JP2002535654A - Method for identifying receptor ligands that can be taken up into cells by internalization - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for recognizing a target that can be taken up into a cell from a bank of peptides, pseudopeptides or non-peptide compounds, a biological extract or a purified tissue extract fraction by internalization induced by binding to a ligand. It relates to an effective method for detecting and / or identifying body ligands. The method of the present invention is characterized in that a labeled form of the receptor is expressed on the cell surface, and the cell is contacted with the bank under conditions that permit intracellular internalization of the receptor-ligand complex. And visualizing the internalization by detecting a label bound to the receptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to screening methods applicable to identify potential ligands for certain receptors that can be taken up into cells by internalization.

More specifically and without limitation, the present invention relates to receptors belonging to the family of receptors having seven transmembrane domains that bind to protein G, and tyrosine kinases having a single transmembrane domain. Receptors belonging to the family of receptors.

[0003] Receptors with seven transmembrane domains that bind to protein G (G
As for PCR), about 800 receptors have been cloned from various eukaryotes to date. As for human GPCR, 240 types of receptors have been isolated. Of these human receptors, only 140 had their corresponding endogenous ligands identified, and the remaining 100 formed pools of so-called orphan receptors whose ligands had not yet been identified. I have.

On the other hand, hundreds of new GPCR agonists have been registered in the last 20 years.

[0005] Thus, orphan receptors that have been cloned very recently are of great importance for pharmaceutical research because of their potential as potential therapeutic targets. However, in order for these receptors to gain therapeutic efficacy, it is necessary that the endogenous ligands of these receptors be isolated in advance and the function of these receptors elucidated. is there.

For all such orphan receptors that bind to protein G, only five endogenous ligands have been isolated so far. They all belong to a new family of peptides, namely orexin / hypocretin, nociceptin / orphanin, PrRP or prolactin releasing peptide, apelin and leukokinin-type peptides (leukokinin-like peptides).

[0007] In each case, methods for screening purified tissue fractions by various tests were used to identify these peptides.

[0008] The test to be used is that when an agonist binds to its receptor, a second messenger is formed, which produces various products according to the signaling pathways used by the receptor, The choice was made based on the fact that change always occurs.

In the case of nociceptin, cAMP accumulation due to stimulation of adenylate cyclase was measured.

In the case of orexin, cytosolic Ca 2+ due to stimulation of phospholipase C
Accumulation was measured.

In the case of PrRP, the formation of arachidonic acid due to the stimulation of phospholipase A2 was tested.

In the case of apelin, changes in the acidity of the extracellular matrix were measured by a “cell sensor”.

However, none of these identification methods can be considered to be perfect methods for the following reasons.

[0014] Identification methods for measuring the production of second messengers require that the signaling pathway of the receptor be known. In the presence of orphan receptors, for which no pathway involved can be inferred, it is necessary to test all known signaling pathways with the expectation that the receptor of interest will not bind to the unknown pathway. This requires obtaining a large number of samples. This method also may, but does not always occur, cause the receptor of interest to bind to the second messenger cascade in a heterologous transfection system expressing the second messenger.

Regarding the method for measuring the acidity of the extracellular matrix, the following problems occur because protons are generated by activated cells. That is, when a peptide bank or a purified tissue extract fraction is co-present with an orphan receptor expressed on the cell surface, a change in extracellular pH is measured. Due to the stimulation of all endogenous receptors present. Therefore, the ligand responsible for orphan receptor activation cannot be easily identified.

More specifically, an object of the present invention is a novel method for detecting and / or identifying a ligand for an orphan receptor, which has higher reliability than the conventional method as described above, It can be performed using small samples and can be performed in the presence of another endogenous receptor and in the presence of a large number of peptidic or non-peptidic ligands for such secondary endogenous receptors. It is to provide a method that can be used.

