JP5586285B2 - Receptor interaction detection method and cyclic AMP sensor protein - Google Patents

Description

  In the present invention, interaction between different receptors in cells (for example, interaction between D1 receptor and 5HT2A receptor), activity of one receptor (for example, D1 receptor) (for example, concentration of cyclic AMP), and the like. The present invention relates to a receptor interaction detection method for non-invasively analyzing and detecting each cell and a cyclic AMP sensor protein for detecting an interaction between different receptors.

  Many hormones and neurotransmitters act on G protein-coupled receptors and cause various cellular activities through intracellular second messengers. Neurotransmitters that act on G protein-coupled receptors are involved in the regulation of brain functions over a wide range of human emotions, moods, dependence, cognition, learning, memory, etc. Among them, monoamines and their receptors are It is targeted for treatment of mental disorders such as manic depression, schizophrenia, mood disorders, autism, attention deficit / hyperactivity disorder. Such higher-order functions of the brain are controlled by the temporal and spatial concentration distributions of transmitter substances and their receptors in the brain. Therefore, it is necessary to capture in real time temporal and spatial changes and interactions of G protein-coupled receptors in the brain when analyzing information transmission mechanisms related to specific emotional activities.

  So far, studies on temporal and spatial changes and interactions of receptors in the brain have revealed the existence of drugs that have an effect on emotional activity, and their action points are monoamine G protein-coupled receptors. There is a background that has been elucidated. Conventional signal transduction studies involving monoamine G protein-coupled receptors have been analyzed mainly by behavioral pharmacological techniques using model animals.

  For example, Body et al. Disclose a method for controlling timing behavior, which is a typical operant conditioning in rats, using selective inhibitors for 5HT2A receptor, D1 receptor, and D2 receptor subtypes (see FIG. Non-patent document 1). Since the effect of d-amphetamine on timing behavior is inhibited by selective inhibitors on 5HT2A and D1 receptors, the action of d-amphetamine on timing behavior involves the 5HT2A and D1 receptors. It is made clear.

  For example, Auclar et al. Disclose a method of controlling d-amphetamine-induced locomotor activity in rats using a selective inhibitor for the 5HT2A receptor (see Non-Patent Document 2). d-Amphetamine causes dopamine release in the brain and causes increased dopamine-induced locomotor activity. The selective inhibitor for 5HT2A receptor has been shown to suppress the increase of dopamine-induced locomotor activity without suppressing dopamine release, revealing the interaction between dopamine receptor and 5HT2A receptor. Yes.

Body S. , Cheung T .; H. C. , Bezzina G. , Asgari K .; Fone K. C. F. Glennon J .; C. , Bradshaw C.I. M.M. Szababi E., et al. , “Effects of d-amphetamine and DOI (2,5-dimetic-4-iodophatamine) on timing behavior: intelligence between D1 and 5-HT2Areceptors, VHT. 189, pp331-343, 2006 Auclar A. Blanc G., et al. , Glowinski J. et al. Tassin J .; -P. , “Role of serotonin2A receptor in d-amphetamine-induced release of dopamine: comparison with precious data on alpha1b-adrenergic. 91, pp 318-326, 2004.

  However, in the conventional method of analyzing temporal and spatial interactions of different G protein-coupled receptors using behavioral pharmacological techniques using model animals, the spatial arrangement of the receptors is unknown. Since the interaction is analyzed as it is, there is a problem that the interaction between receptors is not accurately shown.

  The present invention has been made in view of the above problems, and can clarify the spatial arrangement of receptors to obtain an accurate quantitative result from each cell regarding receptor interaction. An object of the present invention is to provide a receptor interaction detection method capable of reproducibly observing a specific receptor interaction in an individual cell, and a cyclic AMP sensor protein for detecting the receptor interaction. .

  In order to solve the above-described problems and achieve the object, a receptor interaction detection method according to the present invention is a receptor interaction for detecting an interaction between a first receptor and a second receptor in a cell. A detection method comprising a production step of producing the cell containing a reporter serving as an index indicating the activity of the second receptor, and a predetermined method for causing the cell produced in the production step to emit light from the outside of the cell. And a step of adding a predetermined receptor-binding substance that binds to the first receptor and a measurement step of measuring the amount of luminescence of the cells after the treatment and the addition step are performed And an analysis step for analyzing the activity of the second receptor in the cell based on the amount of luminescence measured in the measurement step, and an activity of the second receptor analyzed in the analysis step The first A detection step of detecting the interaction between the the receptor second receptor, characterized in that it comprises a. Here, the reporter includes a fluorescent protein and a luminescent protein that can generate light according to gene expression, and specifically includes a cyclic AMP (adenosine monophosphate) sensor protein. The photoprotein is specifically a bioluminescent protein. The predetermined treatment for causing light emission includes, for example, adding a luminescent substrate (or supplying it in advance to a sample such as a cell) or irradiating excitation light. When measuring fluorescence (for example, GFP, YFP, etc.) by bioluminescence resonance energy transfer (so-called BRET), irradiation of excitation light is not necessary by adding a substrate for bioluminescence.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein in the preparation step, the reporter is a cyclic AMP sensor protein, and in the analysis step, The activity of the two receptors is analyzed by the concentration of cyclic AMP.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the cyclic AMP sensor protein emits light by interacting with the cyclic AMP in the cell. It is characterized by being.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the cyclic AMP sensor protein is a cyclic AMP binding region of firefly luciferase and PKA (protein kinase A). It is characterized by being a fusion protein consisting of

  The receptor interaction detection method according to the present invention is characterized in that, in the receptor interaction detection method described above, the cyclic AMP binding region is derived from mouse PKA-RIα.

  The receptor interaction detection method according to the present invention is characterized in that, in the receptor interaction detection method described above, the cyclic AMP binding region has a length of 220 to 250 amino acid residues. To do.

  The receptor interaction detection method according to the present invention is characterized in that, in the receptor interaction detection method described above, the cyclic AMP binding region includes at least two cyclic AMP binding sites.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the cyclic AMP sensor protein includes an N-terminal fragment of the luciferase, a C-terminal fragment of the luciferase, and The luciferase luminescent enzyme activity is recovered when the N-terminal fragment and the C-terminal fragment are bound within the cyclic AMP sensor protein molecule, including the cyclic AMP binding region.

  The receptor interaction detection method according to the present invention is characterized in that, in the receptor interaction detection method described above, the cyclic AMP binding region includes the amino acid sequence set forth in SEQ ID NO: 37 or 39. To do.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein, in the production step, the cell contains the first receptor and the second receptor. It is what it does.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein, in the preparation step, the cell contains a gene for the first receptor and a gene for the second receptor. It is characterized by having been introduced with a gene.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the measurement step measures the photon amount of the luminescent label depending on the activity of the second receptor. It further includes a photon amount measuring step.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the measurement step includes an imaging step of imaging a luminescent label depending on the activity of the second receptor. It is further characterized by including.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the imaging step is performed simultaneously on a plurality of different cells, and the analysis step includes the imaging The method further includes a collation step of collating the plurality of light-emitting images captured in the step for each cell.

  The receptor interaction detection method according to the present invention is characterized in that, in the receptor interaction detection method described above, the measurement step is repeatedly executed.

  The receptor interaction detection method according to the present invention is the receptor interaction detection method described above, wherein the first receptor is a 5HT2A receptor, and the second receptor is a D1 receptor. It is characterized by being.

  The receptor interaction detection method according to the present invention is characterized in that, in the receptor interaction detection method described above, in the treatment and addition step, the receptor binding substance is serotonin.

  The present invention also relates to a cyclic AMP sensor protein, and the cyclic AMP sensor protein according to the present invention is for detecting an interaction between a first receptor and a second receptor. It is characterized by.

  The cyclic AMP sensor protein according to the present invention is characterized in that, in the cyclic AMP sensor protein described above, it emits light by interacting with cyclic AMP (adenosine monophosphate).

  The cyclic AMP sensor protein according to the present invention is the cyclic AMP sensor protein described above, wherein the first receptor is a 5HT2A receptor and the second receptor is a D1 receptor. It is characterized by.

  According to the present invention, a cell containing a reporter serving as an index indicating the activity of the second receptor is prepared, and the prepared cell is subjected to a predetermined treatment for causing light emission from the outside of the cell and the first receptor. A predetermined receptor-binding substance that binds to the cell is added, the amount of light emitted from the cell after the treatment and addition is measured, and the activity of the second receptor in the cell is analyzed based on the amount of light emitted Since the interaction between the first receptor and the second receptor is detected based on the analyzed activity of the second receptor, the spatial arrangement of the receptors is clarified and the mutual relationship between the receptors is determined. It is possible to obtain an accurate quantitative result from each cell regarding the action, and as a result, it is possible to observe the interaction of a specific receptor with high reproducibility in each individual cell.

  According to this invention, since the reporter is a cyclic AMP sensor protein and the activity of the second receptor is analyzed by the concentration of cyclic AMP, the space of the receptor is utilized using the cyclic AMP detection principle by bioluminescence. Clarification of specific arrangements to obtain accurate quantitative results from each cell regarding receptor interactions, and as a result, reproducible observation of specific receptor interactions in individual cells There is an effect that can be done.

