Patent Application
20070022484
The present invention relates to a pair of probes for analyzing protein-protein interactions that detects where and when a protein-protein interaction occurs, and quantifies the amount of protein-protein interactions in living cells, and relates to a method for analyzing protein-protein interaction using the pair of probes. More specifically, the present invention relates to a pair of probes for analyzing protein-protein interactions that enables high-precision and high-speed screening of an intracellular signal reinforcing/suppressing substance participating in various protein-protein interactions in living cells or individuals, and a method for analyzing protein-protein interactions using the probe.
Protein-protein interactions are known to play key roles in structural and functional organization of living cells. It is also known that protein-protein interactions are engaged in a genetic transcription mechanism or intracellular signal transduction.
As a method for analyzing protein-protein interactions, split enzyme methods have been reported (Rossi, F., Charlton, C. A. and Blau, H. M., Proc. Natl. Acad. Sci. USA, 94, 8405-8410(1997); Remy, I., and Michnick, S. W., Proc. Natl. Acad. Sci. USA, 96, 5394-5399(1999); Pelletier, J. N., Arndt, K. M., Pluckthun, A., and Michnick, S. W., Nature Biotech. 17, 683-690(1999); Karimova, G., Pidoux, J., Ullmann, A., and Ladant, D., Proc. Natl. Acad. Sci. USA, 95, 5752-5756). According to these methods, a cleaved protein is complemented by a protein-protein interaction to restore its enzymatic activity, which is measured by expression type of bacteria or cells, or a fluorescent enzyme substrate; thereby the protein-protein interaction can be detected.
Such split enzyme methods are however accompanied by the drawbacks that not only the enzymatic reaction needs much time but also where or when the protein-protein interaction occurs in a cell cannot be identified because the stable-fluorescent product of the enzymatic reaction freely diffuses in the cell due to the stability thereof.
The present inventors have therefore made, as a probe capable of analyzing protein-protein interactions of any proteins with high precision in a convenient manner, a pair of probes for analyzing protein-protein interactions, in which firefly luciferase or GFP is produced and the enzymatic activity of firefly luciferase or fluorophore of GFP is regenerated by a protein splicing due to the protein-protein interaction (Japanese Patent Application No. 2000-224939; PCT/JP00/09348).
However, it is difficult to identify when and where the protein-protein interaction occurs even by using a pair of probes for analyzing protein-protein interactions, since the fluorescent product of enzymatic reaction or fluorophore of GFP is diffusive.
The purpose of the present invention is to overcome the problems of the conventional technologies and provide a probe for detecting where and when a protein-protein interaction occurs, and quantifying the amount of a protein-protein interaction in living cells; and a method for analyzing protein-protein interactions using the probe.
The first aspect of the present invention is a pair of probes for analyzing protein-protein interactions, which comprises:
a probe A containing at least an N-terminal half polypeptide of split Renilla luciferase; and
a probe B containing at least the remaining C-terminal half polypeptide of split Renilla luciferase.
The second aspect of the present invention is the one related to the above-described pair of probes, wherein the probe A contains an N-terminal half polypeptide of an intein and N-split Renilla luciferase, and the probe B contains a C-terminal half polypeptide of the intein and C-split Renilla luciferase.
The third aspect of the present invention is the one related to the above-described pair of probes, wherein a linker sequence is linked to each of the N-terminal half polypeptide of split Renilla luciferase and the remaining C-terminal half polypeptide of split Renilla luciferase. The fourth aspect of the present invention is the one related to the said pair of probes, wherein the linker sequence consists of 3 to 20 amino acid residues.
The fifth aspect of the present invention is the one related to any one of the above-described pair of probes, wherein the N-terminal half polypeptide of split Renilla luciferase and the remaining C-terminal half polypeptide of split Renilla luciferase are obtained by splitting Renilla luciferase between Ser91 and Tyr92.
The sixth aspect of the present invention is a method for analyzing protein-protein interactions, which comprises
fusing a protein “a” to any one of the above-described probe A, and fusing a protein “b” to any one of the above-described probe B;
making the protein “a” fused to the probe A and the protein “b” fused to the probe B coexist in the presence of coelenterazine and oxygen; and
measuring luminescence thus emitted.