The method developed according to the present invention relates to the properties possessed by a receptor with seven transmembrane domains or a tyrosine kinase type receptor that binds to protein G, Take advantage of the property that it can be taken up by cells internalizing the receptor below. Internalization is a sufficiently universal phenomenon involving many receptors.
When the ligand is labeled with a fluorogenic molecule or an epitope marker, such internalization can be observed by microscopy with confocal, light or electron microscopy. The ligand-receptor complex is taken up into cells according to a well-developed and unique pathway. For example:-a labeled ligand capable of forming a complex with the respective receptor and inducing internalization by activation;-a protein involved in signal transduction, in this case a labeled protein kinase C; In order to track the intracellular transport of proteins involved in receptor desensitization after body activation, in this case, labeled β-arrestin 2, the method of fluorescent labeling or epitope labeling is preferred. It is believed that there is.

However, it involves a method of labeling a ligand, a method of labeling a protein involved in signal transduction, or a desensitization of a receptor such as a labeled β-arrestin 2 after activation of the receptor. No method can be used to label proteins that do not provide complete reliability. In fact, the receptors tracked based on internalization cannot be clearly identified. Furthermore, recent data suggests that various proteins are involved in the mechanism of internalization and desensitization of various receptors. In particular, β-arrestin 2 is not considered to perform an overall function.

For example, following the movement of β-arrestin bound to EGFP, the orphan receptor was stimulated by its ligand, phosphorylation of the receptor occurred, and β-arrestin 2-GFP migrated from the cytoplasm to the membrane. May bind to the receptor. Subsequently, receptor sequestration and internalization occur. Thus, when contacting an orphan receptor with a peptide bank or a bank of purified tissue fractions to search for endogenous ligands of the orphan receptor overexpressed on the surface of eukaryotic cells, Is activated, as well as all endogenous receptors bound to protein G. As a result, both orphan and endogenous receptors trigger the migration of β-arrestin 2-GFP. Therefore, it is not possible to identify the ligand that activated the orphan receptor.

In addition, when the endogenous ligand being searched for is contained in a peptide bank or a bank of a purified tissue fraction, if all the peptides are uniformly labeled, the ligand being searched for may be replaced with the ligand being searched for. Cannot be identified.

The advantage of the screening method of the present invention is exactly that it eliminates the above-mentioned disadvantage that ligands cannot be clearly identified.

More particularly, the invention relates to the intracellular formation of a peptide, pseudopeptide or non-peptide compound from a bank, biological extract or purified tissue extract fraction by internalization induced by binding to a ligand. An effective method for detecting and / or identifying a ligand of a receptor of interest that can be incorporated into a cell, comprising: at least expressing a labeled form of the receptor on a cell surface; A cell comprising at least one peptide capable of being a ligand for the receptor,
Contacting the bank, biological extract and / or purified tissue extract fraction containing the pseudopeptide or non-peptide compound under conditions sufficient to permit intracellular internalization of the receptor-ligand complex And visualizing said internalization by detecting a label bound to said receptor.

It is also an object of the present invention to label orphan receptors to find the corresponding possible agonist.

The method of labeling the orphan receptor itself has distinct advantages over the detection techniques mentioned above. In fact, this method allows direct observation of the receptor to be tested. When the receptor is contacted with an endogenous ligand, the ligand-receptor-label complex is taken up by the internalization into the cell. It is possible to clearly and unambiguously identify the receptor taken up into the cell.

[0025] In the method of the present invention, the cells further constitutively overexpress the labeled receptor. The advantage is that the cytoplasmic tail of the receptor is labeled intracellularly, so that labeling does not interfere with ligand binding.

A marker suitable for the present invention may be an autofluorescent protein or may be an epitope marker detectable by immunohistochemistry.