  According to the present invention, since the cyclic AMP sensor protein emits light by interacting with the cyclic AMP in the cell, the light emission of the cyclic AMP is enhanced by the light emission whose interaction is enhanced in the presence of the cyclic AMP. There is an effect that the concentration can be detected.

  According to the present invention, since the cyclic AMP sensor protein is a fusion protein composed of a firefly luciferase and a cyclic AMP binding region of PKA (protein kinase A), the cyclic AMP concentration is calculated by measuring the amount of luminescence. There is an effect that can be.

  According to this invention, since the cyclic AMP binding region is derived from mouse PKA-RIα, there is an effect that it can be used in cells derived from mammals.

  According to the present invention, since the cyclic AMP binding region has a length of 220 to 250 amino acid residues, the fusion protein can be produced while maintaining the luciferase activity as much as possible.

  According to the present invention, since the cyclic AMP binding region includes at least two cyclic AMP binding sites, there is an effect that the luciferase activity can be changed by changing the cyclic AMP concentration.

  According to this invention, the cyclic AMP sensor protein includes an N-terminal fragment of luciferase, a C-terminal fragment of luciferase and a cyclic AMP binding region, and the N-terminal fragment and the C-terminal fragment are the cyclic AMP. When bound within the sensor protein molecule, the luminescent enzyme activity of luciferase is restored, so that the luminescence intensity can be changed by the structural change of the cyclic AMP binding region.

  According to this invention, since the cyclic AMP binding region includes the amino acid sequence set forth in SEQ ID NO: 37 or 39, there is an effect that an optimal one as a cyclic AMP sensor protein can be selected according to conditions.

  According to this invention, since the cell contains the first receptor and the second receptor, it is easy to conduct an experiment using an easy-to-handle cell artificially including the receptor to be analyzed. There is an effect that can be done.

  According to the present invention, since the cell is a gene into which the first receptor gene and the second receptor gene have been introduced, an easy-to-handle cell artificially including the receptor to be analyzed is used. In addition to facilitating the experiment, there is an effect that a natural response can be obtained.

  According to this invention, since the photon amount of the luminescent label depending on the activity of the second receptor is measured, the luminescence amount from the whole cell or the whole cell population can be obtained using the photon counting technique. There is an effect.

  According to this invention, since the luminescent label depending on the activity of the second receptor is imaged, there is an effect that it is possible to obtain the amount of luminescence from individual cells using a luminescence microscope.

  According to this invention, since imaging is performed simultaneously on a plurality of different cells and the plurality of captured luminescent images are collated for each cell, the amount of luminescence from each cell can be obtained individually and simultaneously. There is an effect that can be done.

  According to the present invention, since the measurement of the light emission amount is repeatedly executed, it is possible to observe the temporal change of the light emission amount.

  According to the present invention, since the first receptor is the 5HT2A receptor and the second receptor is the D1 receptor, the spatial arrangement of the D1 receptor and the 5HT2A receptor is clarified and the D1 receptor Accurate quantitative results from each cell regarding the interaction between HT2A and 5HT2A receptor, and as a result, the interaction between D1 receptor and 5HT2A receptor can be observed with high reproducibility in individual cells. There is an effect.

  According to this invention, since the receptor-binding substance is serotonin, there is an effect that the activity of the 5HT2A receptor can be observed.

  According to this invention, since the cyclic AMP sensor protein is for detecting the interaction between the first receptor and the second receptor, if the cyclic AMP sensor protein is used, the receptor Clarifies the spatial arrangement of cells and obtains accurate quantitative results from each cell regarding receptor interactions, resulting in reproducible observation of specific receptor interactions in individual cells There is an effect that can be.

  According to the present invention, since the cyclic AMP sensor protein interacts with cyclic AMP (adenosine monophosphate) and emits light, if the cyclic AMP sensor protein is used, the cyclic AMP sensor protein interacts in the presence of cyclic AMP. This has the effect that the cyclic AMP concentration can be detected by the enhanced light emission.

  According to the present invention, since the cyclic AMP sensor protein is for detecting the interaction between the D1 receptor and the 5HT2A receptor, if the cyclic AMP sensor protein is used, the D1 receptor and the 5HT2A receptor are used. To obtain accurate quantitative results from each cell regarding the interaction between D1 receptor and 5HT2A receptor, and as a result, the D1 receptor and 5HT2A receptor in individual cells. There is an effect that the interaction can be observed with good reproducibility.

FIG. 1 is a diagram illustrating an example of the overall configuration of the light emission observation system 100. FIG. 2 is a diagram illustrating an example of the configuration of the light emission image capturing unit 106 of the light emission observation system 100. FIG. 3 is a diagram illustrating an example of the configuration of the light emission image capturing unit 106 of the light emission observation system 100. FIG. 4 is a block diagram illustrating an example of the configuration of the image analysis device 110 of the light emission observation system 100. FIG. 5 is a diagram showing the detection principle of cyclic AMP using split luciferase. FIG. 6 is a view showing a luminescence image before stimulation with serotonin including HEK293 cells into which cyclic AMP sensor protein, 5HT2A receptor and D1 receptor are introduced. FIG. 7 is a diagram showing a luminescence image 5 minutes after stimulation with serotonin including HEK293 cells into which cyclic AMP sensor protein, 5HT2A receptor and D1 receptor are introduced. FIG. 8 is a diagram showing a luminescence image 5 minutes after stimulation with dopamine containing HEK293 cells into which cyclic AMP sensor protein, 5HT2A receptor and D1 receptor were introduced. FIG. 9 is a graph showing the change over time in the luminescence intensity of the cyclic AMP sensor protein by serotonin stimulation and dopamine stimulation in selected HEK293 cells into which 5HT2A receptor and D1 receptor have been introduced. FIG. 10 is a view showing a luminescence image before stimulation with serotonin including HEK293 cells into which cyclic AMP sensor protein, 5HT2A receptor and D2 receptor are introduced. FIG. 11 is a diagram showing a luminescence image 5 minutes after stimulation with serotonin including HEK293 cells into which cyclic AMP sensor protein, 5HT2A receptor and D2 receptor are introduced. FIG. 12 is a diagram showing a luminescence image 5 minutes after stimulation with dopamine containing HEK293 cells into which a cyclic AMP sensor protein, 5HT2A receptor and D2 receptor were introduced. FIG. 13 is a diagram showing a luminescence image 5 minutes after stimulation with forskolin including HEK293 cells into which a cyclic AMP sensor protein, 5HT2A receptor and D2 receptor are introduced. FIG. 14 is a graph showing the change over time in the luminescence intensity of the cyclic AMP sensor protein by serotonin stimulation, dopamine stimulation and forskolin stimulation in selected HEK293 cells into which 5HT2A receptor and D2 receptor have been introduced. FIG. 15 is a diagram showing a luminescence image before stimulation with serotonin including HEK293 cells into which a cyclic AMP sensor protein and a 5HT2A receptor are introduced. FIG. 16 is a view showing a luminescence image 5 minutes after stimulation with serotonin including HEK293 cells into which a cyclic AMP sensor protein and a 5HT2A receptor have been introduced. FIG. 17 is a diagram showing a luminescence image 5 minutes after stimulation with forskolin including HEK293 cells into which a cyclic AMP sensor protein and a 5HT2A receptor have been introduced. FIG. 18 is a graph showing changes over time in the luminescence intensity of the cyclic AMP sensor protein induced by serotonin stimulation and dopamine stimulation in selected HEK293 cells into which 5HT2A receptor has been introduced. FIG. 19 is a diagram showing the detection principle of cyclic AMP using a mutant split luciferase. FIG. 20 is a diagram showing a luminescence image before serotonin stimulation in HEK293 cells into which cpGL4_C_RIα_αAB_N, 5HT2A receptor, and D1 receptor were introduced. FIG. 21 is a diagram showing a luminescence image 5 minutes after stimulation with serotonin in HEK293 cells into which cpGL4_C_RIα_αAB_N, 5HT2A receptor, and D1 receptor were introduced. FIG. 22 is a diagram showing a luminescence image 5 minutes after dopamine stimulation in HEK293 cells into which cpGL4_C_RIα_αAB_N, 5HT2A receptor, and D1 receptor were introduced. FIG. 23 is a graph showing temporal changes in the luminescence intensity of cpGL4_C_RIα_αAB_N induced by serotonin and dopamine in selected cells in HEK293 cells into which 5HT2A receptor and D1 receptor have been introduced. FIG. 24 is a diagram showing a luminescence image before serotonin stimulation in HEK293 cells into which cpGL4_C_RIα_αAB_N and 5HT2A receptor have been introduced. FIG. 25 is a view showing a luminescence image 5 minutes after stimulation with serotonin in HEK293 cells into which cpGL4_C_RIα_αAB_N and 5HT2A receptor have been introduced. FIG. 26 is a diagram showing a luminescence image after 5 minutes of dopamine stimulation in HEK293 cells into which cpGL4_C_RIα_αAB_N and 5HT2A receptor have been introduced. FIG. 27 is a graph showing temporal changes in the luminescence intensity of cpGL4_C_RIα_αAB_N by stimulation with serotonin and dopamine in selected cells in HEK293 cells into which 5HT2A receptor has been introduced. FIG. 28 is a table showing details of various cyclic AMP sensor proteins produced. FIG. 29 is a diagram comparing the activities of various cyclic AMP sensor proteins.