The seventh aspect of the present invention is the one related to the above-described method, which comprises introducing a polynucleotide expressing the protein “a” fused to the probe A and a polynucleotide expressing the protein “b” fused to the probe B into cells, thereby making the protein “a” fused to the probe A and the protein “b” fused to the probe B coexist in the presence of coelenterazine and oxygen.
The eighth aspect of the present invention is the one related to the above-described method, which comprises introducing a polynucleotide expressing the protein “a” fused to the probe A and a polynucleotide expressing the protein “b” fused to the probe B into a non-human totipotent cell, causing ontogenesis of the cell to non-human animal, thereby making the protein “a” fused to the probe A and the protein “b” fused to the probe B coexist in the presence of coelenterazine and oxygen in any one of the cells of the animal or offspring animal thereof.
The ninth aspect of the present invention is a non-human animal or offspring animal thereof, which is obtained by
introducing a polynucleotide expressing the protein “a” fused to the probe A and a polynucleotide expressing the protein “b” fused to the probe B into a non-human totipotent cell; and
causing ontogenesis of the cell to non-human animal.
The tenth aspect of the present invention is a method for screening a substance, which comprises:
introducing a test sample into the above-described non-human animal or offspring animal thereof; and
analyzing a protein-protein interaction in the cell of the non-human animal or offspring animal thereof.
The pair of probes for analyzing protein-protein interactions of the present invention is characterized by the use of Renilla luciferase as an indicator substance.
Renilla luciferase (Lorenz, W. W., et al., Proc. Natl. Acad. Sci. USA, 88, 4438-4442(1991)) (SEQ ID NO: 1) is a monomeric protein having a molecular weight of 36 kDa, one half the molecular weight of that of firefly luciferase, and is known to easily express within mammalian cells. Although the crystal structure of Renilla luciferase is unknown, it is known that N-terminal and several cysteine residues are important for its luminescence activity (Paulmurugan, R., et al., Proc. Natl. Acad. Sci. USA, 99, 15608-13(2002), Liu, J., et. al., Gene 203, 141-148(1997)). Different from firefly luciferase, the enzymatic reaction of Renilla luciferase does not require ATP (Liu, J. & Escher, A., Gene 237, 153-159(1999)). Its substrate coelenterazine is known to penetrate through the mammalian cell membrane and rapidly diffuse throughout the cytozol (Contag, C. H. & Bachmann, M. H., Annu. Rev. Biomed Eng., 4, 235-260(2002); Greer, L. F. & Szalay, A. A., Luminescence, 17, 43-74(2002)).
Renilla luciferase catalyzes the oxidation of coelenterazine in the presence of dissolved oxygen to its excited-state product (oxycolenterazine monoanion) to emit luminescence with a broad band (400 nm-630 nm) covering a tissue-transparent near infrared region, thereby yielding coelenteramide and carbon dioxide.
Taking advantage of the above characteristics of the Renilla luciferase, the present inventors have come up with the idea of realizing a probe that is capable of detecting when and where a protein-protein interaction occurs in living cells.
Constitution and working principle of the pair of probes for analyzing protein-protein interactions of the present invention are shown in FIGS. 1 and 2.
As illustrated in
To analyze the protein-protein interactions using the pair of probes of the present invention, an analysis-target protein “a” 3a is fused to probe A 1a and another analysis-target protein “b” 3b is fused to probe B 1b, and then they are co-existed in the presence of coelenterazine and dissolved oxygen (I). Interaction between protein “a” 3a and protein “b” 3b (II), and the consequent jaxtaposioning of N-split Renilla luciferase 2a and C-split Renilla luciferase 2b simultaneously lead to formation of the complemented Renilla luciferase 2 (III). In the presence of coelenterazine and oxygen in the system, the complement Renilla luciferase 2 catalyzed the oxidation of coelenterazine, which is a membrane permeable substrate, occurs (IV) and the energy of the carbonyl group of coelenterazine to the ground state is emitted as bioluminescence 4 spontaneously (V).