The fluorogenic protein that can be used in the method of the present invention is preferably a protein of the family of the wild-type fluorogenic protein GFP and mutants thereof (eg EG)
FP, EBFP and EYFP). Wang S.M. & Halzelri
gg T.G. (1944) Nature, 369: 400-403; Yang T
T et al. (1996) Nucleic Acid Res, 24: 4592-559.
3: Helm R & Tsien RY (1994) Curr. Biol. ,
6: 1, 178-182; Ormo M et al., (1996) Science, 27.
3: 1392-1395.

Non-fluorescent markers which can also be used in the present invention include, in particular, hemagglutinin, polyhistidine, myc protein, flag protein and viral epitopes. For all these strongly immunogenic compounds,
High affinity selection antibodies are already commercially available (eg, anti-myc monoclonal antibody, Clontech; anti-influenza hemagglutinin antibody, Boehrin)
ger; antiviral antibody of vesicular stomatitis virus, Clontech; anti-polyhistidine antibody, In Vitrogen). To detect these antibodies,
One of these antibodies (the so-called first antibody) recognizes the antigenic group, and then the first antibody is detected by a second antibody from another species. The second antibody is either labeled with a fluorophore or is labeled with horseradish peroxidase, which is subsequently reacted with a suitable substrate.

This type of labeling method has the advantage, inter alia, of a high measurement sensitivity which allows the detection of ligands at a concentration of 10 ⁻⁸ M, ie at a concentration of 100 femtomoles (fmole) in 10 μl. When the receptor is highly expressed and the ligand has a sufficient concentration (10 ⁻⁸ M or more), internalization of the complex can be observed by microscopy with a confocal or light microscope.

Preferably, this internalization is detected by microscopy with a light microscope and / or a confocal microscope.

A final advantage is that unlabeled other endogenous receptor present on the surface of the host cell is detected because it is not detected when the receptor is taken up into the cell by internalization. Do not interfere with the measurement of the receptor. Therefore, there is no background noise. This is particularly advantageous for the purpose of obtaining a sensitive measurement method.

In general, according to the method of the present invention, a test subject, despite the presence of multiple peptidic or non-peptidic ligands for other endogenous receptors and such secondary endogenous receptors, is present. It has been found that internalization of the receptor-ligand complex in the minute can be detected with sufficient sensitivity.

An advantage of the screening method of the present invention is that it can be suitably used for testing any receptor as long as the receptor has the ability to be taken up into cells by internalization.

According to a preferred embodiment of the present invention, the receptor of interest is a receptor that binds to protein G.

The method of the present invention comprises a family of GPCR receptors with seven transmembrane domains that bind to protein G, a family of tyrosine kinase receptors with a single transmembrane domain, or a family of receptors for cytokines. It is important to characterize the ligand of the receptor belonging to

In the case of GPCRs, the method of labeling the C-terminus of the receptor has the following advantages: no modification of the post-translational maturation of the receptor occurs, binding of agonists and antagonists and Intracellular signaling takes place in the same way as the native receptor,-no modification of the internalization of the ligand-receptor complex,-rapid detection of expression and unfixed survival The detection method may be performed on the cells.

The receptor to be tested is bound to a marker and then expressed on the surface of a host cell. Of course, these two operations, that is, the operation of labeling the receptor and the operation of expressing the receptor in the host cell, are both within the ordinary knowledge of those skilled in the art.

In general, these operations are carried out in the following procedure: The sequence of the coding part of the receptor of interest is replaced by the coding sequence of the fluorescent protein (GFP, EGFP, EBFP, EYFP) of the expression vector or the coding sequence of the epitope marker. Insert upstream and downstream in phase. These expression vectors (pGFP-N1 or pGFP-C1, where N1 or C1 indicates the location of the receptor for N-terminal or C-terminal proteins) already contain the sequences of these markers. Cells are transfected by conventional methods (eg, liposome method, calcium phosphate method), and then selected based on antibiotic resistance. In the case of the GFP family, cells expressing the receptor bound to the fluorogenic protein can be screened by flow cytometry.

