, a phospholipaseC,
and a G protein that assemble into a transducisome. A cell line
containing Gq-type GPCR receptor that expresses .beta.-lactamase in
response to the addition of the agonist was produced by FACS
selection. The G.alpha.q protein was endogenously expressed. The
activation response is inhibited by an antagonist. Jurkat clones
expressing NFAT-.beta.la, as described in Negulescu et al filed
Jun. 19, 1997 (herein incorporated by reference) were transfected
with expression vectors containing the Gq receptor and neomycin
resistance gene (double transfection). The transfected population
was neo-selected and sorted by FACS for clones responding to the
GPCR agonist. Cells were stimulated for three hours with the
indicated ligands. Cells were then loaded with .beta.-lactamase
substrate CCF2/ac2AM for 1 hour, washed, dispensed into wells of a
microtiter plate (100,000 cells/well) and the blue/green ratio was
recorded by a plate reader.
[0177] A twenty-fold change in signal upon receptor activation with
an agonist (saturating dose 100 .mu.M) was observed. A receptor
antagonist (10 .mu.M) completely inhibited the agonist activation
of the receptor.
[0178] To identify modulators of signal transduction using a
transudcisome these cells are transfected with a polynucleotide
encoding a transducisome protein that comprises individual PDZ
domains that assemble such transducisome. Expression of
transducisome protein enhances signal transduction to provide for
better signals due to increased .beta.-lactamase expression and
activity. Controls for the affect of transducisome protein on
signal transduction can also be used to identify modulators of
transducisome protein/signal transduction protein interactions.
Such controls include mutant or truncated transducisome protein
that fails to functionally bind the signal transduction protein
that is normally part of a transducisome complex or cells not
expressing transducisome proteins from exogenous or mutated
polynucleotides (e.g., non-induced polynucleotide having expression
controlled by an inducable promoter, defective transducisome
proteins due to a mutation or non-transfected cells). Test
compounds are added in the presence or absence of agonist,
antagonist, inverse agonist or other known modulators.
Example 6--An Ion Channel Based Screen for Modulators of Signal
Transduction Function
[0179] A screen for identifying modulators of signal transduction
was designed using cells that can express rhodospin, an ion channel
that binds a transducisome protein, and a phospholipase that binds
a transducisome protein, a PKC that binds a transducisome protein
and a transducisome protein. Cells are maintained in the dark and
exposed for a predetermined time to light at a wavelength that will
activate rhodospin that in turn activates Gq.alpha.. The G-protein
in turn activates PLC.beta. leading to the increase of inositol
triphosphate and diacylgylcerol that lead to the activation of ion
channel, TRP. Ion channel activity is monitored with voltage
sensitive dyes using a fluorimeter. Test compounds can be added
before or after light activation.
Example 7--A FRET Based Screen for Modulators of Transducisome
Protein Binding to Signal Transduction Proteins
[0180] A screen for identifying modulators of transducisone binding
was designed using cells that can express modified GFP FRET
partners fused to transducisome fragments and modified GFP FRET
partners fused to signal transduction proteins that bind to the
corresponding transducisome fragment. Polynucleotides encoding a
first modified GFP partner fused to an INAD protein containing a
PDZ5. The resulting fusion protein is oriented as follows:
N-INAD-C--N-GFP FRET partner-C; wherein N is the n-terminus of each
respective fragment and C is the c-terminus of each respective
fragment. Polynucleotides encoding a second modified GFP partner
fused to a PLC.beta. protein containing a PDZ5. The resulting
fusion protein is oriented as follows: N-GFP
FRET-partner-C--N--PLC.beta. protein-C; wherein N is the n-terminus
of each respective fragment and C is the c-terminus of each
respective fragment. Expression of each modified GFP fusion protein
is accomplished with use of a constitutive promoter and a vector
suitable for expression in CHO or insect cells. After expression of
the GFP fusion proteins, FRET is measured between the modified GFP
FRET partners in the presence and absence of test chemicals using a
fluorimeter.
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[0231] All publications, including patent documents and scientific
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reference in their entirety for all purposes to the same extent as
if each individual publication were individually incorporated by
reference.