  Embodiments of a receptor interaction detection method and a cyclic AMP sensor protein for detecting receptor interaction according to the present invention will be described below in detail with reference to the drawings. In addition, this invention is not limited by this Embodiment. Here, in the present embodiment, a case where an interaction between a D1 receptor and a 5HT2A receptor is detected by a luminescent image using a luminescent protein (specifically, a bioluminescent protein) will be described as an example. The same can be applied to other receptors and photon counting.

[Constitution]
First, the configuration of the luminescence observation system 100 used in the receptor interaction detection method according to the present invention will be described with reference to FIG. 1, FIG. 2, and FIG. FIG. 1 is a diagram illustrating an example of the overall configuration of the light emission observation system 100.

  As shown in FIG. 1, a luminescence observation system 100 includes a container 103 (specifically, a petri dish, a slide glass, a microplate, a gel support, a fine particle carrier, etc.) that contains cells 102, and a stage 104 on which the container 103 is arranged. And a light emission image capturing unit 106 for measuring weak light emission, and an image analysis device 110. The luminescent image capturing unit 106 may be disposed below the stage 104. Thereby, disturbance light from above the sample due to opening and closing of a cover (not shown) can be completely blocked, and as a result, the S / N ratio of the luminescent image can be increased. The light emission image capturing unit 106 may be a laser scanning optical system.

  The cell 102 is, for example, a living cell that is luminescence-labeled so as to emit light depending on a cyclic AMP (adenosine monophosphate) concentration. Here, as a protein that emits light depending on the cyclic AMP concentration (photoprotein), in addition to a fusion protein of an N-terminal luminescent enzyme, a C-terminal luminescent enzyme, and a cyclic AMP-binding protein, for example, International Publication No. You may use the fusion protein currently disclosed by 2007/120522 or Japanese translations of PCT publication No. 2007-508014 gazette. Here, the fusion protein is expressed in the cell 102 by introducing into the cell 102 an expression vector containing a polynucleotide encoding the fusion protein, which is configured so that the fusion protein is expressed in the cell 102. You may comprise. The cell 102 is given a predetermined luminescent substrate (for example, luciferin) and a predetermined stimulus (for example, drug stimulus) from outside the cell 102.

The luminescence image capturing unit 106 is specifically an upright luminescence microscope and captures a luminescence image of the cell 102. As shown in the figure, the luminescent image capturing unit 106 includes an objective lens 106a, a dichroic mirror 106b, a CCD (charge-coupled device) camera 106c, and an imaging lens 106f. Specifically, it is preferable that the objective lens 106a has a value of (numerical aperture / magnification) ² of 0.01 or more. The dichroic mirror 106b is used when the light emitted from the cell 102 is separated by color and the light emission amount and light emission intensity are measured by color using two colors of light emission. The CCD camera 106c takes a light emission image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the objective lens 106a, the dichroic mirror 106b, and the imaging lens 106f. The CCD camera 106c is connected to the image analysis device 110 so as to be able to communicate with each other by wire or wirelessly. Here, when there are a plurality of cells 102 in the imaging range, the CCD camera 106c may capture a light-emitting image and a bright-field image of the plurality of cells 102 included in the imaging range. The imaging lens 106f forms an image (specifically, an image including the cell 102) incident on the imaging lens 106f via the objective lens 106a and the dichroic mirror 106b. Note that FIG. 1 shows an example in which a light emission image corresponding to two light emissions separated by the dichroic mirror 106b is separately captured by the two CCD cameras 106c. The image capturing unit 106 may include an objective lens 106a, a single CCD camera 106c, and an imaging lens 106f.

  Here, when measuring the light emission amount and the light emission intensity for each color using the light emission of two colors, as shown in FIG. 2, the light emission image pickup unit 106 includes an objective lens 106a, a CCD camera 106c, and a split image unit 106d. And the imaging lens 106f. The CCD camera 106c may capture a light emission image (split image) and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the split image unit 106d and the imaging lens 106f. The split image unit 106d separates the light emitted from the cell 102 by color, and is used when measuring the light emission amount and light emission intensity by color using two colors of light, similar to the dichroic mirror 106b.

  Further, when measuring the emission amount and emission intensity for each color using light emission of a plurality of colors (that is, when using multicolor light emission), the light emission image pickup unit 106 includes an objective lens 106a and an objective lens 106a as shown in FIG. The CCD camera 106c, the filter wheel 106e, and the imaging lens 106f may be used. Then, the CCD camera 106c may capture a light emission image and a bright field image of the cell 102 projected onto the chip surface of the CCD camera 106c via the filter wheel 106e and the imaging lens 106f. The filter wheel 106e is used when light emitted from the cells 102 is separated by color by exchanging filters, and a light emission amount and light emission intensity are measured for each color using light emission of a plurality of colors.

  Returning to FIG. 1, the image analysis apparatus 110 is specifically a personal computer. As shown in FIG. 4, the image analysis device 110 is roughly divided into a control unit 112, a clock generation unit 114 that measures the system time, a storage unit 116, a communication interface unit 118, and an input / output interface unit. 120, an input device 122, and an output device 124. These units are connected via a bus.

  The storage unit 116 is a storage unit, and specifically, a memory device such as a random access memory (RAM) or a read-only memory (ROM), a fixed disk device such as a hard disk, a flexible disk, an optical disk, or the like is used. Can do. And the memory | storage part 116 memorize | stores the data etc. which were obtained by the process of each part of the control part 112. FIG. The communication interface unit 118 mediates communication between the image analysis device 110 and the CCD camera 106c. That is, the communication interface unit 118 has a function of communicating data with other terminals via a wired or wireless communication line. The input / output interface unit 120 is connected to the input device 122 and the output device 124. Here, in addition to a monitor (including a home television), a speaker or a printer can be used as the output device 124 (hereinafter, the output device 124 may be described as a monitor). In addition to the keyboard, mouse, and microphone, the input device 122 can be a monitor that realizes a pointing device function in cooperation with the mouse.

  The control unit 112 has an internal memory for storing a control program such as an OS (Operating System), a program that defines various processing procedures, and necessary data, and executes various processes based on these programs. . The control unit 112 is roughly composed of a light emission image capturing instruction unit 112a, a light emission image acquisition unit 112b, an image analysis unit 112c, and an analysis result output unit 112d.

  The luminescent image capturing instruction unit 112a instructs the CCD camera 106c to capture a luminescent image and a bright field image via the communication interface unit 118. The luminescent image acquisition unit 112b acquires the luminescent image and bright field image captured by the CCD camera 106c via the communication interface unit 118. The control unit 112 controls the emission image capturing instruction unit 112a to cause the CCD camera 106c to repeatedly capture the emission image and the bright field image of the cell 102, and controls the emission image acquisition unit 112b to control the captured emission image and the bright image. A field-of-view image is acquired every time it is imaged or after all imaging is completed.

  The image analysis unit 112c measures the luminescence intensity of the luminescence emitted from the cells 102 over time based on the plurality of luminescence images acquired by the luminescence image acquisition unit 112b. The analysis result output unit 112d outputs the analysis result from the image analysis unit 112c to the output device 124. Specifically, the analysis result output unit 112d graphs and displays on the output device 124 time-series data regarding the emission intensity of the luminescence emitted from the cell 102 obtained by the image analysis unit 112c.

[Receptor interaction detection method]
The present invention is a receptor interaction detection method for detecting an interaction between a first receptor and a second receptor in a cell, and includes a reporter serving as an index indicating the activity of the second receptor. A production step for producing the cells, and a predetermined treatment for causing the cells produced in the production step to emit light from outside the cells and adding a predetermined receptor-binding substance that binds to the first receptor A treatment step and an addition step, a measurement step for measuring the luminescence amount of the cell after the treatment and the addition step are performed, and the second in the cell based on the luminescence amount measured in the measurement step Based on the analysis step of analyzing the activity of the receptor and the activity of the second receptor analyzed in the analysis step, the interaction between the first receptor and the second receptor is detected. Including a detection step. It relates receptor interaction detecting method according to.

  In the production process, a cell containing a reporter is produced. This reporter may be a cyclic AMP sensor protein as described below. There are no limitations on the types of cells to be used, and bacterial cells, yeast cells, plant cells, animal cells, and the like can be used. When animal cells are used, particularly mammalian cells are used, for example mouse cells, monkey cells and human cells. There is no particular limitation on the method for introducing the reporter into the cell, and a known introduction method can be used. The cell may be a cell aggregate cultured as a part of the living tissue, or may be in the form of an organ or the like to which the living tissue belongs or a living individual. When the reporter is a protein, one method is a method of introducing a nucleic acid containing or consisting of a base sequence encoding the protein into a cell, and then expressing the protein in the cell. For example, an expression vector containing the nucleic acid can be introduced into cells by the calcium phosphate method, lipofection method, electroporation method, or the like, and the protein can be expressed from the expression vector. Instead of expressing from a vector, the protein can also be expressed from the genome after preparing a cell in which the nucleic acid is integrated into the genome. Another method is to introduce the protein purified outside the cell directly into the cell. For example, the protein can be directly injected into cells by a microinjection method. Alternatively, cells can be incubated in a culture solution containing the protein, and the protein can be taken into the cell by endocytosis.