As the transformed cells, for example, eukaryotic cells such as monkey renal epithelial cell COS-7, hamster ovary cell CHO, and human embryonic kidney cell HEK293 are used.

According to a variant embodiment of the method of the invention, it is possible to express two or more different receptors, each labeled with a different marker, on the surface of one cell. The two receptors can of course be taken up by internalization into the cell. Thus, if this embodiment is selected, it is possible to simultaneously screen potential ligands for multiple receptors in the same sample.

In the method of the present invention, a host cell is placed in the presence of a potential ligand. For this,
The host cells are contacted with a bank of peptides, pseudopeptides or non-peptide compounds, a biological extract or a purified tissue extract fraction.

The contacting is performed under conditions sufficient to permit intracellular internalization of one or more receptor-marker-ligand complexes. Sufficient conditions for temperature, time and concentration will of course be apparent to those skilled in the art. It may also be necessary to repeat the experiment.

In general, the optimal temperature for the internalization of a ligand-receptor complex into a mammalian cell is 37 ° C. The average half-life of the internalization process (the time required for 50% of surface receptors to be taken up into cells) is about 10 minutes. Therefore, usually, when the time of contact with the ligand is 20 to 40 minutes, the receptor incorporated into the cell can be optimally detected.
The optimal concentration of ligand is on the order of 10-20 times the affinity of the ligand for the receptor (Kd).

The internalization of the ligand-receptor complex can be followed by microscopy with confocal microscopy, or microscopy with light microscopy when the labeled receptor is highly expressed. Chase. Internalization is specifically
It means that the marker (fluorescent marker or immunohistochemical marker) on the cell membrane is transported inside the cell in vesicle form.

When the receptor is labeled with a fluorogenic label, the receptor is detected directly by observing the transfer of fluorescence.

If the receptor is labeled with a non-fluorescent antigen, internalization is performed by microscopy with a confocal microscope after fixing the cells and detecting the antigenic epitope with a second antibody conjugated to a fluorophore. Observe. Alternatively, the antigenic epitope may be detected by an enzymatic reaction or by using a radioactive antibody. This detection is specifically carried out by detecting the accumulation of an opaque precipitate that can be observed by either an optical microscope or an electron microscope.

As described above, internalization can be observed in about 10 cells, and can be observed in a very small amount, ie, an incubation volume on the order of μl. These two features are particularly important when very small samples are available.

Accordingly, the method of the present invention may be used to detect and / or identify one or more endogenous ligands of an orphan receptor, or alternatively, to a known receptor, ie, a new type of receptor whose endogenous ligand is known. Obviously, it would be very advantageous to identify agonists or antagonists.

The method of the present invention is based on the direct observation of biological phenomena involving many GPCR receptors or tyrosine kinase receptors, ie, internalization, and is therefore performed using biological preparations such as tissue extracts. Obviously, this is a particularly effective method for finding ligands for fan receptors, especially for peptide receptors.

Also, the use of the method of the present invention in conjunction with a labeled receptor identified and characterized as a “biosensor” capable of detecting toxic substances present in trace amounts in biological fluids or substances that cannot bind to the receptor. Is also possible.

The present invention also includes all ligands of the receptor identified by the method of the present invention.

Representative examples of the present invention will be described below without limitation based on the drawings and examples.

FIG. 1: FIG. 1a: Neurotensin bound to fluorogenic EGFP on the surface of CHO cells incubated with buffer alone or in the presence of low concentrations (1 nM) of murine or frog neurotensin that do not induce internalization Confocal micrograph of type 1 receptor (NTR1-EGFP).

FIG. 1 b: The formation of a large number of 0.6 μm-diameter fluorescent vesicles in the cytoplasm of CHO cells incubated in the presence of neurotensin at a concentration of 10 nM and 100 nM, and thus the receptor NTR1-EGFP A micrograph showing that internalization has occurred.