As described above, in the pair of probes 1 of the present invention, probe A 1a and probe B 1b may consist only of N— and C-split Renilla luciferases, respectively. And in addition, probe A 1a and probe B 1b may contain an N-terminal polypeptide of an intein 6a and a C-terminal polypeptide of the intein 6b, respectively.
Described specifically, as illustrated in
To analyze the protein-protein interactions using such a pair of probes, probe A′ 1c and probe B′ 1d are fused to the target proteins of analysis “a” 3a and “b” 3b, respectively (I). Interaction between the proteins 3a and 3b (II) induces the folding of N— and C-terminal halves of the intein and protein splicing reaction by the intein occurs. The split Renilla luciferases 2a and 2b are linked together by a peptide bond to reconstitute (splice) Renilla luciferase 2 (III′).
In the presence of coelenterazine and oxygen in the system, the Renilla luciferase 2 catalyzed oxidation of coelenterazine, which is a membrane permeable substrate, occurs (IV) and the energy of the carbonyl group of coelenterazine to the ground state is emitted as bioluminescence 4 spontaneously (V). By detecting this bioluminescence 4, the protein-protein interaction can be analyzed.
When the interaction (II) does not occur between the protein “a” 3a and protein “b” 3b, the splicing reaction by the intein does not occur, Renilla luciferase 2 is not reconstituted (spliced) (III′) and no marked change in bioluminescence 4 appears.
As the intein to be used in the pair of probes in the present invention, various known substances of biological origin can be used. Examples include substances derived from eukaryotic organisms typified by Saccharomyces cerevisiae (yeast) Sce VMA and Candida tropiallis (candida) Ctr VMA, those derived from eubacteria such as Mycobacterium tuberculosis (tubercle bacilli) Mtu recA, and those derived from archaebacteria such as Thermoplasma asidophilum Tac VMA.
In the pair of probes 1c and 1d of the present invention, the intein is preferably a site-specific endonuclease in order that autocatalytic excision of the intein occurs by the interaction between protein “a” 3a and protein “b” 3b. Moreover, in the pair of probes 1c and 1d in the present invention, two sites participating in the splicing in a protein precursor must be folded properly and juxtaposed precisely to each other in order that the splicing reaction by the intein occurs effectively (Duan. X., Gimble, F. S and Quiocho, F. A., Cell, 89, 555-564(1997)). An intein of biological origin may be used as it is, but it may be designed to facilitate splicing by substituting or deleting some amino acid residues or introducing a proper linker sequence.
The pair of probes in the present invention, probe A 1a and probe B 1b may consist only of N— and C-split Renilla luciferases, respectively, and in addition, probe A 1a and probe B 1b may have linker sequences 5a and 5b at their terminals, respectively. In the case of having linker sequences 5a and 5b, probe A 1a and probe B 1b are fused to proteins 3a and 3b via these linker sequences 5a and 5b, respectively. Although various sequences are given as examples of the linker sequences 5a and 5b and no particular limitation is imposed on them. Examples include those consisted of 3 to 20 amino acids sequences, more specifically, those having a glycine-alanine repeated sequence (
Similarly, probe A′ 1c may consist only of the N-terminal half polypeptides of an intein 6a and N-split Renilla luciferases 2a, and it may further contain a linker sequence 5a similar to those describer above. Probe B′ 1d may also consist only of the C-terminal half polypeptides of an intein 6b and C-split Renilla luciferases 2b, and it may further contain a linker sequence 5a. In other words, the N-terminal half polypeptides of intein 6a and N-split Renilla luciferases 2a may be linked directly or may be linked via linker sequences 5a in probe A′ 1c, and the C-terminal half polypeptides of intein 6b and C-split Renilla luciferases 2b may be linked directly or may be linked via linker sequences 5b in probe B′ 1d (
The N— and C-split Renilla luciferases 2a and 2b to be used in the pair of probes 1 of the present invention are the N-terminal half polypeptide and the remaining C-terminal half polypeptide obtained by splitting Renilla luciferase at a proper site. These N— and C-split Renilla luciferases 2a and 2b are complemented (III) by the interaction between protein “a” 3a and protein “b” 3b (II) (
In case of protein complementation, it is required that N— and C-split Renilla luciferases 2a and 2b do not exhibit luminescence activity before the complementation (III) (they exist individually) and restore enzymatic activity by the complementation. In the case of protein splicing, it is required that N— and C-split Renilla luciferases 2a and 2b do not exhibit luminescence activity before the reconstitution (splicing) (III′) (they exist individually) and restore enzymatic activity by the re-constitution. For this purpose, the Renilla luciferase must be split at the position of cysteine (Cys), serine (Ser), or tyrosine (Tyr) so that the active centers are divided into two.