  In the treatment and addition step, a predetermined treatment for causing light emission and addition of a predetermined receptor-binding substance that binds to the first receptor are performed. The predetermined treatment for causing light emission includes, for example, adding a luminescent substrate or irradiating excitation light. There is no special limitation in the method of adding a luminescent substrate, and it can select suitably. Preferably, the luminescent substrate is added immediately before the measurement. There is no particular limitation on the amount of the luminescent substrate to be added, but preferably the luminescent enzyme is added in an amount sufficient to cause a luminescent reaction. The receptor is activated by the addition of a predetermined receptor binding substance that binds to the first receptor. The first receptor can be, for example, a 5HT2A receptor, and the receptor binding agent can be a serotonin, such as 5HT.

  In the measurement step, luminescence generated from the cells by the treatment and addition steps is measured. Measurements can be made by imaging or photocounting. When imaging is performed, an image of a cell in which a luminescent reaction occurs is acquired, which is convenient for measuring luminescence for each cell. Imaging can be performed at an arbitrary timing, but is preferably performed after a certain time from the addition of the substrate. In particular, when a plurality of measurements are repeated, it is preferable to capture images at the same timing through a plurality of measurements in order to suppress variations in measurement results. The best imaging time is appropriately selected according to the conditions. When the emission intensity is low, the imaging time (exposure time when using a microscope) is lengthened, and conversely when the emission intensity is too high, the imaging time is shortened. Imaging can be performed by acquiring only light having a wavelength related to the luminescence reaction, but at the same time, a bright field image or an image using light having another wavelength may be acquired. For example, a specific protein in which a probe (for example, fluorescent or luminescent protein) is fused simultaneously with a cyclic AMP sensor protein is introduced into a cell, and light from the specific protein is detected simultaneously with light derived from the cyclic AMP sensor protein. May be.

  In the analysis step, the activity of the second receptor is analyzed. Based on the measurement result obtained in the measurement process, it is determined whether light emission has occurred. When luminescence occurs, it is determined that the second receptor is activated. Preferably, a D1 receptor or a D2 receptor can be selected as the second receptor. The D1 receptor has the effect of activating adenylate cyclase when the ligand is bound, and increasing the intracellular cyclic AMP concentration. On the other hand, the D2 receptor has an effect of activating phosphodiesterase when the ligand is bound, and reducing the intracellular cyclic AMP concentration. The second receptor is preferably a receptor that is known to have an action of activating adenylate cyclase.

  In the detection step, the interaction between the first receptor and the second receptor is detected based on the analysis result of the activity of the second receptor. When the receptor binding substance is given to the first receptor in the treatment and addition step and the activity of the second receptor is recognized in the analysis step, the mutual relationship between the first receptor and the second receptor is obtained. A positive conclusion is obtained for the action. On the other hand, when the activity of the second receptor is not recognized in the analysis step, a negative conclusion is obtained for the interaction between the first receptor and the second receptor.

[Cyclic AMP sensor protein]
Next, the cyclic AMP sensor protein will be supplementarily described.

  In the present invention, a cyclic AMP sensor protein can be used as a reporter serving as an index indicating the activity of the second receptor. As the cyclic AMP sensor protein, a fusion protein comprising a firefly luciferase and a cyclic AMP binding region of PKA can be used.

  As the cyclic AMP binding region, a mouse derived from PKA-RIα can be used. Here, PKA-RIα means PKA control subunit α. PKA-RIα has a cyclic AMP binding domain and has the ability to bind to cyclic AMP.

  Regarding the length of the cyclic AMP binding region, the number of amino acid residues may be 120 to 250. The number of amino acid residues may be 200 to 250, 220 to 250, 230 to 250, 230 to 245, or 230 to 240. For example, the number of amino acid residues may be 230 or 241.

  As the cyclic AMP binding region, one containing at least two cyclic AMP binding sites can be used. A “cyclic AMP binding site” is defined as a site in a cyclic AMP binding region to which a cyclic AMP1 molecule can bind. The cyclic AMP binding site includes the aforementioned cyclic AMP binding domain of PKA-RIα. If the above definition is satisfied, even a site that is not specified or recognized as a cyclic AMP binding domain is included in the cyclic AMP binding site.

  In the method according to the present invention, a cyclic AMP sensor protein comprising an N-terminal fragment of luciferase, a C-terminal fragment of luciferase and a cyclic AMP binding region, wherein the N-terminal fragment and the C-terminal fragment are cyclic A cyclic AMP sensor protein that recovers the luminescent enzyme activity of luciferase when bound in the AMP sensor protein molecule can be used. The “N-terminal fragment of luciferase” means a luciferase fragment from which a plurality of amino acids have been deleted from the C-terminus, and has lost luminescent enzyme activity. “The C-terminal fragment of luciferase” means a luciferase fragment from which a plurality of amino acids have been deleted from the N-terminus, and has lost the luminescent enzyme activity. The cyclic AMP sensor protein “includes” a fragment thereof and a cyclic AMP binding region means that the cyclic AMP sensor protein includes an N-terminal fragment of luciferase, a C-terminal fragment of luciferase, a cyclic AMP binding region, and an arbitrary It means a fusion protein composed of other polypeptides. Thus, these components are not included as side chains or modifying groups. As an example of such a cyclic AMP sensor protein, as shown in FIG. 5, an N-terminal fragment of luciferase is present on the N-terminal side of the cyclic AMP sensor protein, and the cyclic AMP sensor protein is present on the C-terminal side. It is a cyclic AMP sensor protein in which a C-terminal fragment of luciferase is present and a cyclic AMP binding region derived from PKA-RIα is present between these fragments. As another example, as shown in FIG. 19, the C-terminal fragment of luciferase is present on the N-terminal side of the cyclic AMP sensor protein, and the N-terminal side of luciferase is present on the C-terminal side of the cyclic AMP sensor protein. It is a cyclic AMP sensor protein in which fragments exist and a cyclic AMP binding region derived from PKA-RIα is present between these fragments.

  The N-terminal fragment may be, for example, a fragment containing the first to 400th amino acids of luciferase, and the C-terminal fragment may be, for example, those containing the 401st to 550th amino acids of luciferase. In particular, it is preferable that the N-terminal fragment contains the first to 416th amino acids of luciferase, and the C-terminal fragment contains the 399th to 550th amino acids of luciferase. Thus, the N-terminal side fragment and the C-terminal side fragment may partially overlap their sequences. There may also be a luciferase sequence portion that is not contained in the N-terminal fragment or the C-terminal fragment.

  In the cyclic AMP sensor protein, the luciferase is divided into an N-terminal fragment and a C-terminal fragment, so that in a normal environment (for example, an environment in which no cyclic AMP exists), the cyclic AMP sensor protein Has no luciferase activity. However, under certain circumstances (for example, in the presence of cyclic AMP), the cyclic AMP sensor protein restores luciferase activity. This recovery is due to the function of the cyclic AMP binding region. For example, when a cyclic AMP binding region derived from mouse PKA-RIα is selected, when cyclic AMP binds to the cyclic AMP binding region, as shown in FIGS. The structure changes, and as a result, the N-terminal fragment and the C-terminal fragment approach each other. These two fragments cooperate to exert luciferase activity.

  In the expression that “the luminescent enzyme activity is recovered when the N-terminal fragment and the C-terminal fragment are bound within the cyclic AMP sensor protein molecule”, the N-terminal fragment and the C-terminal fragment are “bound”. There is no limitation, and a chemical bond such as a covalent bond or a non-covalent bond may be used, but it is “close” or “contact” to such an extent that the N-terminal fragment and the C-terminal fragment can perform the original function of luciferase. "Is preferable. Alternatively, the N-terminal fragment and the C-terminal fragment may be “associated”. That is, the term “binding” used in the expression includes the meaning of “approach”, “contact”, or “meeting”.

  In the above expression, the bond between the N-terminal fragment and the C-terminal fragment may be a bond generated in response to cyclic AMP. More specifically, the cyclic AMP molecule binds to the cyclic AMP binding region, resulting in a change in the structure of the cyclic AMP binding region, and the resulting binding between the N-terminal fragment and the C-terminal fragment or It may be close.

  The cyclic AMP binding region is preferably a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 37 or 39.

[Example 1]
Examples of the present embodiment will be described below with reference to FIGS. Here, FIG. 5 is a diagram showing the detection principle of cyclic AMP using the split luciferase assay method.