FIG. 2: Receptors when treated with or without acid washing in the presence of 10 nM neurotensin (FIG. 2a) or in the purified fraction of frog brain extract (FIG. 2b) Photomicrograph showing the internalization of NTR1-EGFP. Using the same experimental conditions as in FIG. 1 and, if an acid wash is used, the endogenous ligand still bound to the cell surface after the end of the addition period is removed by acid wash.

FIG. 3: Receptor NTR1-E in the presence of 10 prepurified fractions of frog brain extract
Internalization of GFP.

FIG. 4: Radioimmunoassay of prepurified fractions of frog brain extract performed using antibodies against conserved regions of neurotensin.

[0058]Materials and methods  (1) Construction of receptor NTR1-EGFP Complete coding sequence of receptor NT1 (K. Tanaka, M. Masua)
nd S.D. Nakanishi (1990) Structure and fu
nctional expression of the cloned ra
t neurotensin receptor. Neuron 4: 847-
54) was PCR amplified. For amplification, 5 'of the coding sequence of this cDNA was used.
Specific to the termini and the 3 'terminus and also restriction sites HindIII or BamH1
5'-CTT AAG CTT ATG CAC CT
CAAC AGC TCC GTG-3 '(SEQ ID NO: 1) and 5'-TTT
GGA TCC GCG TAC AGG GTC TCC CGG GT-3
'(SEQ ID NO: 2) was used. Purification by digestion with enzymes HindIII and BamH1
After that, the amplified sequence was converted into the expression vector pEGFP-N1 (Clontech).
It was inserted at the HindIII site and the BamH1 site. Fluorescent ddNTP
Used, fully automatic sequencer AbiPrism377 (Perkin-Elm)
er) to confirm the construct.

(2) Stable Transfection in CHO Cells CHO-K1 cells (American Type Culture Coll
action: ATCC) in a humidified atmosphere of 5% CO ₂ , 7.5% fetal calf serum, 1 mM glutamine, 100 units / ml penicillin and 100 units / m
The cells were cultured in F12 medium supplemented with 1 streptomycin (Boehringer Mannheim). In order to establish a stable system that expresses this receptor,
CHO-K1 cells (about 2.6 × 10 ⁶ cells) were converted into cationic liposomes (Dos
per, Boehringer). Transfected cells are transferred to Geneticin (geneti).
cine) Selected based on resistance. The resulting clones were screened using flow cytometry based on measuring the intensity of the fluorescence of each cell. The second selection was performed using the fluorogenic receptor NTR1-E localized on the membrane surface of the cells of each clone.
It was performed by observing the expression level of GFP with a fluorescence microscope.

(3) Preliminary purification of frog brain extract −Collecting tissue  Newly killed male European frog (Rana ridib) in the laboratory
2541 samples of brain corresponding to 215 g of tissue weight before drying
Was collected. The brain immediately after collection was frozen on dry ice and stored at -80 ° C. −Extract tissue  Brain is immersed in 0.5 M boiling acetic acid (2 liters) for 15 minutes and then mixed
And mixed homogeneously. The homogenate was centrifuged at 4000 xg for 30 minutes at 4 ° C. Next
Then, 10 Sep-Pak C18 column assemblies (Wa
ters Associates, Milford, Mass.) at 2 ml / min.
Pre-filtered at a flow rate of The substance retained in the Sep-Pak column was
Elution was performed with a 0% acetonitrile solution. This process was repeated twice. −Purification of frog brain extract by semi-preparative HPLC  The substance eluted from the Sep-Pak column was partially evaporated to remove acetonitrile.
And then centrifuged at 13000 xg for 5 minutes. Collect supernatant and split into two
. Each of the two pools was then treated with water / trifluoroacetic acid (99.9: 0.1; vo
l: vol) semi-preparative column Vydac C18 218TP1 equilibrated with solution
010 (1 × 25 cm) at a flow rate of 2 ml / min. Adsorbed on the matrix
Increased peptidic material to 14-42% in 40 minutes at a flow rate of 2 ml / min,
The acetonitrile gradient then rises to 42-56% over 60 minutes at a flow rate of 1 ml / min.
And eluted using a gradient. The eluate was collected per 1 min fraction and collected at 215 nm and 2
The absorbance at 80 nm was measured. The eluted fraction was stored at -20C. Internalize
Before performing the dilution experiments, dilute each fraction with water and partially evaporate to acetonitrile.
Was removed and the required volume of water was added to a final volume of 50 μl.

