JP2004248506A - Constitutively translocating cell line - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying compounds altering internalization of a GPCR (G protein-coupled receptor). <P>SOLUTION: An agonist-independent method for screening for compounds altering the desensitization of the GPCR is provided. A GRK (G protein-coupled receptor kinase) desensitizing the GPCR in the absence of an agonist and a cell line modifying the GRK are included. The method for identifying whether or not the GPCR is expressed with a plasmic membrane and the GPCR has an affinity for arrestin is provided. Furthermore, a modified GPCR having an increased affinity for the arrestin is included. The modified GPCR is useful for methods for screening for the compounds altering the desensitization comprising the agonist-independent method and the agonist-dependent method described in the specification. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【表１（つづき）】

【図面の簡単な説明】
【図１Ａ】本発明で使用することができるＧＰＣＲのリストである。
【図１Ｂ】本発明で使用することができるＧＰＣＲのリストである。
【図１Ｃ】本発明で使用することができるＧＰＣＲのリストである。
【図２】本発明で使用することができるＧＲＫのリストである。一定のＧＲＫのアミノ酸配列および核酸配列を示す。ＧＲＫ２−Ｃ２０（改変ＧＲＫ）のアミノ酸配列および核酸配列を示す。
【図３】アレスチンに対する親和性が増強されたＧＰＣＲのリストである。アミノ酸配列および核酸配列を示す。
【図４】ＧＲＫ２−Ｃ２０の存在下でのアレスチンＧＦＰのＧＰＣＲへのアゴニスト非依存的トランスロケーションを示す図である。
【図５】ＧＲＫ２−Ｃ２０の存在下でのアレスチンＧＦＰのＧＰＣＲへのアゴニスト非依存的トランスロケーションを示す図である。
【図６】ＧＲＫ２−Ｃ２０の存在下でのアレスチンＧＦＰのＧＰＣＲへのアゴニスト非依存的トランスロケーションを示す図である。
【図７】ＧＲＫ２−Ｃ２０の存在下でのアレスチンＧＦＰのＧＰＣＲへのアゴニスト非依存的トランスロケーションを示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to assays for GPCR desensitization in an agonist-independent manner, host cells useful for such methods, methods for identifying compounds that alter GPCR desensitization, identified compounds, and diseases. It relates to the use of these compounds in therapy.
[0002]
[Prior art]
G protein-coupled receptors (GPCRs) are cell surface proteins that translate hormone or ligand binding into intracellular signals. GPCRs are found in all animals, insects, and plants. GPCR signaling plays a central role in regulating a variety of physiological functions including light signaling, smell, neurotransmission, vascular tone, cardiac output, digestion, pain, fluid and electrolyte balance. Despite these being related to many physiological functions, GPCRs share many common features. These include seven membrane domains that are cross-linked by alternating intracellular and extracellular loops and intracellular carboxyl-terminal tails of various lengths.
[0003]
GPCRs are used in a number of disease states (angina, essential hypertension, myocardial infarction, supraventricular and ventricular arrhythmias, congestive heart failure, atherosclerosis, renal failure, diabetes, respiratory indications (such as asthma) ), Chronic bronchitis, bronchospasm, emphysema, airway obstruction, upper respiratory indications (rhinitis, etc.), seasonal allergies, inflammatory diseases, inflammation in the injury reaction, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, anti- Gastrinemia, gastrointestinal indications (acid / digestive disorders, etc.), erosive esophagitis, gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux, peptic ulcer, Zollinger-Ellison syndrome, pain, obesity, nervous gluttony Disorders, depression, threatening disorders, organ malformations (eg, heart malformations), neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), multiple sclerosis, Epstein-Barr infection, and cancer. Murrell is associated with but not limited to).
[0004]
The magnitude of the physiological response regulated by a GPCR is related to the balance between GPCR signaling and signal termination. GPCR signaling is regulated by a family of intracellular proteins called arrestins. Arrestin binds to activated GPCRs, including those that are agonist activated and especially those that are phosphorylated by G protein-coupled receptor kinase (GRK).
[0005]
Receptors, including GPCRs, have historically been targeted for drug discovery and therapeutics because they bind ligands, hormones, and drugs with high specificity. Approximately 15% of the therapeutics used today are directly targeted or interact with GPCRs. See, for example, Jurgen Drews, 2000, "Drug Discovery: A Historical Perspective", Science, 287, 1960-1964.
[0006]
There is a need for an accurate and easy to interpret method of G protein-coupled receptor activity and an assay for GPCR activity. One method disclosed in U.S. Patent Nos. 5,891,646 and 6,110,693 to Barak et al., Which is incorporated herein by reference, A conjugate of arrestin and a detectable molecule is used.