  The split luciferase assay is a fragment (fragment) of the N-terminal luminescent enzyme that is obtained by inactivating luciferase such as firefly luciferase or Renilla luciferase at a specific position to inactivate luminescent enzyme activity (bioluminescent ability). And the C-terminal luminescent enzyme are reconstituted to recover the luminescent enzyme activity, and are used for detecting protein-protein interactions and detecting intracellular organelle migration of proteins.

  FIG. 5 shows an example in which a cyclic AMP binding protein is used as a protein that causes a non-covalent interaction between the N-terminal luminescent enzyme (NLuc) and the C-terminal luminescent enzyme (CLuc). That is, when cyclic AMP binds to the cyclic AMP-binding protein in the fusion protein of N-terminal luminescent enzyme, C-terminal luminescent enzyme and cyclic AMP-binding protein expressed in cells, the cyclic AMP binding The three-dimensional structure of the protein changes, and the N-terminal luminescent enzyme and the C-terminal luminescent enzyme bind to each other, so that the luminescent enzyme activity is recovered and a detectable signal is emitted. In this example, an example in which the cyclic AMP binding domain of the regulatory subunit Iα (PKA-RIα) of PKA (protein kinase A) is used as the cyclic AMP binding protein in the split luciferase assay system will be described. Here, the flow (procedure) of the experiment in the present embodiment is as follows.

[Preparation 1: Cloning of N-terminal luminescent enzyme gene and C-terminal luminescent enzyme gene and cyclic AMP binding domain gene of PKA-RIα and preparation of fusion protein expression plasmid]
[Procedure 1]
In order to prepare the N-terminal luminescent enzyme (NLuc) gene and the C-terminal luminescent enzyme (CLuc) gene, a synthetic oligo DNA (deoxyribonucleic acid) used for PCR (polymerase chain reaction) was prepared with the following sequence.
[Synthetic oligo DNA sequence for NLuc gene preparation]
NLuc_Fw (SEQ ID NO: 1): 5'-TGTGGATCCCAGCCACCCATGAGATAGGCCAA-3 '
NLuc_Rv (SEQ ID NO: 2): 5′-CAGCTCGAGGTCCTTGTCGATGAGAGCGTT-3 ′
[Synthetic oligo DNA sequence for CLuc gene preparation]
CLuc_Fw (SEQ ID NO: 3): 5′-ATCAGATCTGGCTACGTTAACAACCCCGAG-3 ′
CLuc_Rv (SEQ ID NO: 4): 5′-CTAGAATTCTTACACCGGCGATCTGGCCGCC-3 ′

In addition, for the cloning of the PAMP-RIα cAMP binding domain gene, a synthetic oligo DNA used for PCR was prepared with the sequence shown below.
PKA_RIA_β1_Fw (SEQ ID NO: 5): 5′-ATTCTCGAGGCTATGTTCCAGTCTCTCTT-3 ′
PKA_RIA_B_Rv (SEQ ID NO: 6): 5′-GGAAGATCTGACGGACGGGACACGAAGCT-3 ′

[Procedure 2]
Using pGL4.10 (manufactured by Promega Corp.) as a template, the N-terminal luciferase gene (NLuc: including amino acids 1 to 416 of the GL4.10 gene) and the C-terminal luciferase gene (CLuc: GL4.10) The 399th to 550th amino acids of the gene) are synthesized with the above synthetic oligo DNA (a pair of the DNA sequence of SEQ ID NO: 1 and the DNA sequence of SEQ ID NO: 2, and the DNA sequence of SEQ ID NO: 3 and the DNA sequence of SEQ ID NO: 4). The pair) was amplified by PCR using primers.

[Procedure 3]
The region containing both A and B of the cyclic AMP binding domain in the PKA regulatory subunit Iα (PKA-RIα) is used as a template with the mouse PKA regulatory subunit Iα (PKA-RIα) cDNA as a template. Amplified by PCR using synthetic oligo DNA as a primer. Using the DNA sequence of SEQ ID NO: 5 and the primer of the DNA sequence of SEQ ID NO: 6, the RIα_β1AB gene (including the region corresponding to the amino acid sequence of positions 152 to 381 of the mouse PKA-RIα gene, SEQ ID NO: 39) was PCR-encoded. Amplified by

[Procedure 4: Preparation of fusion protein gene expression plasmid]
The NLuc gene, CLuc gene and RIα_β1AB gene amplified by PCR are inserted between the BamHI site and EcoRI site of pcDNA3.1 (manufactured by Invitrogen), which is a plasmid for animal cell expression, and the plasmid pcDNA / GL4_N_RIα_β1AB_C for animal cell expression is used. Was made.

[Preparation Part 2: Cloning of human D1 receptor gene, D2 receptor gene and 5HT2A receptor gene]
[Procedure 1]
For the production of human D1 receptor gene, D2 receptor gene and 5HT2A receptor gene, synthetic oligo DNAs used for PCR were prepared with the sequences shown below.
[Synthetic oligo DNA sequence for preparing human D1 receptor gene]
HU_D1R_Fw (SEQ ID NO: 7): 5′-GCCACATGAGGACTCTGAACACCCTCTCC-3 ′
HU_D1R_Rv (SEQ ID NO: 8): 5′-TCAGGTTGGGTGCTGACCGTTTTGTGTGAT-3 ′
[Synthetic oligo DNA sequence for preparing human D2 receptor gene]
HU_D2R_Fw (SEQ ID NO: 9): 5′-GCCACATGGATCCACTGAATCTGCTCGG-3 ′
HU_D2R_Rv (SEQ ID NO: 10): 5′-TCAGCAGTGGAGGATCTTCAGGAAGGCCT-3 ′
[Synthetic oligo DNA sequence for preparing human 5HT2A receptor gene]
HU — 5HT2AR_Fw (SEQ ID NO: 11): 5′-GCCACATGGGAATTTCTTGTGAAGAAAA-3 ′
HU — 5HT2AR_Rv (SEQ ID NO: 12): 5′-TCACACACAGCTCACCTTTTCATTCACTCC-3 ′

[Procedure 2]
D1 receptor gene (including the region corresponding to the full length of human D1 receptor gene) D2 receptor gene (full length of human D2 receptor gene) using human brain cDNA library (manufactured by Takara Bio Inc.) as a template And a 5HT2A receptor gene (including a region corresponding to the full length of the human 5HT2A receptor gene) were amplified by PCR using the synthetic oligo DNA as a primer.

[Procedure 3]
The D1 receptor gene, D2 receptor gene, and 5HT2A receptor gene amplified by PCR were subcloned into the pBluescript II vector, and then the sequence of the prepared DNA was confirmed by DNA sequencing.

[Procedure 4]
The respective genes are incorporated into mammalian cell expression vector pcDNA3.1 (+) (manufactured by Invitrogen Corp.), human D1 receptor expression plasmid pcDNA / D1R, human D2 receptor expression plasmid pcDNA / D2R and human 5HT2A reception. A plasmid pcDNA / 5HT2AR for body expression was prepared.

[Experiment: Luminescent Imaging of cAMP Concentration Variation in HEK293 Cells Containing Human D1 Receptor, D2 Receptor and 5HT2A Receptor]
[Procedure 1: Culture of HEK293 cells]
HEK293 cells obtained from ATCC (American Type Culture Collection) were cultured in Earle's MEM / Medium (GI) supplemented with 10% Fetal Bovine Serum and 1 × Nonesential amino acids in a 5% CO2 incubator. .

[Procedure 2: Introduction of protein expression plasmid]
HEK293 cells were seeded at a cell density of 2 × 10 ⁵ / dish in a 35 mm diameter glass bottom dish, cultured overnight in a 5% CO 2 incubator, and a plasmid pcDNA / GL4_N_RIα_β1AB_C for expression of cyclic AMP sensor protein, human D1 receptor The expression plasmid pcDNA / D1R (or human D2 receptor expression plasmid pcDNA / D2R) and the human 5HT2A receptor expression plasmid pcDNA / 5HT2AR are mixed, and transfected using FuGENE HD (Roche). Cultured overnight in a% CO2 incubator. The amino acid sequence of the human D1 receptor is specifically the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of the human 5HT2A receptor is specifically the amino acid sequence of SEQ ID NO: 30.

[Procedure 3: Shooting a luminescent image]
After adding 2 mM luciferin (Promega) in the medium and allowing to stand for 1 hour, the culture dish was set on a luminescence microscope “LV (LUMINOVIEW) -200” (Olympus), and the luminescence images were displayed at 20-second intervals. Time-lapse photography was performed. As the light emission observation conditions, the magnification of the objective lens was 40 times, the exposure time was 10 seconds, and the binning was 2 × 2. An EM-CCD camera iXon (manufactured by Andor Co.) was used as the CCD camera 106c, and the luminescence image was taken into a personal computer configured as the image analysis device 110.

[Procedure 4: Shooting Luminescent Image by Dopamine Stimulation]
After 1 minute from the start of time-lapse photography, stimulation was performed sequentially with serotonin (5HT: final concentration 10 μM), dopamine (DA: final concentration 10 μM) and forskolin (FSK: final concentration 5 μM), and then time-lapse photography of the luminescent image was performed. went.