(4) Internalization Cells were distributed in a multiwell plate (LabTek, Nunc) pretreated with polyallylamine (0.1 mg / ml, Aldrich) (50% confluency, 20,000 cells / well) And then overnight. The incubation volume (excluding the addition period) and wash volume of these plates is 2 per well.
50 μl. Ninety minutes before the start of the experiment, the cell culture medium was changed to cycloheximide (70
Replace with a medium supplemented with μM, Sigma). Then, a low temperature buffer (
NaCl, pH 7.4, 140 mM, KCl of 5 mM, CaCl ₂ of 1.8 mM, MgCl ₂ of 3.6 mM, 0.1% bovine serum albumin, 0.01% glucose and 0.8mM 1,10 Cells on ice in phenanthroline)
Pre-incubate for 5 minutes. The cells are then incubated with the diluted ligand for 30 minutes at 4 ° C. in 50 μl R buffer (addition period). At this stage, the batch of cells is washed with a hypertonic acid (0.5 mL in Earl's buffer, pH 4).
2M acetic acid and 0.5 mM NaCl at 4 ° C. for 2 minutes) to dissociate the ligand from its surface receptor. The medium was changed to 37 ° C. R buffer, and the plate was
Induction is induced by incubation at 20 ° C. for 20-30 minutes (transfer period). At the end of the incubation, cells are washed with cold R buffer, fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer, pH 7.4, washed again with cold R buffer, and then washed with Ve.
Inflate using ctashield (Vector).

(5) Confocal microscope Consisting of an inverted filter (Leica DM IRBE) equipped with an excitation filter having an excitation wavelength of 488 nm and an emission wavelength of 530-600 nm, respectively, and an argon / krypton laser with an emission filter. The cells are observed with a confocal microscope Leica TCS NT. The use of an oil immersion objective with a 63 × magnification gives an image of 1024-1024 pixels for each cell.

(6) Radioimmunoassay for quantifying neurotensin in a prepurified fraction of frog brain extract A 5 μl sample of each fraction collected at the outlet of semi-preparative HPLC is treated by radioimmunoassay to quantify neurotensin I do. For the determination of neurotensin,
An antibody against the C-terminal fragment of porcine neurotensin was used (Ma
rcos et al., Peptides, 1996, 17: 139-146).

The antibodies were used at a final dilution of 1: 50,000 and the assay sensitivity was 10 pg. 4% bovine serum albumin and 7000 cpm of (3- [125I] iodotyrosyl) neurotensin (Amersham, Buckingamshire,
Radioimmunoassay at 4 ° C. in 0.02 M veronal buffer (pH 8.6) containing UK). Samples and antibodies were incubated at 4 ° C. for 48 hours. Each sample contains a γ-globulin solution (1 in 0.02 M veronal buffer).
%) And polyethylene glycol solution (0.1% bovine serum albumin and 0.1%
The tracer fraction bound to the antibody was separated by addition and precipitation of 20% in 0.02 M veronal buffer containing Triton X-100. After incubation for 20 minutes at room temperature, the samples are centrifuged (3000 × g, 30 minutes) and the pellet is counted in a gamma counter.