As described later in examples, according to the studies of the inventors, high-accuracy detection was possible by using N-split Renilla luciferase of probe A (or A′) and C-split Renilla luciferase of probe B (or B′) available by splitting the luciferase between Ser91 and Tyr92.
In the present invention, a protein-protein interaction is analyzed by using the pair of probes in the following manner: by fusing one probe (for example, probe A) to protein “a” which is one of the protein in the pair for analysis and fusing the other probe (probe B) to the other protein “b” which is the other protein in the pair, causing them to co-exist and detecting and measuring bioluminescence 4. Any method can be adopted in order to fuse each protein 3a and 3b to probe 1a, 1b, 1c and 1d insofar as it does not have any influence on the pair of target proteins or the pair of probes. For example, ordinarily employed chemical, biochemical or genetic engineering method can be employed.
In the present invention, an example of methods for making the pair of probes exist in the presence of coelenterazine and oxygen is that the pair of probes (1a and 1b, or 1c and 1d) fused to proteins “a” 3a and “b” 3b is added to a solution containing coelenterazine. Using such a method, the protein-protein interaction can be detected and determined in vitro.
In the present invention, it is also possible to make the pair of probes exist in the presence of coelenterazine and oxygen by transfecting the expression vectors in cells. The expression vectors encode protein “a” which is fused to probe A 1a and protein “b” which is fused to probe B 1b, or encode protein “a” 3a which is fused to probe A′ 1c and protein “b” 3b which is fused to probe B′ 1d. In this case, addition of oxygen is not necessary, because oxygen is there enough in the cells.
As the expression vector, a plasmid vector for the expression of animal cells is preferably employed. Such a plasmid vector can be introduced into a cell with a known methods, for example, electroporation, phosphorylated calcium method, liposome method or DEAE dextran method. By introducing the expression vectors for expressing proteins of analysis (3a and 3b) which is fused to probes (1a and 1b, or 1c and 1d) into the cells, it is possible to make the pair of probes (1a and 1b, or 1c and 1d), proteins (3a and 3b), coelenterazine and oxygen exist together in the cell. Using such a method, the protein-protein interaction can be detected and determined in vivo without damaging the cell.
Moreover, according to the method for analyzing protein-protein interaction of the present invention, a polynucleotide expressing protein “a” 3a fused to probe A 1a and protein “b” 3b fused to probe B 1b, or a polynucleotide expressing protein “a” 3a fused to probe A′ 1c and protein “b” 3b fused to probe B′ 1d may be introduced into a non-human totipotent cell, and be caused ontogenesis of the cell to a non-human animal. By this method, the pair of probes 1, proteins 3, coelenterazine and oxygen are made coexist in all cells of the animal or offspring animal thereof. In this case, addition of oxygen is not necessary, because oxygen exists in the living body at a sufficient concentration.
The present invention also provides the thus available transgenic non-human animal. The transgenic non-human animal can be prepared in accordance with a known preparation method (for example, Proc. Natl. Acad. Sci. USA, 77, 7380-7384(1980)). Such a transgenic non-human animal has, in all the somatic cells thereof, the pair of probes 1 and proteins 3 so that an intracellular signal reinforcing/suppressive substance relating to the protein-protein interaction can be screened with high accuracy by introducing a sample substance such as pharmaceutical or toxic substance in the body of the animal and analyzing the protein-protein interaction in its cells and tissues.