[0007]
Although only a few hundred human GPCRs are known, it is estimated that thousands of GPCRs are present in the human genome. Of these known GPCRs, many are orphan receptors that are not yet associated with a ligand.
[0008]
Most existing methods of identifying GPCR antagonists rely on the presence of an agonist. Compound identification assays that prevent the action of GPCRs typically require that the GPCRs be first activated to identify interfering compounds. For receptors with known agonists, these agonists are currently being used to activate these receptors. However, many GPCRs are orphan receptors without known ligands or agonists.
[0009]
The agonist dependence of GPCR assays continues to be problematic, as antagonist discovery of orphan receptors typically relies on prior discovery of agonists or ligands. An agonist-independent screening method for a compound that alters GPCR desensitization can eliminate (1) an agonist addition step in the screening method and (2) identify a compound that alters orphan receptor desensitization It becomes. Agonist-independent methods eliminate the step of identifying an orphan receptor agonist prior to screening for compounds that alter orphan receptor desensitization.
[0010]
[Problems to be solved by the invention]
The present invention relates to methods for identifying compounds that alter GPCR internalization.
[0011]
[Means for Solving the Problems]
A first aspect of the present invention is a method for identifying a compound that alters the internalization of a GPCR, comprising: (a) a cell containing a GPCR, an arrestin, and a modified GRK, wherein the GPCR expresses the GRK; Obtaining a cell characterized in that it is at least partially internalized in an agonist-independent manner; (b) exposing the cell to the compound; and (c) exposing the GPCR, arrestin, or Determining the cellular distribution of the modified GRK; (d) (1) the cellular distribution of the GPCR, arrestin, or modified GRK in cells in the presence of the compound and (2) the cellular distribution of the modified GRK in the absence of the compound. Monitoring the difference of the GPCR, arrestin, or modified GRK from the cell distribution in the cells. An agonist cannot be obtained by the above method. In this way, the difference between (1) and (2) in step (d) may indicate a modulation of GPCR internalization.
[0012]
GRKs can be overexpressed, the expression of which is inducible and can include a CAAX motif. The GRK can be GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, or a biologically active fragment thereof.
[0013]
GPCRs can be modified such that phosphorylation is enhanced by GRK. GPCR is β ₂ AR (Y326A), a GPCR listed in FIG. 1, an orphan GPCR, a modified GPCR, a taste receptor, a class A GPCR, a class B GPCR, a mutant GPCR, or a biologically active fragment thereof.
[0014]
The arrestin may be visual arrestin, cone arrestin, β arrestin 1, β arrestin 2, or a biologically active fragment thereof.
[0015]
GPCRs, GRKs, and arrestins can be detectably labeled. Molecules associated with desensitization can be detectably labeled, or molecules that interact with molecules associated with desensitization can be detectably labeled.
[0016]
In a further aspect, the present invention is a method for identifying a compound that alters phosphorylation of a GPCR, comprising: (a) obtaining a cell comprising a GPCR and GRK; and (b) exposing the cell to the compound. (C) identifying whether GRK phosphorylation of a GPCR is altered in the presence of said compound.
[0017]
The cellular distribution of the GPCR or GRK can be determined. The difference between (1) the cellular distribution of GPCR or GRK in cells in the presence of the compound and (2) the cellular distribution of GPCR or GRK in cells in the absence of the compound can be monitored. The difference between (1) and (2) can be correlated with GPCR phosphorylation.
[0018]
GRK was not present in the plasma membrane, which may indicate that GRK phosphorylation of the GPCR has changed. The phosphorylation status of the GPCR can be identified. GRK activity can be identified. The internalization ability of the GPCR can be identified.
[0019]
In a further aspect, the invention provides a method of identifying whether a GPCR is expressed at the plasma membrane, comprising: (a) a cell comprising a GPCR, arrestin, and GRK, wherein said arrestin is detectably labeled. (B) determining the cell distribution of the arrestin; and (c) correlating the cell distribution of the arrestin with the ability of the GPCR to express at the plasma membrane. A method comprising: Arrestin can be located in vesicles, pits, endosomes, or elsewhere in the desensitization pathway.
[0020]
Further, the present invention is a method for identifying whether a GPCR is expressed at the plasma membrane, comprising: (a) a GPCR, a GRK, and a cell containing the GRK, wherein the GRK is detectably labeled. Obtaining a cell characterized by the following steps: (b) determining the cell distribution of the GRK; and (c) correlating the cell distribution of the GRK with the ability of the GPCR to be expressed at the plasma membrane. For further methods. GRK may be located at the plasma membrane.
[0021]
In a further aspect, the present invention is a method for analyzing the arrestin binding capacity of a GPCR, comprising: (a) a cell comprising a GPCR, arrestin, and GRK, wherein said arrestin is detectably labeled. (B) determining the cell distribution of the arrestin; and (c) correlating the cell distribution of the arrestin with the arrestin binding ability of a GPCR. Arrestin or GPCRs can be located in vesicles, pits, or endosomes.