Example 1 Receptor NTR1 in the Presence of Frog or Murine Neurotensin
-Internalization of EGFP (1) Characterization of Receptor NTR1-EGFP The receptor NTR1-EGFP was stably expressed in a CHO cell line, and its binding properties and intracellular signaling properties were measured. Neurotensin is the receptor NTR
It was found that both 1-EGFP and wild-type NT1 receptor have the same affinity (0.3 nM). Similarly, the labeled receptor produced inositol phosphate with an EC50 (1 nM), which is also comparable to the value obtained with the wild-type NT1 receptor (results not shown).

(2) Receptor NTR1-E in the Presence of Frog or Murine Neurotensin
Internalization of GFP In cells incubated with buffer alone or low concentrations (less than 1 nM) of murine or frog neurotensin, labeled fluorescence of the receptor NTR1-EGFP is localized to the cell membrane (FIG. 1a). ).

Incubation of CHO-NTR1-EGFP cells with increasing concentrations of neurotensin (concentration range ≧ 10 nM) triggers internalization of the receptor NTR1-EGFP. This internalization is evidenced by a decrease in fluorescent labeling of the membrane and the formation of numerous fluorescent vesicles with a diameter of 0.6 μm in the cytoplasm (FIG. 1b). This type of label is not detected when the ligand has been predissociated from the surface receptor by treatment with an acidic wash after the end of the addition period (FIG. 2a). These results indicate that the internalization observed resulted from the binding of neurotensin to the membrane receptor during the addition period.

Example 2 Receptor NTR1-EGF in the Presence of a Prepurified Fraction of Frog Brain Extract
Internalization of P In the first series of experiments, 10 0.5 μl samples of each of the 120 eluted fractions of the frog brain extract (preliminary purification on the semi-preparative column) were pooled in 10 pools each consisting of 10 fractions Were prepared, and each fraction was supplemented with R buffer to make up to 50 μl. Only pool 2 containing fractions 11-20 elicits internalization of the receptor NTR1-EGFP.

In the second series of experiments, the fractions of pool 2, ie fractions 11-20 (initial volume 0
. (5 μl fractions diluted to 50 μl with R buffer) were tested individually. Fraction 1
Only 5, 16, 17 and 18 induce internalization of the receptor NTR1-EGFP (FIG. 3). If the cells were not acid washed after the end of the addition period, this internalization would not be detected (FIG. 2b). This indicates that the internalization observed resulted from the binding of the specific ligand to the receptor NTR1-EGFP during the addition period.

Example 3 Radioimmunoassay for Quantifying Neurotensin in a Prepurified Fraction of Frog Brain Extract Radioimmunoassay of a prepurified fraction of frog brain extract using an antibody against a conserved region of neurotensin It shows that the reactive substance is contained only in fractions 15, 16, 17 and 18 (FIG. 4). The apparent total amount of neurotensin measured in the eluted fraction of the semi-preparative HPLC is 894 ng in 16 ml, which corresponds to an amount of 352 pg peptide per frog brain. Therefore, fraction 15,
The substances that induce the internalization of the receptor NTR1-EGFP contained in 16, 17 and 18 correspond to endogenous neurotensin in the frog brain.

In conclusion, the above internalization method is a direct, simple, high specificity and high reliability means that can detect as little as 500 femtomoles of neurotensin in 50 μl by observing single cells. This method can be used for pure neurotensin solutions and for fractionation of tissue extracts containing not only neurotensin but also many other neuropeptides (the minimum number per fraction is estimated to be 50 peptides). And a similar detection sensitivity, ie, a detection sensitivity of about 250 femtomoles by the RIA assay.

[Sequence list]

[Brief description of the drawings]

FIG. 1a: Low concentrations of buffer alone (1
Confocal micrographs of neurotensin type 1 receptor (NTR1-EGFP) bound to fluorogenic EGFP on the surface of CHO cells incubated in the presence of nM) murine or frog neurotensin.