In the method for analyzing protein-protein interaction according to the present invention, the protein-protein interaction can be confirmed by detecting luminescence (bioluminescence) 4 emitted by the above-described operation based on its working principle. The bioluminescence from Renilla luciferase has tissue permeability, so not only the timing but also the site of the protein-protein interaction can be identified by the observation and imaging of the luminescence in the cell. The protein-protein interaction can also be quantitatively determined by measuring a change of the luminescence intensity while varying conditions such as concentration or adding a specific sample. Moreover, it is possible to trace the protein-protein interaction from start to finish by measuring a time course of the luminescence intensity.
Embodiments of the invention will next be described in further detail in the following examples with reference to the accompanying drawings. It is needless to say that the invention is not limited to or by these examples, and details can be changed or modified.
A known protein-protein interaction (Ozawa, T. et al., Anal. Chem. 73, 2516-2521(2001); Sato, M., et al., Nat. Biotechnol., 20, 287-294(2002); Sato, M., et al., Anal. Chem., 71, 3948-3954(1999)) between Y941 peptide (SEQ ID NO: 2) and N-terminal SH2 domain (SH2n) upon protein phosphorylation was confirmed using the pair of probes of the present invention.
In the following examples, reagents employed were as follows:
Restriction enzymes, modification enzymes and ligases were purchased from Takara Biomedicals (Tokyo, Japan). A synthetic renilla luciferase gene vector (hRL-CMV) coding renilla luciferase with most frequently used codons in mammals and renilla luciferase assay kits were purchased from Promega Co (Madison, Wis.). Mammalian expression vectors, pcDNA3.1(+) and pIRES, were obtained from Invitrogen (Groningen, Netherlands) and Clonetech (Palo Alto, Calif.), respectively. Ham's F-12 media, fetal bovine serum (FBS) and LipofectAMINE were obtained from Gibco BRL (Rockville, Md.). Anti-myc antibody, anti-flag antibody and anti-phosphotyrosine antibody (PY20) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Alkaline-phosphatase labeled anti-rabbit and anti-mouse antibodies were purchased from Jacson ImmunoResearch Lab., Inc. (Pennsylvania, Pa.). A nitrocellulose membrane was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Chemiluminescent substrates of CDP-STAR were purchased from New England Biolabs, Inc. (Beverly, Mass.).
The 941 tyrosine residue in Y941 peptide derived from insulin receptor substrate-1 (IRS1) is phosphorylated by the insulin receptor upon insulin stimulation and dephosphorylated by a protein tyrosine phosphatase (Sato, M., et al., Anal. Chem. 71, 3948-3954(1999); Pratipanawatr, W., et al., Diabetes, 50, 2572-2578(2001)). The SH2n from p85 subunit (p85 330-429) of phosphatidylinositol 3-kinase binds with the phosphorylated 941-tyrosine residue within the IRS1 (Yoshimura, R., et al., Diabetes, 46, 929-936(1997); Golstein, B. J., et al., J. Biol. Chem. 1275, 4283-4289(2000)).
(1) Plasmid Construction
The gene of Renilla luciferase was split at eight sites near cysteine, serine and tyrosine to obtain two inactive fragments (N-split Renilla luciferase and C-split Renilla luciferase).
SEQ ID NO: 1 and
The N-split Renilla luciferase (hRLn) and C-split Renilla luciferase (hRLc) were fused to the proteins (Y941 and SH2n), respectively. The plasmid construct of each split Renilla luciferase-fused protein (sRL) is shown in
The amino acid sequence of Y941 is TEEAYMKMDLGPG (SEQ ID NO: 2) that is a tyrosine phosphorylated domain in IRS1. The SH2n is an N-terminal SH2 domain from p85 subunit of bovine phosphatidylinositol. In
To ensure that N— and C-split Renilla luciferases are spatially proximal when a protein-protein interaction occurs, a linker sequence (SEQ ID NO: 4) made of flexible 18 amino acid residues containing G-A repeat was inserted between the N-split Renilla luciferase and Y941 and another linker sequence (SEQ ID NO: 5) was inserted between the SH2n and C-split Renilla luciferase. Moreover, Myc and FLAG epitopes were inserted into the downstream of the N— and C-split Renilla luciferases.