[0022]
In a further aspect, the invention relates to a compound identified by the method of the invention.
[0023]
In a further aspect, the invention provides a method of treating a disease by modulating the desensitization of a GPCR in a host cell, comprising: (a) obtaining a compound identified by the method of the invention; Administering the compound to a host.
[0024]
Another aspect of the invention pertains to a host cell comprising a GPCR and a modified GRK. GRKs can be inducible or overexpressed. The host cell can further include arrestin, and the arrestin can be detectably labeled. A GPCR, GRK, another molecule associated with desensitization, or a molecule that interacts with a molecule associated with desensitization can be detectably labeled.
[0025]
A further aspect of the present invention is a method of modifying a nucleic acid encoding GRK that is constitutively internalized by a GPCR, comprising: (a) obtaining a nucleic acid encoding GRK; and (b) a nucleic acid encoding the GRK. Modifying the encoded GRK to include a CAAX motif, wherein the modified GRK phosphorylates a GPCR in the presence of an agonist, and (c) expressing the modified GRK in a cell. Involving methods. The nucleic acid encoding GRK may include SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
[0026]
The invention also relates to kits for identifying compounds that modulate GPCR internalization, including host cells containing GPCRs and modified GRKs.
[0027]
In a further aspect, the invention includes a NPXXXY motif and a carboxyl terminal tail, wherein the carboxyl terminal tail comprises a putative palmitoylation site and one or more phosphorylation clusters, and wherein the carboxyl terminal tail is derived from a second GPCR. A second GPCR comprising one or more phosphorylation clusters, wherein the second GPCR comprises one or more phosphorylation clusters, wherein the second GPCR comprises one or more phosphorylation clusters, wherein the second GPCR comprises one or more phosphorylation clusters; A modified GPCR further comprising a second putative palmitoylation site 25 amino acid residues downstream. The first GPCR may be a class A receptor. The first GPCR can be hGPR3, hGPR6, hGPR12, hSREB2, hSREB3, hGPR8, or hGPR22. The second GPCR may be a class B receptor. The class B receptor can be selected from the group consisting of a vasopressin V2 receptor, a neurotensin 1 receptor, a substrate P receptor, and an oxytocin receptor.
[0028]
The present invention relates to nucleic acids encoding modified GPCRs. The present invention relates to SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. The invention also includes an expression vector comprising the nucleic acid. Also included are host cells containing the vector or nucleic acid.
[0029]
In a further aspect, the invention is a method of screening a compound for GPCR activity, comprising: (a) obtaining a cell expressing at least one modified GPCR, wherein the cell is conjugated to a detectable molecule ( conjugate to) arrestin, (b) exposing the cells to the compound, (c) detecting the location of arrestin in the cells, and (d) intracellularly in the presence of the compounds. Comparing the position of arrestin with the position of arrestin in the cell in the absence of the compound; (e) (1) the position of arrestin in the cell in the presence of the compound; Correlating the difference with the location of arrestin in the cell in the absence of the compound. Arrestin can be detected in endosomes, endocytic vesicles, or pits.
[0030]
A further aspect of the invention modulates the activity of a GPCR, comprising a cell expressing at least one modified GPCR, wherein said cell further comprises a molecule associated with desensitization conjugated to a detectable molecule. It is a kit for identifying molecules.
[0031]
BEST MODE FOR CARRYING OUT THE INVENTION
The objects and advantages of the present invention will be understood by reading the following detailed description in conjunction with the accompanying drawings.
[0032]
According to the present invention, conventional molecular biology, microbiology, immunology, and recombinant DNA techniques within the skill of the art can be used. Such techniques are explained fully in the literature. See, for example, Sambrook et al., "Molecular Cloning: An Experimental Manual", Third Edition, 2001; "Modern Molecular Biology Protocols", Volumes I-IV, Ausubel, R .; M. Ed., 2002 (revised every other month); Cell Biology: An Experimental Handbook, Volumes I-III, J. Am. E. FIG. Ed. Cellis, 1994; "Modern Immunology Protocol", Volumes I-IV, Coligan, J. et al. E. FIG. Ed., 2002 (revised every other month); J. Gait, 1984; "Nucleic acid hybridization", B.A. D. Hames & S.M. J. Higgins eds., 1985; D. Hames & S. J. Higgins eds., 1984; "Animal Cell Culture, 4th Edition", R.M. I. Freshney ed., 2000; "Fixed cells and enzymes", IRL Press, 1986; Perbal, "Molecular Cloning Practice Guide", 1988; "Using Antibodies: Experimental Manual: Portable Protocol", Volume 1, Harlow Ed and Lane, David, Cold Spring Harbor Press, 1998; "Using Antibodies: Experimental Manual", Harlow Ed and Lane, David, Cold Spring Harbor Press, 1999; "G-protein coupled receptors"; See Haga et al., 1999.