[Procedure 5: Graphical display of change in light emission over time]
A plurality of ROIs (Region of Interest) are designated for each luminescent image (see FIGS. 6, 10 and 15) taken before stimulation, and each luminescent image taken after stimulation. A plurality of ROIs are designated as shown in FIG. 7, FIG. 8, FIG. 11 to FIG. 13 and FIG. The emission intensity of each designated ROI was measured based on each emission image, and the change over time of the measured emission intensity was displayed as a graph (see FIGS. 9, 14, and 18). The analysis of the luminescence image was performed using Metamorph software (made by Universal Imaging) that functions as the image analysis unit 112c. In addition, in FIG. 9, FIG. 14, and FIG. 18, each graph corresponding to each of Cell1 to Cell4 is shown in each of numbers 1 to 4 in FIGS. 6 to 8, FIGS. 10 to 13, and FIGS. The time-dependent change of the emission intensity of each corresponding ROI is shown.

[results of the experiment]
As a result of the above experiment, as shown in FIG. 6 to FIG. 9, changes in intracellular cyclic AMP response to stimulation to 5HT2A receptor, D1 receptor, and D2 receptor are observed at a single cell level (single cell level). I was able to detect it. In addition, when the change in luminescence intensity in individual cells shown in FIG. 9 is observed, an increase in intracellular cyclic AMP concentration in response to stimulation with 5HT2A receptor is observed in the combination of 5HT2A receptor and D1 receptor. It was done. However, as shown in FIGS. 11 to 14 and FIGS. 16 to 18, intracellular cyclic AMP concentration in response to stimulation to 5HT2A receptor in the combination of 5HT2A receptor and D2 receptor and in 5HT2A receptor alone No increase was observed. From previous studies of the interaction between serotonin receptors and dopamine receptors, it is known that behaviors that are thought to be mediated through D1 receptors are induced through 5HT2A receptors in individuals. The results obtained by this experiment indicate that the response observed in individuals can be explained at the cellular level.

[Summary of this example]
As described above, according to this example, it is possible to obtain an accurate quantitative result from each cell without affecting intracellular information transmission. As a result, 5HT2A receptor and D1 receptor in each cell are obtained. The interaction with the body can be observed over time with good reproducibility.

[Other embodiments]
In the embodiment described above, a fusion mainly composed of an N-terminal luminescent enzyme, a C-terminal luminescent enzyme, and a cyclic AMP-binding domain of PKA-RIα, which are divided so that the luminescent enzyme activity is recovered by binding to each other. Although the protein has been described as a cyclic AMP sensor protein, the present invention is not limited to this, and is replaced with a fusion protein comprising an N-terminal luminescent enzyme, a C-terminal luminescent enzyme, and a cyclic AMP-binding domain of PKA-RIα. In addition, the cyclic AMP sensor protein disclosed in International Publication No. 2007/120522 and Japanese Translation of PCT International Publication No. 2007-508014 may be used.

[Example 2]
A cyclic AMP sensor protein according to an embodiment different from that of Example 1 was prepared, and its usefulness was examined. Specifically, as shown in FIG. 19, a C-terminal fragment is arranged on the N-terminal side of the cyclic AMP sensor protein, an N-terminal fragment is arranged on the C-terminal side, and cyclic AMP is interposed between them. A cyclic AMP sensor protein in which a binding region was arranged was prepared. That is, in short, a cyclic AMP sensor protein in which the C-terminal fragment and the N-terminal fragment were replaced with the cyclic AMP sensor protein according to Example 1 was prepared. In addition, the cyclic AMP sensor protein according to this example and the genes and primers used for the production thereof were distinguished by attaching “mutant” or “cp (circular permutation)” to the names thereof. .

[Preparation: Cloning of N-terminal luminescent enzyme gene and C-terminal luminescent enzyme gene for creating mutant luciferase and cyclic AMP binding domain gene of PKA-RIα, and preparation of mutant luciferase fusion protein expression plasmid]
[Procedure 1]
Synthetic oligo DNA (deoxyribonucleic acid) used for PCR (polymerase chain reaction) for the production of mutant luciferase N-terminal luminescent enzyme (cpNLuc) gene and C-terminal luminescent enzyme (cpCLuc) gene is shown below Prepared in sequence.
[Synthetic oligo DNA sequence for cpNLuc gene preparation]
cpNLuc_Fw (SEQ ID NO: 13): 5′-ATCAGATCTGAAGATCGCAAAAAACATTAAG-3 ′
cpNLuc_Rv (SEQ ID NO: 14): 5′-CTAGAATTCTTAGTCCTTGTCGATGGAGAGC-3 ′
[Synthetic oligo DNA sequence for cpCLuc gene preparation]
cpCLuc_Fw (SEQ ID NO: 15): 5'-TGTGGATCCCCACCCACATGAGCGGCTACGTTAACAAACCCC-3 '
cpCLuc_Rv (SEQ ID NO: 16): 5′-CAGCTCGAGCACGGCGATCTTGCCGCCCT-3 ′

In addition, for the cloning of the PAMP-RIα cAMP binding domain gene, a synthetic oligo DNA used for PCR was prepared with the sequence shown below.
PKA_RIA_α_Fw (SEQ ID NO: 17): 5′-TTTCTCGAGCTTGATGATAACGAGAGAAGT-3 ′
PKA_RIA_B_Rv (SEQ ID NO: 18): 5′-GGAAGATCTGACGGACGGGACACGAAGCT-3 ′

[Procedure 2]
Using a commercially available luciferase gene pGL4.10 (manufactured by Promega Corp.) as a template, a mutant N-terminal luciferase gene (cpNLuc: including amino acids 1 to 416 of the GL4.10 gene) and a mutant type. Of the C-terminal luciferase gene (cpCLuc: including amino acids 399 to 550 of the GL 4.10 gene) and the synthetic oligo DNA (a pair of the DNA sequence of SEQ ID NO: 13 and the DNA sequence of SEQ ID NO: 14, and the sequence) PCR was carried out using the DNA sequence of No. 15 and the DNA sequence of SEQ ID No. 16) as primers.

[Procedure 3]
The region containing both A and B of the cyclic AMP binding domain in the PKA regulatory subunit Iα (PKA-RIα) is used as a template with the mouse PKA regulatory subunit Iα (PKA-RIα) cDNA as a template. Amplified by PCR using synthetic oligo DNA as a primer. RIα_αAB gene (including a region corresponding to the 141st to 381th amino acid sequences of the mouse PKA-RIα gene) using primers of the DNA sequence of SEQ ID NO: 17 and the DNA sequence of SEQ ID NO: 18 (the base sequence is the sequence) No. 37) was amplified by PCR.

[Procedure 4: Preparation of fusion protein gene expression plasmid]
Nucleic acids each containing cpNLuc gene, cpCLuc gene and RIα_αAB gene amplified by PCR are appropriately treated with restriction enzymes, and then BamHI site and EcoRI site of pcDNA3.1 (manufactured by Invitrogen), an animal cell expression plasmid. To insert an animal cell expression plasmid pcDNA / cpGL4_C_RIα_αAB_N.

[Experiment: Luminescent Imaging of cAMP Concentration Variation in HEK293 Cells Containing Human D1 Receptor, D2 Receptor and 5HT2A Receptor]
[Procedure 1: Culture of HEK293 cells]
HEK293 cells obtained from ATCC (American Type Culture Collection) were cultured in Earle's MEM / Medium (GI) supplemented with 10% Fetal Bovine Serum and 1 × Nonesential amino acids in a 5% CO2 incubator. .

[Procedure 2: Introduction of protein expression plasmid]
HEK293 cells were seeded at a cell density of 2 × 10 ⁵ / dish in a 35 mm diameter glass bottom dish, cultured overnight in a 5% CO 2 incubator, and a plasmid pcDNA / cpGL4_C_RIα_αAB_N for expression of cyclic AMP sensor protein, human D1 receptor The expression plasmid pcDNA / D1R (or human D2 receptor expression plasmid pcDNA / D2R) and the human 5HT2A receptor expression plasmid pcDNA / 5HT2AR are mixed, and transfected using FuGENE HD (Roche). Cultured overnight in a% CO2 incubator. For pcDNA / D1R, pcDNA / D2R and pcDNA / 5HT2AR, those prepared in the same manner as in Example 1 were used.

[Procedure 3: Shooting a luminescent image]
After adding 2 mM luciferin (Promega) in the medium and allowing to stand for 1 hour, the culture dish was set on a luminescence microscope “LV (LUMINOVIEW) -200” (Olympus), and the luminescence images were displayed at 20-second intervals. Time-lapse photography was performed. As the light emission observation conditions, the magnification of the objective lens was 40 times, the exposure time was 10 seconds, and the binning was 2 × 2. An EM-CCD camera iXon (manufactured by Andor Co.) was used as the CCD camera 106c, and the luminescence image was taken into a personal computer configured as the image analysis device 110.

[Procedure 4: Taking a luminescent image by stimulation with serotonin and dopamine]
One minute after the start of time-lapse photography, stimulation was sequentially performed with serotonin (5HT: final concentration 10 μM) and dopamine (DA: final concentration 10 μM), and then time-lapse photography of the luminescence image was performed.