Escherichia coli strain DH5a was used as a bacterial host for all plasmid construction subcloning. All plasmids were verified by sequencing with a genetic analyzer AB1 prism310 (PE Biosystems, Tokyo, Japan).
(2) Cell Culture and Transfection
CHO-HIR cells were grown in Ham's F-12 supplemented with 10% heat-inactivated FBS (Filtron), 100 unitlml penicillin and 100 pg/mL streptomycin. Transfection was performed using LipofectAMlNE 2000. The cells seeded in 6-well culture plates were transfected with 2 ng of the reporter plasmid. 6 h after the transfection, the medium containing LipofectAMINE was replaced with the Ham's F-12 supplemented with FBS, penicillin and streptomycin and incubated for 40 h to express the proteins.
(3) Immunoprecipitation and Immunoblotting Analysis
CHO-HIR cells were stimulated with 100 nM insulin for 5 min at 37° C. and were lysed mechanically with an ice-cold 2× immunoprecipitation buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 0.2 mM sodium ortho-vanadate, 10 yg/mL leupeptin, 10 pg/ml pepstatin, 0.2 mlM phenylmethylsulfonyl fluoride (PMSF), 2% IGEPALCA630, I % Triton X-100). The n-terminal fusion protein was immunogrecipitated from the whole cell lysates of the CHO-HIR cells with anti-myc antibody for 1 h at 4° C. Protein G-Sepharose 4 FF beads were used to absorb the immunoprecipitates and then washed fives times with an ice-cold immunoprecipitation buffer. The samples were separated by SDS-polyacrylamide gel electrophoresis and analyzed with the following antibodies: for Y941 phosphorylation, anti-phosphotyrosine antibody (1:500); for n-terminal protein assessment, anti-myc antibody (1:500); for c-terminal protein assessment, anti-flag anti body (1:1000).
(4) Luminescence Assay
CHO-HIR cells were stimulated with insulin at 37° C. Luminescence intensity was measured with a Renilla luciferase assay kit. Then the cells were washed twice with a cold PBS and lysed. The lysate was subjected to centrifugation at 15,000 g for 30 s at 4° C. A portion of the supernatant was measured for luminescence intensity with a Minilumat LB9506 luminometer (Berthold GmbH & Co. KG, Wildbad, Germany) for 10 s. The protein concentration in the supernatant was assessed by the Bradford method.
(5) Results
The CHO-HIR cells expressing the probe (SRL91), in which Renilla luciferase was split between Ser91 and Tyr92, exhibited a luminescence activity of 25-fold higher with 10−7 M insulin than with its absence.
On the other hand, the probes having other split Renilla luciferase exhibited only a 2-4 fold increase with 10−7 M insulin.
To confirm that the complementation of Renilla luciferase in the CHO-HIR cells expressing sRL91, was triggered by the protein-protein interaction due to protein phosphorylation, a sRL91 mutant (sRL91F) was prepared by replacing the phosphorylated site of the 941-tyrosin residue in the Y941 peptide in sRL91 with a phenylalanine residue. The N-terminal and C-terminal probes were indicated by SEQ ID NO: 6 and NO: 7, respectively.
In a similar procedure as Example 1, the luminescence intensities of the resulting sRL91F were assessed with and without insulin stimulation. Similarly, N-terminal and C-terminal sRL91 were respectively expressed from CHO-HIR cells by using the plasmids in
The results are shown in
It has thus been confirmed that complementation of Renilla luciferase is induced from the protein-protein interaction between Y941 and SH2n. Accordingly, the sRL91 containing split Renilla luciferases obtained by splitting Renilla luciferase between Ser91 and Tr92 is effective as a probe for analyzing protein-protein interaction.