[0033]
Unless defined otherwise, the following terms used in the specification and claims have the following meanings.
[0034]
A “replicon” is any genetic element (eg, a plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo (ie, capable of replicating under its own control).
[0035]
A “vector” is a replicon, such as a plasmid, phage, cosmid, or the like, into which another DNA segment can be ligated so that the ligated segment can replicate.
[0036]
"DNA molecule" refers to a polymeric deoxyribonucleotide (adenine, guanine, thymine, or cytosine), either in single-stranded form or double-stranded helix. The term refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary structure. Thus, the term includes double-stranded DNA found, inter alia, in linear DNA (eg, restriction fragments), viruses, plasmids, and chromosomes. Consideration of a particular double-stranded DNA molecule follows the general convention of showing only the 5 ′ → 3 ′ sequence along the non-transcribed strand of DNA (ie, the strand having a sequence homologous to mRNA). The sequence can be described therein.
[0037]
"Origin of replication" refers to a DNA sequence that is involved in the initiation of DNA synthesis.
[0038]
A “coding sequence” of DNA is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (eg, mammalian) DNA, and even synthetic DNA sequences. The polyadenylation signal and transcription termination sequence are usually located 3 'to the coding sequence.
[0039]
Transcriptional and translational regulatory sequences are DNA regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, etc. that express the coding sequence in a host cell.
[0040]
Expression of the coding sequence in the host cell may be inducible. "Inducible" means that expression can be regulated. For example, a nucleic acid can be present in a cell but is not expressed until the required signal is obtained. Typically, inducible expression of a protein is regulated by a promoter that requires the signals necessary for inducible transcription of the protein. However, expression can also be induced by processes or sequences that increase the number of DNA sequences of interest in the cell. Such processes or sequences include the origin of replication and the physical addition of DNA to cells.
[0041]
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 'direction) coding sequence. For the purposes of defining the present invention, a promoter sequence is linked to the 3 'end by a transcription initiation site and upstream (5') so as to induce the minimum number of bases or elements required to initiate transcription at detectable levels above background. 'Direction). The promoter sequence contains a transcription initiation site (conveniently defined by mapping with nuclease S1) and a protein binding domain (consensus sequence) responsible for RNA polymerase binding. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalcarno sequences in addition to the -10 and -35 consensus sequences.
[0042]
An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under control" of the transcriptional and translational regulatory sequences when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
[0043]
A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide (the N-terminus of the polypeptide) that directs the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the medium, which signal peptide causes the protein to be released from the cell. Before being cut off by the host cell. Signal sequences related to various prokaryotic and eukaryotic proteins can be found.
[0044]
As used herein, the term "oligonucleotide" refers to a probe of the invention and is defined as a molecule comprising two or more, preferably more than three, ribonucleotides. Its exact size depends on a number of factors, in other words, on the ultimate function and use of the oligonucleotide.
[0045]
As used herein, the term "primer" refers to a condition that is naturally occurring or synthesized as a purified restriction digest, and that induces the synthesis of a primer extension product complementary to a nucleic acid strand (ie, nucleotides and Oligonucleotide that can act as a starting point for synthesis when placed in the presence of an inducer such as a DNA polymerase and at the appropriate temperature and pH). Primers, which can be single-stranded or double-stranded, must be sufficiently long to prime the synthesis of desired extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. For example, for diagnostics that depend on the complexity of the target sequence, oligonucleotide primers typically contain 15-25 or more nucleotides, but can contain fewer nucleotides.
[0046]
The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primer must be sufficiently complementary to hybridize with each of its strands. Thus, the primer sequence need not accurately reflect the template sequence. For example, a non-complementary nucleotide fragment can be attached to the 5 'end of the primer, making the remaining sequence of the primer complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed with the primer if the primer sequence has sufficient complementarity with the sequence of the hybridizing strand to form a template for the synthesis of extension products.
[0047]
As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
[0048]
A cell has been "transformed" by exogenous or endogenous DNA when such DNA has been transferred inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells, for example, transforming DNA can be maintained with episomal elements such as plasmids. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome such that it is passed on to daughter cells via chromosomal replication. This stability is evidenced by the ability to establish cell lines or clones comprised of a population of daughter cells containing eukaryotic transforming DNA. A "clone" is a population of cells from a single cell or mitotic common ancestor. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
[0049]
Two DNA sequences are "substantially homologous" if at least about 65% (preferably at least about 80%, most preferably at least about 90% or 95%) of the nucleotides match the DNA sequence of the defined length. is there. Substantially homologous sequences can be identified by sequence comparison using standard software available in the sequence databank or by, for example, Southern hybridization experiments under stringent conditions defined in the particular system. . Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., Supra, "DNA Cloning", Volumes I and II, supra, "Nucleic Acid Hybridization", supra.