[Procedure 5: Graphical display of change in light emission over time]
As shown in the figure, a plurality of ROIs (Region of Interest) are designated for each light emission image (see FIGS. 20 and 24) taken before the stimulation, and each light emission image (FIG. 21) taken after the stimulation. 22, 25 and 26), a plurality of ROIs are designated as shown. The emission intensity of each designated ROI was measured based on each emission image, and the change over time of the measured emission intensity was displayed in a graph (see FIGS. 23 and 27). The analysis of the luminescence image was performed using Metamorph software (made by Universal Imaging) that functions as the image analysis unit 112c. In FIGS. 23 and 27, each graph corresponding to each of Cell1 to Cell4 is a time-dependent emission intensity of each ROI corresponding to each of numbers 1 to 4 in FIGS. 20 to 22 and FIGS. It shows a change.

[results of the experiment]
As a result of the above experiment, as shown in FIG. 6 to FIG. 9 and FIG. 20 to FIG. 23, the change in intracellular cyclic AMP response to stimulation to 5HT2A receptor, D1 receptor, or D2 receptor is at a single cell level. Detection was possible at (one cell level). In addition, when the change in luminescence intensity in individual cells shown in FIG. 9 and FIG. 23 is observed, in the combination of 5HT2A receptor and D1 receptor, the intracellular cyclic AMP concentration with respect to stimulation to 5HT2A receptor An increase was observed. However, as shown in FIGS. 11 to 14 and FIGS. 16 to 18 and FIGS. 24 to 27, in the combination of 5HT2A receptor and D2 receptor and in the case of 5HT2A receptor alone, against stimulation to 5HT2A receptor. No increase in intracellular cyclic AMP concentration was observed. From previous studies of the interaction between serotonin receptors and dopamine receptors, it is known that behaviors that are thought to be mediated through D1 receptors are induced through 5HT2A receptors in individuals. The results obtained by this experiment indicate that the response observed in individuals can be explained at the cellular level.

[Summary of this example]
As described above, according to this example, it is possible to obtain an accurate quantitative result from each cell without affecting intracellular information transmission. As a result, 5HT2A receptor and D1 receptor in each cell are obtained. The interaction with the body can be observed over time with good reproducibility. The luciferase gene, luciferin, etc. used in this example may be other commercially available reagents, or those extracted from organisms that generate bioluminescence and the like.

[Other embodiments]
In the above-described embodiment, mainly from the N-terminal luminescent enzyme and the C-terminal luminescent enzyme which are separated so that the luminescent enzyme activity is recovered by binding to each other, and the cyclic AMP binding domain of PKA-RIα. The fusion protein is described as a cyclic AMP sensor protein, but the present invention is not limited to this, and a fusion comprising an N-terminal luminescent enzyme, a C-terminal luminescent enzyme, and a cyclic AMP binding domain of PKA-RIα. The cyclic AMP sensor protein disclosed in Patent Document 1 and Patent Document 2 may be used instead of the protein.

[Example 3]
Various cyclic AMP sensor proteins were prepared and their usefulness was examined. Specifically, 16 cyclic AMP binding regions having different lengths and ranges were prepared, and for each, an N-terminal fragment, a cyclic AMP binding region and a C-terminal fragment were arranged in this order, and A total of 32 types of cyclic AMP sensor proteins were prepared, in which the C-terminal fragment, the cyclic AMP binding region, and the N-terminal fragment were arranged in this order. All of them were measured for enzyme activity in the presence and absence of cyclic AMP.

[Procedure 1]
N-terminal luminescent enzyme (NLuc) gene and C-terminal luminescent enzyme (CLuc) gene for creating split luciferase, and N-terminal luminescent enzyme (cpNLuc) gene and C-terminal luminescent enzyme for generating mutant split luciferase ( For the preparation of the (cpCLuc) gene, a synthetic oligo DNA (deoxyribonucleic acid) used for PCR (polymerase chain reaction) was prepared with the following sequence.
[Synthetic oligo DNA sequence for NLuc gene preparation]
NLuc_Fw (SEQ ID NO: 1): 5'-TGTGGATCCCAGCCACCCATGAAGATGCCCAA-3 '
NLuc_Rv (SEQ ID NO: 2): 5′-CAGCTCGAGGTCCTTGTCGATGAGAGCGTT-3 ′
[Synthetic oligo DNA sequence for CLuc gene preparation]
CLuc_Fw (SEQ ID NO: 3): 5′-ATCAGATCTGGCTACGTTAACAAACCCCGAG-3 ′
CLuc_Rv (SEQ ID NO: 4): 5′-CTAGAATTCTTACACGGCGATTCTGCCGCC-3 ′
[Synthetic oligo DNA sequence for cpNLuc gene preparation]
cpNLuc_Fw (SEQ ID NO: 13): 5′-ATCAGATCTGAAGATCGCAAAAAACATTAAG-3 ′
cpNLuc_Rv (SEQ ID NO: 14): 5′-CTAGAATTCTTAGTCCTTGTCGATGGAGAGC-3 ′
[Synthetic oligo DNA sequence for cpCLuc gene preparation]
cpCLuc_Fw (SEQ ID NO: 15): 5'-TGTGGATCCCCACCCACATGAGCGGCTACGTTAACAAACCCC-3 '
cpCLuc_Rv (SEQ ID NO: 16): 5′-CAGCTCGAGCACGGCGATCTTGCCGCCCT-3 ′

In addition, for the cloning of the PAMP-RIα cAMP binding domain gene, a synthetic oligo DNA used for PCR was prepared with the sequence shown below.
PKA_RIA_Xn1_Fw (SEQ ID NO: 19): 5'-ATTCTCGAGGATTATAAGACAATGGCTGCT-3 '
PKA_RIA_Xn2_Fw (SEQ ID NO: 20): 5′-GCCCTCGAGAAGAATGTGCTGTTTTCACAC-3 ′
PKA_RIA_α_Fw (SEQ ID NO: 21): 5′-TTTCTCGAGCTTGATGATAACGAGAGAAGT-3 ′
PKA_RIA_β1_Fw (SEQ ID NO: 22): 5′-ATTCTCGAGGCTAGTTTCCAGTCTCCTTT-3 ′
PKA_RIA_β2_Fw (SEQ ID NO: 23): 5′-TTTCTCGAGGGAGAGACGGTATTCAGCAA-3 ′
PKA_RIA_β3_Fw (SEQ ID NO: 24): 5′-GGTCTCGAGGGGGATAACTTCTATGTGATT-3 ′
PKA_RIA_β4_Fw (SEQ ID NO: 25): 5′-ATTCTCGAGGGAGAAATGGATGTCTATGTC-3 ′
PKA_RIA_β5_Fw (SEQ ID NO: 26): 5′-AATCTCGAGTGGGCAACCAGTGTTGGGGAA-3 ′
PKA_RIA_A_Rv (SEQ ID NO: 27): 5′-CTCAGATCTGTCCAGAGACTCTAAAAATA-3 ′
PKA_RIA_B_Rv (SEQ ID NO: 28): 5′-GGAAGATCTGACGGACAGGGAACGAAGCT-3 ′

[Procedure 2]
Then, using pGL4.10 (manufactured by Promega Corp.) as a template, the nucleic acid containing a luciferase gene fragment was amplified by PCR using the primer set shown in Table 1 below.

［手順３］
ＰＫＡの制御サブユニットＩα（ＰＫＡ−ＲＩα）にはｃＡＭＰ結合ドメインがＡとＢの２ヶ所あり、Ａのみを含む領域、または、ＡおよびＢの両方を含む領域を、マウスのＰＫＡ制御サブユニットＩα（ＰＫＡ−ＲＩα）のｃＤＮＡを鋳型とし、上記合成オリゴＤＮＡをプライマーとしてＰＣＲにより増幅した。使用したプライマーと増幅された遺伝子との関係を図２８に示す（図中、「遺伝子名」「使用したプライマー」「ＰＫＡ−ＲＩα中の対するアミノ酸の位置」「アミノ酸残基の数」が参照される）。

[Procedure 3]
The PKA regulatory subunit Iα (PKA-RIα) has two cAMP-binding domains, A and B. A region containing only A or a region containing both A and B is designated as the mouse PKA regulatory subunit Iα. Amplification was performed by PCR using (PKA-RIα) cDNA as a template and the above synthetic oligo DNA as a primer. The relationship between the used primer and the amplified gene is shown in FIG. 28 (see “gene name”, “primer used”, “position of amino acid in PKA-RIα”, “number of amino acid residues” in the figure). )

  SEQ ID NO: 31 shows the amino acid sequence of the entire PKA regulatory subunit Iα (PKA-RIα). SEQ ID NO: 32 describes the amino acid sequence corresponding to the RIα-Xn1A gene. SEQ ID NO: 33 describes the amino acid sequence corresponding to the RIα-Xn1AB gene. SEQ ID NO: 34 describes the amino acid sequence corresponding to the RIα-Xn2A gene. SEQ ID NO: 35 describes the amino acid sequence corresponding to the RIα-Xn2AB gene. SEQ ID NO: 36 describes the amino acid sequence corresponding to the RIα-αA gene. SEQ ID NO: 37 describes the amino acid sequence corresponding to the RIα-αAB gene. SEQ ID NO: 38 describes the amino acid sequence corresponding to the RIα-β1A gene. SEQ ID NO: 39 describes the amino acid sequence corresponding to the RIα-β1AB gene. SEQ ID NO: 40 describes the amino acid sequence corresponding to the RIα-β2A gene. SEQ ID NO: 41 describes the amino acid sequence corresponding to the RIα-β2AB gene. SEQ ID NO: 42 describes the amino acid sequence corresponding to the RIα-β3A gene. SEQ ID NO: 43 describes the amino acid sequence corresponding to the RIα-β3AB gene. SEQ ID NO: 44 describes the amino acid sequence corresponding to the RIα-β4A gene. SEQ ID NO: 45 describes the amino acid sequence corresponding to the RIα-β4AB gene. SEQ ID NO: 46 describes the amino acid sequence corresponding to the RIα-β5A gene. SEQ ID NO: 47 describes the amino acid sequence corresponding to the RIα-β5AB gene.