Sequence CWU 1
1
16 1 674 PRT Drosophila melanogaster 1 Met Val Gln Phe Leu Gly Lys
Gln Gly Thr Ala Gly Glu Leu Ile His 1 5 10 15 Met Val Thr Leu Asp
Lys Thr Gly Lys Lys Ser Phe Gly Ile Cys Ile 20 25 30 Val Arg Gly
Glu Val Lys Asp Ser Pro Asn Thr Lys Thr Thr Gly Ile 35 40 45 Phe
Ile Lys Gly Ile Val Pro Asp Ser Pro Ala His Leu Cys Gly Arg 50 55
60 Leu Lys Val Gly Asp Arg Ile Leu Ser Leu Asn Gly Lys Asp Val Arg
65 70 75 80 Asn Ser Thr Glu Gln Ala Val Ile Asp Leu Ile Lys Glu Ala
Asp Phe 85 90 95 Lys Ile Glu Leu Glu Ile Gln Thr Phe Asp Lys Ser
Asp Glu Gln Gln 100 105 110 Ala Lys Ser Asp Pro Arg Ser Asn Gly Tyr
Met Gln Ala Lys Asn Lys 115 120 125 Phe Asn Gln Glu Gln Thr Thr Asn
Asn Asn Ala Ser Gly Gly Gln Gly 130 135 140 Met Gly Gln Gly Gln Gly
Gln Gly Gln Gly Met Ala Gly Met Asn Arg 145 150 155 160 Gln Gln Ser
Met Gln Lys Arg Asn Thr Thr Phe Thr Ala Ser Met Arg 165 170 175 Gln
Lys His Ser Asn Tyr Ala Asp Glu Asp Asp Glu Asp Thr Arg Asp 180 185
190 Met Thr Gly Arg Ile Arg Thr Glu Ala Gly Tyr Glu Ile Asp Arg Ala
195 200 205 Ser Ala Gly Asn Cys Lys Leu Asn Lys Gln Glu Lys Asp Arg
Asp Lys 210 215 220 Glu Gln Glu Asp Glu Phe Gly Tyr Thr Met Ala Lys
Ile Asn Lys Arg 225 230 235 240 Tyr Asn Met Met Lys Asp Leu Arg Arg
Ile Glu Val Gln Arg Asp Ala 245 250 255 Ser Lys Pro Leu Gly Leu Ala
Leu Ala Gly His Lys Asp Arg Gln Lys 260 265 270 Met Ala Cys Phe Val
Ala Gly Val Asp Pro Asn Gly Ala Leu Gly Ser 275 280 285 Val Asp Ile
Lys Pro Gly Asp Glu Ile Val Glu Val Asn Gly Asn Val 290 295 300 Leu
Lys Asn Arg Cys His Leu Asn Ala Ser Ala Val Phe Lys Asn Val 305 310
315 320 Asp Gly Asp Lys Leu Val Met Ile Thr Ser Arg Arg Lys Pro Asn
Asp 325 330 335 Glu Gly Met Cys Val Lys Pro Ile Lys Lys Phe Pro Thr
Ala Ser Asp 340 345 350 Glu Thr Lys Phe Ile Phe Asp Gln Phe Pro Lys
Ala Arg Thr Val Gln 355 360 365 Val Arg Lys Glu Gly Phe Leu Gly Ile
Met Val Ile Tyr Gly Lys His 370 375 380 Ala Glu Val Gly Ser Gly Ile
Phe Ile Ser Asp Leu Arg Glu Gly Ser 385 390 395 400 Asn Ala Glu Leu
Ala Gly Val Lys Val Gly Asp Met Leu Leu Ala Val 405 410 415 Asn Gln
Asp Val Thr Leu Glu Ser Asn Tyr Asp Asp Ala Thr Gly Leu 420 425 430
Leu Lys Arg Ala Glu Gly Val Val Thr Met Ile Leu Leu Thr Leu Lys 435
440 445 Ser Glu Glu Ala Ile Lys Ala Glu Lys Ala Ala Glu Glu Lys Lys
Lys 450 455 460 Glu Glu Ala Lys Lys Glu Glu Glu Lys Pro Gln Glu Pro
Ala Thr Ala 465 470 475 480 Glu Ile Lys Pro Asn Lys Lys Ile Leu Ile
Glu Leu Lys Val Glu Lys 485 490 495 Lys Pro Met Gly Cys His Arg Leu
Arg Arg Gln Lys Gln Pro Cys His 500 505 510 Asp Trp Leu Cys Asn His
Pro Arg Leu Ser Gly Gly Gln Val Ala Ala 515 520 525 Asp Lys Arg Leu
Lys Ile Phe Asp His Ile Cys Asp Ile Asn Gly Thr 530 535 540 Pro Ile