[0050]
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, Encodes the same amino acid sequence as 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89 Not only the DNA sequence but also SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90 It should be appreciated that overlapping sequences are also within the scope of the invention. "Degenerate to" means identifying a particular amino acid using different three letter codons.
[0051]
"Arrestin" means all forms of naturally occurring arrestin or arrestin variants created by genetic engineering, including visual arrestin (sometimes called arrestin 1), retinal cone arrestin (arrestin 4 and arrestin 4). Arrestin 1 (sometimes called arrestin 2), and β arrestin 2 (sometimes called arrestin 3).
[0052]
“ΒARK1” is GRK (also called GRK2) for β-adrenergic receptor kinase 1.
[0053]
“ΒAR” is a GPCR that refers to a β-adrenergic receptor.
[0054]
“Internalization” of GPCR is translocation from a cell surface membrane to an intracellular vesicle membrane where a substance remaining outside the cell is difficult to access.
[0055]
"Carboxy-terminal tail" means the carboxy-terminal tail of the GPCR after membrane span 7. The carboxy-terminal tail of many GPCRs begins immediately after the conserved NPXXXY motif, which marks the end of the seven transmembrane domain (ie, the sequence following the NPXXXY motif is the carboxy-terminal tail of the GPCR). The carboxy-terminal tail can be relatively long (about thousands of amino acids), relatively short (about tens of amino acids), or virtually non-existent (less than about 10 amino acids). As used herein, “carboxyl-terminal tail” refers to all three variants (longer, shorter, or substantially non-existent) and includes palmitoylated cysteine residues. It may or may not be included.
[0056]
Preferably, the "class A receptor" is not translocated to the intracellular vesicles or endosomes associated with arrestin-GFP with the arrestin protein in HEK-293 cells.
[0057]
"Class B receptors" are preferably translocated with arrestin proteins to intracellular vesicles or endosomes associated with arrestin-GFP in HEK-293 cells.
[0058]
"DAC" means any desensitizing active compound. A desensitizing active compound is any compound that affects a GPCR desensitizing mechanism by stimulating or inhibiting a process. DACs include any cellular component of the process and any cellular structure associated with the process, including but not limited to arrestin, GRK, GPCR, phosphoinositide 3-kinase, AP-2 protein, clathrin, protein phosphatase, and the like. ) May affect the GPCR desensitization pathway. DACs include compounds that inhibit arrestin translocation to GPCR, compounds that inhibit arrestin binding to GPCR, compounds that stimulate arrestin translocation to GPCR, compounds that stimulate arrestin binding to GPCR, GPCR Compound that inhibits GRK phosphorylation of GPCR, compound that stimulates GRK phosphorylation of GPCR, compound that stimulates or inhibits binding of GRK to GPCR, compound that inhibits protein phosphatase desensitization of GPCR, protein phosphatase desensitization of GPCR Stimulation may include, but is not limited to, compounds that prevent GPCR internalization or recirculation to the cell surface, release of arrestin from GPCRs, compounds that modulate the release of GPCR antagonists, inverse agonists, and the like. A DAC can inhibit or stimulate the GPCR desensitization process and need not bind to the same GPCR ligand binding site as the GPCR transient agonist and antagonist. A DAC can act independently of a GPCR (ie, the DAC does not exhibit high affinity for one particular GPCR or one particular type of GPCR). DACs can bind to the same sites as agonists or antagonists, but do not desensitize the receptor (perhaps by not phosphorylating the receptor at all or binding to arrestin or any other protein). DACs can bind to the allosteric site of the receptor to inhibit or enhance desensitization.
[0059]
"Detectable molecule" refers to spectroscopic, photochemical, biochemical, immunochemical, electrical, radioactive, and optical means, including, but not limited to, fluorescence, phosphorescence, and bioluminescence, radioactive decay. Means any molecule that can be detected by Detectable molecules include, but are not limited to, GFP, luciferase, β-galactosidase, rhodamine binding antibodies, and the like. Detectable molecules include radioisotopes, epitope tags, affinity labels, enzymes, fluorophores, chemiluminescers, and the like. Detectable molecules include those that are detected indirectly or directly as a function of interaction with another molecule.
[0060]
"GFP" means green fluorescent protein and refers to various naturally occurring forms of GFP that can be isolated or engineered from natural sources as well as artificially modified GFP. GFP is well known in the art. See, for example, U.S. Patent Nos. 5,625,048, 5,777,079, and 6,066,476. Other fluorescent proteins in which GFP is isolated or engineered from natural sources (yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), blue fluorescent protein, luciferin, UV excitable It is well understood in the art that they are readily interchangeable with, but not limited to, fluorescent proteins, or proteins of a wavelength in between. As used herein, "GFP" refers to all fluorescent proteins known in the art.