FIG. 1b. The formation of a large number of fluorescent vesicles 0.6 μm in diameter in the cytoplasm of CHO cells incubated in the presence of neurotensin at a concentration of 10 nM and 100 nM, and thus the internalization of the receptor NTR1-EGFP A micrograph showing that the zation has occurred.

FIG. 2a is a photomicrograph depicting the internalization of the receptor NTR1-EGFP when treated with or without an acid wash in the presence of 10 nM neurotensin.

FIG. 2b is a photomicrograph depicting the internalization of the receptor NTR1-EGFP when treated with or without acid washing in the presence of a purified fraction of a frog brain extract.

FIG. 3. Receptor NTR1-EGFP in the presence of 10 pre-purified fractions of frog brain extract
Internalization.

FIG. 4. Radioimmunoassay of pre-purified fractions of frog brain extract performed using antibodies against conserved regions of neurotensin.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Schiartourel, Nicolas France, F-76113 Saint-Pierre-de-Maneville, Ludou de Cubilon, 17 (72) Inventor Baudet, Allan Canada, Québec-Aciyu 3-Pa-1-B-2, Lieumont-Mont-Royal, Promenade-Soulay, 39 (72) Inventor Renkei, Zort France, F-75012 Paris, Liu-dou-Cialanton, 119- 121 (72) Inventor Loran-Corte, Catoulinne F-91440, Biy ス l-sur-Yurbet, Ab ニ nil-d ラ ou-la-Prommnat, 9F term (reference) 2G045 AA40 DA36 FA16 FB03 FB12 GC15 4H045 AA30 BA41 DA50 DA51

Claims (15)

[Claims] 1. Receptors that can be taken up into cells by internalization induced by binding to a ligand from a bank of peptides, pseudopeptides or non-peptide compounds, biological extracts or purified tissue extract fractions. An effective method for detecting and / or identifying a somatic ligand, comprising: at least expressing a labeled form of the receptor on the cell surface; and causing the cell to become a ligand for the receptor. At least one peptide obtained,
Contacting said bank, biological extract and / or purified tissue extract fraction containing a pseudopeptide or non-peptide compound under conditions permitting intracellular internalization of a receptor-ligand complex. And visualizing said internalization by detecting a label bound to said receptor. 2. The method according to claim 1, wherein a concentration of the ligand on the order of 10 ⁻⁸ M can be detected. 3. The method according to claim 1, wherein the expressed receptor is an orphan receptor. 4. The method according to claim 1, wherein the expressed receptor is a receptor whose endogenous ligand is known. 5. The method according to claim 1, wherein the receptor is labeled with an autofluorescent protein. 6. The method according to claim 1, wherein the fluorescent protein is a protein of the family of the wild-type fluorescent protein GFP or one of its mutants. 7. The fluorescent protein is selected from the group consisting of proteins EGFP, EBFP and EY.
The method according to claim 1, wherein the method is selected from FP. 8. The method according to claim 1, wherein the receptor is labeled with an epitope marker that can be detected by immunohistochemistry. 9. The method according to claim 8, wherein the epitope marker is selected from hemagglutinin, polyhistidine, myc protein and flag protein and a viral epitope. 10. The method according to claim 1, wherein the internalization is detected by microscopy with a light microscope and / or a confocal microscope. 11. The labeled receptor may be a family of GPCR receptors with seven transmembrane domains that bind to protein G, a family of tyrosine kinase receptors with a single transmembrane domain, or a family of cytokines. The method according to any one of claims 1 to 10, wherein the method belongs to the family of receptors. 12. The method according to claim 1, wherein two or more different receptors, each labeled with a different marker, are expressed on the cell surface. 13. Use of a method according to any one of claims 1 to 12 for detecting and / or identifying one or more endogenous ligands of an orphan receptor. 14. Use of a method according to any one of claims 1 to 13 for identifying new agonists or antagonists of a receptor whose endogenous ligand is known. A ligand for a receptor identified by the method according to any one of claims 1 to 12.