His Val Gly Ser Met Thr Thr Leu Lys Val His Gln Leu Phe 545 550 555
560 His Thr Thr Tyr Glu Lys Ala Val Thr Leu Thr Val Phe Arg Ala Asp
565 570 575 Pro Pro Glu Leu Glu Lys Phe Asn Val Asp Leu Met Lys Lys
Ala Gly 580 585 590 Lys Glu Leu Gly Leu Ser Leu Ser Pro Asn Glu Ile
Gly Cys Thr Ile 595 600 605 Ala Asp Leu Ile Gln Gly Gln Tyr Pro Glu
Ile Asp Ser Lys Leu Gln 610 615 620 Arg Gly Asp Ile Ile Thr Lys Phe
Asn Gly Asp Ala Leu Glu Gly Leu 625 630 635 640 Pro Phe Gln Val Cys
Tyr Ala Leu Phe Lys Gly Ala Asn Gly Lys Val 645 650 655 Ser Met Glu
Val Thr Arg Pro Lys Pro Thr Leu Arg Thr Glu Ala Pro 660 665 670 Lys
Ala 2 2059 DNA Drosophila melanogaster 2 atggttcagt tcctgggcaa
acagggcacc gcgggtgagc tcattcacat ggtgaccctg 60 gacaagacgg
gcaagaagtc cttcggcatc tgcatagtgc gcggcgaggt gaaggattcg 120
cccaacacca agacaaccgg catcttcatc aagggcattg tgcccgacag tcccgcgcat
180 ctgtgtggtc gcctaaaggt tggcgatcgg atcctctcgc tcaacggaaa
ggatgtgcgc 240 aactccaccg aacaggcggt catcgatctc atcaaggagg
cggacttcaa gatcgagctg 300 gagattcaga ccttcgacaa gagcgatgag
cagcaggcca agtcagatcc gcggagcaat 360 ggctacatgc aggccaagaa
caagttcaat caggagcaga ccaccaacaa caatgcgtcc 420 ggaggtcagg
gaatggggca aggtcagggt cagggtcagg gaatggctgg catgaaccgg 480
cagcaatcga tgcagaagcg gaataccaca ttcacggcct cgatgcgtca gaagcatagt
540 aactacgccg acgaggatga cgaggacacc cgggacatga ccggtcgcat
tcgcacggag 600 gcgggttatg agatcgatcg agcctccgcc ggtaattgca
aacttaataa gcaggaaaag 660 gatcgcgaca aggagcagga agatgaattt
ggctacacga tggctaagat caacaagcgg 720 tacaacatga tgaaggatct
gcgcaggatc gaggtccaga gggacgccag caagccactg 780 ggactcgcac
tcgctggcca caaggaccgc cagaagatgg cctgctttgt tgccggtgtg 840
gatcccaacg gagcattggg cagcgtggac attaagccgg gcgacgagat cgtcgaggtc
900 aacggcaatg tgcttaagaa tcgctgccac ttgaacgcct ccgccgtgtt
caagagcgtg 960 gatggggata agctcgtgat gatcacctcg cgacgcaagc
ccaacgatga gggcatgtgc 1020 gtcaagccca tcaaaaagtt ccccaccgcg
tctgatgaga ctaagtttat cttcgaccag 1080 tttcccaagg cgcgcacggt
gcaggtgcgc aaggagggtt cctgggcatc atggtcatct 1140 atggcaagca
cgctgaggtg ggcagtggca ttttcatctc ggatctgaga gagggatcga 1200
atgccgagtt ggcgggcgtg aaagtgggcg acatgctgct ggccgttaat caggatgtaa
1260 cactggaatc caactacgat gatgctactg gactgcttaa acgtgccgag
ggcgtagtga 1320 ccatgattct attgactctc aagagcgagg aggcgataaa
ggctgagaag gcagcggaag 1380 agaaaaagaa ggaggaggcc aagaaagagg
aggaaaagcc acaggaaccc gccacagccg 1440 agatcaagcc gaacaaaaag
atactcattg agttgaaggt ggaaaagaag ccaatgggcg 1500 tcatcgtctg
cggcggcaag aacaaccatg tcacgactgg ctgtgtaatc acccacgttt 1560
atccggaggg acaagtggca gccgacaagc gcctcaagat ctttgaccac atttgtgata
1620 taaatggtac gccaatccac gtgggatcca tgacgacact gaaggtccat
cagttattcc 1680 acaccacata cgagaaggcg gtcaccctaa cggtcttccg