[0061]
"Unknown or orphan receptor" means a GPCR whose ligand is unknown.
[0062]
"Downstream" means the carboxyl-terminal side of the amino acid sequence with respect to the amino terminus.
[0063]
"Upstream" means the amino-terminal side of the amino acid sequence with respect to the carboxyl terminus.
[0064]
Amino acid substitutions for substituting amino acids with particularly preferred properties can be introduced. For example, a Cys can be introduced at a potential site that forms a disulfide bond with another Cys. His can be introduced as a specific “catalytic” residue (ie, His can act as an acid or base and is the most common amino acid in biocatalysis). Pro can be introduced for certain planar structures that include β-turns in the protein structure.
[0065]
Two amino acid sequences are "substantially homologous" if at least about 70% (preferably at least about 80%, most preferably at least about 90% or 95%) of the amino acid residues are identical or exhibit conservative substitutions. ".
[0066]
A "heterologous" region of a DNA construct is an identifiable DNA segment within a larger DNA molecule that is not originally found in association with the larger molecule. Thus, when the heterologous region encodes a mammalian gene, the gene is usually flanked by DNA that is not adjacent to mammalian genomic DNA in the genome of the original organism. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (eg, a genomic coding sequence that contains introns or a synthetic sequence that has codons that differ from the native gene). Allelic variation or naturally occurring mutational events do not result in a heterologous region of DNA as defined herein.
[0067]
The term "pharmaceutically acceptable" refers to molecular components that are physiologically resistant or typically do not produce an allergic or similar untoward reaction (such as gastric upset) when administered to a human and Refers to a composition.
[0068]
As used herein, the term “therapeutically effective amount” refers to an amount sufficient to prevent and preferably alleviate some pathological features (eg, increased blood pressure, respiratory output, etc.). means.
[0069]
A DNA sequence is "operably linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of the DNA sequence. The term "operably linked" includes having an appropriate initiation signal (eg, ATG) before the DNA sequence to be expressed and expressing the DNA sequence under the control of expression control sequences to encode the DNA sequence. And maintaining the correct reading frame to produce the desired product. If the gene desired to be inserted into the recombinant DNA molecule does not contain a suitable start signal, such a start signal can be inserted before this gene.
[0070]
"Hybridization" refers to hydrogen bonds that may be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds between complementary nucleosides or nucleotide bases. For example, adenine (A) and thymine (T) are complementary nucleotide bases that pair via hydrogen bonds.
[0071]
The term “standard hybridization conditions” refers to salt and temperature conditions substantially equal to 5 × SSC and 65 ° C. for both hybridization and washing. However, those skilled in the art will recognize that such "standard hybridization conditions" will depend on particular conditions, including sodium and magnesium concentrations in the buffer, nucleotide sequence length and concentration, mismatch rates, formamide percentages, and the like. Recognize that. Whether the two sequences hybridizing are RNA-RNA, DNA-DNA, or RNA-DNA is also important in determining "standard hybridization conditions". One of ordinary skill in the art will appreciate that such standard hybridization conditions can be hybridized using a higher stringency wash, if desired, at a temperature typically 10-20 ° C below the expected or determined Tm. Determine easily according to the format.
[0072]
"Animal" means any component of the animal kingdom, including vertebrates (eg, frogs, salamanders, chickens, or horses) and invertebrates (eg, helminths, etc.). "Animal" is also meant to include "mammals." Preferred mammals include domestic animals (eg, ungulates such as cows, buffalo, horses, sheep, pigs, and goats), rodents (eg, mice, hamsters, rats, and guinea pigs), dogs, cats, primates. Includes species, wolves, camels, deer, rodents, birds, and fish.
[0073]
"Antagonist" includes wild-type and / or modified GPCR binding to agonist, wild-type and / or modified GPCR desensitization, wild-type and / or modified GPCR-binding arrestin, wild-type and / or modified GPCR endosomal localization , All factors that inhibit internalization, etc., including those that affect wild-type and / or modified GPCRs and wild-type and / or modified GPCR signaling, desensitization, endosomal localization, Factors affecting other proteins involved in resensitization and the like are included.
[0074]
"Modified GPCR" means a GPCR having one or more modifications in the amino acid sequence of its carboxyl-terminal tail. Thus, all or part of the carboxy terminal tail can be modified. These alterations in amino acid sequence include mutations in one or more amino acids, insertion of one or more amino acids, deletion of one or more amino acids, and deletion and deletion of one or more amino acids. It includes one or more amino acid substitutions in which one or more amino acids are added in place of missing amino acids. Such modified GPCRs are described herein and in US patent application Ser. No. 09 / 993,844, which is hereby incorporated by reference.