The cells expressed with sRL91 were incubated in medium supplemented with 100 nm or 10 pM insulin for 1 minute, 5 minutes, 10 minutes, 30 minutes and 60 minutes at 37° C. The luminescence of these cells was immediately assessed. The time course of interaction between Y941 and SH2n was shown in
Further, the cell expressed with sRL91 was incubated in medium supplemented with 10−7 M insulin for 1 minute, 5 minutes, 10 minutes, 30 minutes and 60 minutes at 37° C. The whole cell lysate were immunoprecipitated with anti-myc antibody. The immunoblot analysis was made using anti-phosphorylated tyrosine antibody and anti-myc antibody. Antibody staining was visualized with CDP-STAR and analyzed with “LAS-1000 Image Analyzer” (Fujifilm Co., Tokyo, Japan).
The results are shown in
It has been confirmed that the luminescence activity of sRL91 directly reflects the ongoing protein-protein interaction in living cells.
Spontaneous emission of luminescence by protein-protein interaction-induced complementation of the split Renilla luciferase together with its membrane permeable substrate, coelenterazine. Therefore, noninvasive imaging of the sites and time of its occurrence of a particular protein-protein interaction in living cells was investigated by using the pair of probes of the present invention.
The protein-protein interaction between Y941 and SH2n in sRL91 expressed CHO-HIR cells was imaged. The cells were imaged at room temperature on a Carl Zeiss Axioverts 100 microscope with a cooled charged camera Micromax (Roper Scientific Inc, Tucson, Ariz.) controlled by Till Vision V3.02 (PHOTONICS, Planegg, Germany) with a 40× oil immersion objective.
The CHO-HIR cells expressing sRL91 were thereby imaged with (b) and without (a) 10−7 M insulin stimulation as shown in
The luminescence microscopic images were taken with a CCD camera for 300 seconds (a, b) and 60 seconds (c) as exposure times, respectively in PBS supplemented with a 20% coelenterazine substrate buffer. The luminescence intensity is represented with a color scale.
Upon insulin stimulation, luminescence emitted by complemented Renilla luciferase increased only beneath the cell membrane, whereas such bright contrast was not observed in the absence of insulin (
This indicates that with insulin stimulation, the interaction between Y941 and SH2n occurred beneath the plasma membrane. CHO-HIR cells expressed with full-length Renilla luciferase (hRL124C/A) emitted luminescence uniformly throughout the cells (
The quantitative detection of the interaction between phosphorylated adaptor protein Shc and Grb2 (
As a result, there are much specificity at the interaction sites between the Shc phosphorylated sites and Grb2 when stimulated by epidermal growth factor (EGF), 17β-estradiol (E2), dihydrotestosterone (DHT) or diethylstilbestrol (DES) from the outside of the cells.
The results suggest that when EGF, stimulation to a receptor type kinase, is added, the 317-tyrosine residue is phosphorylated and interacted with Grb2. This interaction requires serine in the PTB domain, presumably because the Shc is interacted with the receptor type kinase in the presence of serine in the PTB domain.
On the other hand, it has been revealed that when E2 is added, existence of serine in the PTB domain is necessary for the phosphorylation of the 239- and 240-tyrosine residues and interaction between the thus phosphorylated tyrosine residues and Grb2. The interaction between the phosphorylated 317-tyrosine residue and Grb2 does not occur in the presence of serine.
In addition, similar results were obtained (
As described in full detail, the present invention provides a pair of probes for analyzing protein-protein interactions by which a protein-protein interaction in living cells can be visualized directly. The pair of probes relies on the complementation of split Renilla luciferase triggered by a protein-protein interaction. Unlike diffusive products involved in other complement enzyme systems (Blakely, B. T., et al., Nat. Biotechnol., 18, 218-22(2000). Rossi, F., et al., Proc. Natl. Acad. Sci. USA, 94, 8405-8410(1997); Remy, I. & Michnick, S. W., Proc. Natl. Acad. Sci. USA, 96, 5394-5399(1999); Galarneau, A., et al., Nat. Biotechnol. 20, 619-622(2002); Wehrman, T. et al., Proc. Natl. Acad. Sci. USA, 99, 3469-3474(2002)), the present bioluminescence readout by the protein-protein interaction-induced complement Renilla luciferase promises locating protein-protein interactions at the sites and time of its occurrence, thereby allowing analysis of protein-protein interactions in vivo in living cells and organisms.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-145466 | May 2003 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/07245 | 5/20/2004 | WO | 1/9/2006 |