cgctgatcct ccggaactgg 1740 aaaagtttaa cgttgacctt atgaaaaaag
caggcaagga gctgggcctg tcgctgtctc 1800 ccaacgaaat tggatgcacc
atcgcggact tgattcaagg acaatacccg gagattgaca 1860 gcaaactgca
gcgcggcgat attatcacca attcaatggc gatgccttgg agggtcttcc 1920
gttccaggtg tgctacgcct tgttcaaggg agccaacggc aaggtatcga tggaagtgac
1980 acgacccaag cccactctac gtacggaggc acccaaggcc tagagacgat
cctcattctc 2040 ctctccgtag cgaagcagt 2059 3 93 PRT artificial PSD-1
3 Met Glu Tyr Glu Glu Ile Thr Leu Glu Arg Gly Asn Ser Gly Leu Gly 1
5 10 15 Phe Ser Ile Ala Gly Gly Thr Asp Asn Pro His Ile Gly Asp Asp
Pro 20 25 30 Ser Ile Phe Ile Thr Lys Ile Ile Pro Gly Gly Ala Ala
Ala Gln Asp 35 40 45 Gly Arg Leu Arg Val Asn Asp Ser Ile Leu Phe
Val Asn Glu Val Asp 50 55 60 Val Arg Glu Val Thr His Ser Ala Ala
Val Glu Ala Leu Lys Glu Ala 65 70 75 80 Gly Ser Ile Val Arg Leu Tyr
Val Met Arg Arg Lys Pro 85 90 4 93 PRT Artificial PSD95-2 4 Glu Lys
Val Met Glu Ile Lys Leu Ile Lys Gly Pro Lys Gly Leu Gly 1 5 10 15
Phe Ser Ile Ala Gly Gly Val Gly Asn Gln His Ile Pro Gly Asp Asn 20
25 30 Ser Ile Tyr Val Thr Lys Ile Ile Glu Gly Gly Ala Ala His Lys
Asp 35 40 45 Gly Arg Leu Gln Ile Gly Asp Lys Ile Leu Ala Val Asn
Ser Val Gly 50 55 60 Leu Glu Asp Val Met His Glu Asp Ala Val Ala
Ala Leu Lys Asn Thr 65 70 75 80 Tyr Asp Val Val Tyr Leu Lys Val Ala
Lys Pro Ser Asn 85 90 5 87 PRT artificial PSD95-3 5 Arg Glu Pro Arg
Arg Ile Val Ile His Arg Gly Ser Thr Gly Leu Gly 1 5 10 15 Phe Asn
Ile Val Gly Gly Glu Asp Gly Glu Gly Ile Phe Ile Ser Phe 20 25 30
Ile Leu Ala Gly Gly Pro Ala Asp Leu Ser Gly Glu Leu Arg Lys Gly 35
40 45 Asp Gln Ile Leu Ser Val Asn Gly Val Asp Leu Arg Asn Ala Ser
His 50 55 60 Glu Gln Ala Ala Ile Ala Leu Lys Asn Ala Gly Gln Thr
Val Thr Ile 65 70 75 80 Ile Ala Gln Tyr Lys Pro Glu 85 6 87 PRT
artificial dlg-3 6 Arg Glu Pro Arg Thr Ile Thr Ile Gln Lys Gly Pro
Gln Gly Leu Gly 1 5 10 15 Phe Asn Ile Val Gly Gly Glu Asp Gly Gln
Gly Ile Tyr Val Ser Phe 20 25 30 Ile Leu Ala Gly Gly Pro Ala Asp
Leu Gly Ser Glu Leu Lys Arg Gly 35 40 45 Asp Gln Leu Leu Ser Val
Asn Asn Val Asn Leu Thr His Ala Thr His 50 55 60 Glu Glu Ala Ala
Gln Ala Leu Lys Thr Ser Gly Gly Val Val Thr Leu 65 70 75 80 Leu Ala
Gln Tyr Arg Pro Glu 85 7 88 PRT artificial nNOS 7 Pro Asn Val Ile
Ser Val Arg Leu Phe Lys Arg Lys Val Gly Gly Leu 1 5 10 15 Gly Phe
Leu Val Lys Glu Arg Val Ser Lys Pro Pro Val Ile Ile Ser 20 25 30
Asp Leu Ile Arg Gly Gly Ala Ala Glu Gln Ser Gly Leu Ile Gln Ala 35
40 45 Gly Asp Ile Ile Leu Ala Val Asn Asp Arg Pro Leu Val Asp Leu
Ser 50 55 60 Tyr Asp Ser Ala Leu Glu Val Leu Arg Gly Ile Ala Ser
Glu Thr His 65 70 75 80 Val Val Leu Ile Leu Arg Gly Pro 85 8 88 PRT