[Procedure 4] (Preparation of prokaryotic expression plasmids pRSET / GL4_N_C and pRSET / cpGL4_C_N)
The fragment containing the NLuc gene amplified by PCR was inserted between the BamHI site and the XhoI site in pRSET2B (manufactured by Invitrogen), a prokaryotic expression plasmid, and then the fragment containing the CLuc gene was inserted into the BglII site and EcoRI site. A prokaryotic expression plasmid (pRSET / GL4_N_C) was prepared by inserting between the sites.

  In addition, a fragment containing the cpCLuc gene amplified by PCR was inserted between the BamHI site and the XhoI site in pRSET2B (manufactured by Invitrogen), a prokaryotic expression plasmid, and then the fragment containing the cpNLuc gene was inserted into the BglII site. And a prokaryotic expression plasmid (pRSET / cpGL4_C_N).

[Procedure 5] (Preparation of fusion protein gene expression plasmid)
Nucleic acids containing various genes derived from PKA-RIα prepared by Procedure 3 were inserted between the XhoI site and the BglII site in the pRSET / GL4_N_C vector or pRSET / cpGL4_C_N vector prepared in Procedure 4. The plasmid thus obtained is shown in FIG. 28 (refer to “name of the resulting plasmid” in the figure).

[Procedure 6] (Expression and purification of fusion protein gene in E. coli)
The fusion protein expression plasmid produced as described above was transformed into E. coli JM109 (DE3) strain, and the fusion protein was purified using The QIA expressionist (manufactured by Qiagen) according to the manual of the kit. Briefly explaining the purification procedure, the prokaryotic expression plasmid was transformed into E. coli JM109 (DE3) strain and inoculated into 5 mL of LB medium. After overnight culture, the cells were collected by centrifugation, and the collected E. coli cells were resuspended in 500 μL of lysis buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysozyme was added (1 mg / mL). Incubated at 4 ° C for 60 minutes. Cells were gently vortexed and lysed, centrifuged at 15,000 xg for 10 minutes and the supernatant transferred to a new tube. 50 μL of 50% Ni-NTA resin suspension was added and gently mixed at 4 ° C. for 60 minutes. The resin was precipitated by centrifugation at 15,000 × g for 10 seconds, and the resin was washed twice with 500 μL of washing buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 20 mM imidazole, pH 8.0). The fusion protein was eluted twice with 50 μL of elution buffer (50 mM NaH 2 PO 4, 300 mM NaCl, 250 mM imidazole, pH 8.0) to obtain a fusion protein. The obtained fusion protein is shown in FIG. 28 (refer to “name of the obtained cyclic AMP sensor protein” in the figure).

[Procedure 7] (Characteristic measurement of fusion protein gene)
Luminescent activity of the fusion protein was measured in the presence and absence of cAMP, respectively. Briefly describing the measurement method, 20 μL of phosphate buffer (PBS) was added to 20 μL of the above fusion protein solution. Furthermore, 1 μL of 4 mM cAMP solution was added (final concentration 100 μM), and the same amount of PBS was added to the negative control. 40 μL of Bright-Glo (manufactured by Promega) was added, and the amount of luminescence (integrated for 10 seconds) was measured with Luminescence JNR-II (manufactured by ATTO).

[result]
As described above, the purified fusion protein was diluted in PBS, and the amount of luminescence with and without cAMP (final concentration 100 μM) was measured (FIG. 29). As a result, it was found that there exists a fusion protein whose luminescence activity increases due to the presence of cAMP. The difference in activity due to the presence or absence of cAMP was particularly large in GL4_N_RIα_β1AB_C (“β1AB” in FIG. 29a) and cpGL4_C_RIα_αAB_N (“αAB” in FIG. 29b).

  As described above, the receptor interaction detection method and the cyclic AMP sensor protein according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.

  In addition, as described above, the photoprotein and analysis method for detecting the interaction between the 5HT2A receptor and the D1 receptor according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine.

100 Luminescence observation system
103 container (petri dish)
104 stages
106 Luminous image pickup unit
106a Objective lens (for light emission observation)
106b Dichroic mirror
106c CCD camera
106d Split image unit
106e Filter wheel
106f Imaging lens
110 Image analyzer
112 Control unit
112a Luminous image capturing instruction unit
112b Luminescent image acquisition unit
112c Image analysis unit
112d Analysis result output part
114 Clock generator
116 storage unit
118 Communication interface
120 I / O interface section
122 Input device
124 Output device

Claims (17)

A receptor interaction detection method for detecting an interaction between a first receptor and a second receptor in a cell, comprising:
A production step of producing the cell containing a reporter serving as an index indicating the activity of the second receptor;
A treatment and an addition step of performing a predetermined treatment for causing the cells produced in the production step to emit light from the outside of the cell and adding a predetermined receptor-binding substance that binds to the first receptor;
A measurement step of measuring the amount of luminescence of the cells after the treatment and the addition step are performed;
An analysis step of analyzing the activity of the second receptor in the cell based on the amount of luminescence measured in the measurement step;
A detection step of detecting the interaction between the first receptor and the second receptor based on the activity of the second receptor analyzed in the analysis step;
A receptor interaction detection method comprising: In the production step, the reporter is a cyclic AMP sensor protein,
In the analyzing step, the activity of the second receptor is a concentration of cyclic AMP.
The method for detecting receptor interaction according to claim 1. The cyclic AMP sensor protein emits light by interacting with the cyclic AMP in the cell,
The method for detecting a receptor interaction according to claim 2. The cyclic AMP sensor protein is a fusion protein comprising a firefly luciferase and a cyclic AMP binding region of PKA (protein kinase A);
The method for detecting a receptor interaction according to claim 2 or 3, wherein:   5. The receptor interaction detection method according to claim 4, wherein the cyclic AMP binding region is derived from mouse PKA-RIα.   6. The receptor interaction detection method according to claim 4 or 5, wherein the cyclic AMP binding region has a length of 220 to 250 amino acid residues.   The receptor interaction detection method according to any one of claims 4 to 6, wherein the cyclic AMP binding region includes at least two cyclic AMP binding sites.   The cyclic AMP sensor protein includes an N-terminal fragment of the luciferase, a C-terminal fragment of the luciferase, and the cyclic AMP binding region, and the N-terminal fragment and the C-terminal fragment are the cyclic AMP. The method for detecting a receptor interaction according to any one of claims 4 to 7, wherein the luminescent enzyme activity of the luciferase recovers when bound in a sensor protein molecule.   The method for detecting a receptor interaction according to any one of claims 4 to 8, wherein the cyclic AMP-binding region comprises the amino acid sequence shown in SEQ ID NO: 37 or 39. In the production step, the cell contains the first receptor and the second receptor;
The method for detecting a receptor interaction according to any one of claims 1 to 9. In the production step, the cell is a cell into which the first receptor gene and the second receptor gene have been introduced;
The method for detecting a receptor interaction according to any one of claims 1 to 9. The measuring step further includes a photon amount measuring step of measuring a photon amount of the luminescent label depending on the activity of the second receptor;
The method for detecting a receptor interaction according to any one of claims 1 to 11, wherein: The measuring step further includes an imaging step of imaging a luminescent label depending on the activity of the second receptor;
The method for detecting a receptor interaction according to any one of claims 1 to 11, wherein: The imaging step is performed simultaneously on a plurality of different cells,
The analysis step further includes a collation step of collating a plurality of luminescent images captured in the imaging step for each cell;
The method for detecting a receptor interaction according to claim 13. The measurement step is repeatedly performed;
The method for detecting a receptor interaction according to any one of claims 1 to 14, wherein: The first receptor is a 5HT2A receptor and the second receptor is a D1 receptor;
The method for detecting a receptor interaction according to any one of claims 1 to 15, wherein: In the treatment and addition step, the receptor binding substance is serotonin;
The receptor interaction detection method according to claim 16.

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