[0075]
"GPCR" means a G protein-coupled receptor, which includes naturally occurring GPCRs and modified GPCRs.
[0076]
"Putative palmitoylation site" means a predicted palmitic acid addition site (preferably a cysteine residue). In the GPCRs used in the present invention, the putative palmitoylation site is located preferably 10-25 amino acid residues downstream of the NPXXXY motif, preferably 15-20 amino acid residues.
[0077]
“Cluster of phosphorylation sites” means a cluster of amino acid residues that can easily function as phosphorylation sites through efficient phosphorylation. Amino acid clusters were identified in the carboxyl-terminal tail of the GPCR as 2, 2, 3, 3, 4, 4, 4, 4, 5, 5 out of 5, in the carboxyl-terminal tail of the GPCR. Occupies five or more consecutive amino acid positions. The cluster of these phosphorylation sites is preferably a cluster of serine (S) and / or threonine (T) residues. A cluster of phosphorylation sites can be substituted, inserted, or added to a GPCR sequence such that the resulting modified GPCR binds arrestin with sufficient affinity to utilize arrestin in the endosome.
[0078]
"NPXXXY motif" means a conserved amino acid motif that marks the end of a seven transmembrane domain. Conserved amino acid motifs most often begin with asparagine and proline, followed by two unspecified amino acids and continuing to tyrosine. The two unspecified amino acids can vary greatly between GPCRs, but the entire NPXXXY motif is conserved.
[0079]
“Abnormal GPCR desensitization” and “abnormal desensitization” are caused by disruption of the GPCR desensitization pathway such that the balance between active and desensitized receptors changes in wild-type conditions. Means that Some receptors are more active than normal, or some are more desensitized than wild-type conditions. Aberrant GPCR desensitization is constitutively active or constitutively desensitized, resulting in increased signaling of the receptor or decreased signaling of the receptor than normal GPCR results.
[0080]
A "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids that are present in a subject. It can be a sample, a stool sample, a tumor sample, a cell wash, an oral sample, saliva, a biological fluid, a tissue extract, freshly harvested cells, or cells incubated in tissue culture.
[0081]
"Immediate administration", "combination administration", "co-administration", or "administer simultaneously" results in the compounds being administered simultaneously or essentially as if two or more compounds were administered simultaneously. Administration in a sufficiently short time.
[0082]
“Conservative abnormalities” include, but are not limited to, abnormalities in the GPCR pathway, such as those that can be caused by abnormal CPGR signaling, such as GPCRs, GRKs, arrestins, AP-2 proteins, clathrins, protein phosphatases ). This abnormal GPCR signaling can contribute to GPCR-related diseases.
[0083]
"Desensitized GPCR" means a GPCR that no longer has the ability to respond to agonists and activate conventional G protein signaling.
[0084]
"Desensitization pathway" means any cellular component of the desensitization process and the cellular structure involved in the desensitization and subsequent processes, including arrestin, GRK, GPCR, AP-2 protein, Include, but are not limited to, clathrin, protein phosphatase, and the like. In the assay of the present invention, the polypeptide can be detected at any stage between them, such as in the cytoplasm, in the cell membrane, in clathrin-coated pits, in intracellular formulations, in endosomes, and the like.
[0085]
"GPCR signaling" means GPCR-induced activation of a G protein. Thereby, for example, cAMP can be produced.
[0086]
"G protein-coupled receptor kinase" (GRK) includes any kinase capable of phosphorylating a GPCR. Certain GRKs that can be used in the present invention are listed in FIG. Includes splice variants, biologically active fragments, modified GRKs, and GRKs from animals and other organisms.
[0087]
"Homo sapiens GPCR" means a GPCR that occurs naturally in Homo sapiens.
[0088]
"Inverse agonist" refers to a compound that, upon binding to a GPCR, inhibits the basic intrinsic activity of the GPCR. Inverse agonists are certain antagonists.
[0089]
"Modified GRK" means a GRK that has been modified to alter desensitization.
[0090]
"Naturally occurring GPCR" means a GPCR that occurs in nature.
[0091]
By "odorant ligand" is meant a ligand compound that perceives an odor, including synthetic and / or recombinantly produced compounds, upon binding to the receptor, including agonist and antagonist molecules.
[0092]
"Odorant receptor" means a receptor that is normally found on the surface of olfactory neurons that, when activated (usually due to the binding of an odorant ligand), senses odor.
[0093]
As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or excipient, such as a liquid or solid charge associated with the delivery or delivery of a chemical. Agents, diluents, excipients, solvents or encapsulating materials).