artificial inaD-3 8 Pro Lys Ala Arg Thr Val Gln Val Arg Lys Glu Gly
Phe Leu Gly Ile 1 5 10 15 Met Val Ile Tyr Gly Lys His Ala Glu Val
Gly Ser Gly Ile Phe Ile 20 25 30 Ser Asp Leu Arg Glu Gly Ser Asn
Ala Glu Leu Ala Gly Val Lys Val 35 40 45 Gly Asp Met Leu Leu Ala
Val Asn Gln Asp Val Thr Leu Glu Ser Asn 50 55 60 Tyr Asp Asp Ala
Thr Gly Leu Leu Lys Arg Ala Glu Gly Val Val Thr 65 70 75 80 Met Ile
Leu Leu Thr Leu Lys Ser 85 9 95 PRT artificial inaD-1 9 Glu Leu Ile
His Met Val Thr Leu Asp Lys Thr Gly Lys Lys Ser Phe 1 5 10 15 Gly
Ile Cys Ile Val Arg Gly Glu Val Lys Asp Ser Pro Asn Thr Lys 20 25
30 Thr Thr Gly Ile Phe Ile Lys Gly Ile Val Pro Asp Ser Pro Ala His
35 40 45 Leu Cys Gly Arg Leu Lys Val Gly Asp Arg Ile Leu Ser Leu
Asn Gly 50 55 60 Lys Asp Val Arg Asn Ser Thr Glu Gln Ala Val Ile
Asp Leu Ile Lys 65 70 75 80 Glu Ala Asp Phe Lys Ile Glu Leu Glu Ile
Gln Thr Phe Asp Lys 85 90 95 10 86 PRT artificial inaD-5 10 Leu Glu
Lys Phe Asn Val Asp Leu Met Lys Lys Ala Gly Lys Glu Leu 1 5 10 15
Gly Leu Ser Leu Ser Pro Asn Glu Ile Gly Cys Thr Ile Ala Asp Leu 20
25 30 Ile Gln Gly Gln Tyr Pro Glu Ile Asp Ser Lys Leu Gln Arg Gly
Asp 35 40 45 Ile Ile Thr Lys Phe Asn Gly Asp Ala Leu Glu Gly Leu
Pro Phe Gln 50 55 60 Val Cys Tyr Ala Leu Phe Lys Gly Ala Asn Gly
Lys Val Ser Met Glu 65 70 75 80 Val Thr Arg Pro Lys Pro 85 11 89
PRT artificial inaD-2 11 Lys Asp Leu Arg Arg Ile Glu Val Gln Arg
Asp Ala Ser Lys Pro Leu 1 5 10 15 Gly Leu Ala Leu Ala Gly His Lys
Asp Arg Gln Lys Met Ala Cys Phe 20 25 30 Val Ala Gly Val Asp Pro
Asn Gly Ala Leu Gly Ser Val Asp Ile Lys 35 40 45 Pro Gly Asp Glu
Ile Val Glu Val Asn Gly Asn Val Leu Lys Asn Arg 50 55 60 Cys His
Leu Asn Ala Ser Ala Val Phe Lys Ser Val Asp Gly Asp Lys 65 70 75 80
Leu Val Met Ile Thr Ser Arg Arg Lys 85 12 80 PRT artificial inaD-4
12 Pro Met Gly Val Ile Val Cys Gly Gly Lys Asn Asn His Val Thr Thr
1 5 10 15 Gly Cys Val Ile Thr His Val Tyr Pro Glu Gly Gln Val Ala
Ala Asp 20 25 30 Lys Arg Leu Lys Ile Phe Asp His Ile Cys Asp Ile
Asn Gly Thr Pro 35 40 45 Ile His Val Gly Ser Met Thr Thr Leu Lys
Val His Gln Leu Phe His 50 55 60 Thr Thr Tyr Glu Lys Ala Val Thr
Leu Thr Val Phe Arg Ala Asp Pro 65 70 75 80 13 5 PRT artificial
import locolization sequence targeting nucleus 13 Lys Lys Lys Arg
Lys 1 5 14 26 PRT artificial import locolization sequence targeting
mitochondrion 14 Met Leu Arg Thr Ser Ser Leu Phe Thr Arg Arg Val
Gln Pro Ser Leu 1 5 10 15 Phe Arg Asn Ile Leu Arg Leu Gln Ser Thr
20 25 15 4 PRT artificial import locolization sequence targeting
endoplasmic reticulum 15 Lys Asp Glu Leu 1 16 4 PRT artificial
insertion into plasma membrane VARIANT (1)..(4) x = any amino acid
16 Cys Cys Xaa Xaa 1
* * * * *