[0094]
“Sensitized GPCR” means a GPCR that has the ability to activate conventional G protein signaling at present in response to an agonist.
[0095]
"Modulation" includes at least up-regulation or down-regulation of expression or an increase or decrease in protein activity. Modulation of a protein includes up-regulating, down-regulating, increasing, or decreasing the activity of the protein or a compound that regulates the protein. Modulation also includes control of the gene, mRNA, or any other step in the synthesis of the protein of interest.
[0096]
An “overexpressed” protein refers to a protein that is expressed at a higher level than the wild-type expression level.
[0097]
"Modified GRK" means a GRK having one or more modifications in the amino acid sequence at the C-terminus of GRK. The modified GRK is constitutively localized at the plasma membrane. Preferably, the GRK is modified by the addition of a CAAX motif.
[0098]
The "CAAX" motif refers to a four amino acid sequence where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid of the protein.
[0099]
"Constitutive" activity refers to the activity obtained in the absence of an agonist. For example, constitutive localization of the modified GRK to the plasma membrane means that the modified GRK is localized to the plasma membrane in the absence of an agonist.
[0100]
"GRK-C20" refers to a modified GRK that has the ability to have a geranylgeranyl group added to a GRK. GRK2-C20 is GRK2 modified in this manner. Preferably, GRK-C20 contains a CAAX motif.
[0101]
The present inventors have developed an independent screening method for compounds that alter GPCR desensitization. This has developed a cell line in which the GPCR is desensitized in the absence of an agonist. These cell lines include a GRK that can be modified. Using these cell lines, a method for screening compounds that alter GPCR desensitization in the absence of agonist was developed. These methods eliminate the agonist addition step from the screening method. By omitting this step, (1) a more effective screening method for compounds that alter GPCR desensitization using known agonists can be obtained, and (2) desensitization of orphan GPCRs without known agonists. Is obtained. Methods have been developed to identify whether the GPCR is expressed at the plasma membrane and to identify whether the GPCR has an affinity for arrestin, preferably using host cells containing orphan GPCRs and GRKs. However, the GPCR is at least partially internalized in an agonist-independent manner upon GRK expression, thus eliminating the need for agonist addition. The GPCR was modified to increase affinity for arrestin. These modified GPCRs are useful in agonist-independent screening methods for compounds that alter desensitization.
[0102]
[GPCR and desensitization]
Exposure of the GPCR to the agonist rapidly diminishes its signaling capabilities, including uncoupling of receptors from the cognate heterotrimeric G protein. The cellular mechanism that regulates agonist-specific or homologous desensitization is a two-step process in which an agonist-coupled receptor is phosphorylated by a G protein-coupled receptor kinase (GRK) and binds to an arrestin protein.
[0103]
After an agonist binds to a GPCR, G protein-coupled receptor kinase (GRK) is known to phosphorylate the intracellular domain of the GPCR. After phosphorylation, the arrestin protein associates with the GRK phosphorylation state, separating the receptor from the cognate G protein. The interaction of arrestin with the phosphorylated GPCR terminates GPCR signaling and results in a desensitized receptor that does not signal.
[0104]
Arrestin bound to the desensitized GPCR allows the GPCR to function as an adapter protein that binds to the adapter protein 2 (AP-2) and a component of the endocytic machinery such as clathrin, thereby allowing the GPCR to bind to clathrin-coated pits or other Target cellular mechanisms for endocytosis (ie, internalization). The internalized GPCR is dephosphorylated and reused on the resensitized cell surface and is retained or degraded inside the cell. The ability of arrestin to interact with GPCRs is one factor that predicts the rate of GPCR dephosphorylation, recycling, and resensitization. GPCR phosphorylation and dephosphorylation in the desensitization process are described in US patent application Ser. No. 09 / 933,844, filed Nov. 5, 2001, which is hereby incorporated by reference. Is exemplified.
[0105]
Using the methods described herein, the present inventors have identified certain GPCRs that show no affinity for arrestin. These GPCRs were modified to include one or more phosphorylations, preferably clusters of phosphorylation sites, properly located in the carboxyl-terminal tail. With this modification, the modified GPCR forms a stable complex with arrestin and is internalized as a unit into the endosome. These modified GPCRs may be useful in GPCR activity assays. These modified GPCRs may be useful for identifying agonists of GPCRs. These modified GPCRs may be useful in the agonist-independent screening methods described herein.
[0106]
An agonist-independent screening method using GPCRs altered to include a DRY motif is described in US Patent Application No. 10 / 054,616, which is incorporated herein by reference. Has been described. GPCR modifications are included in this screening method, and each GPCR utilized must be modified in such a manner.
[0107]
The present inventors have developed an agonist-independent screening method using GRKs that can be modified. These GRKs phosphorylate GPCRs in the absence of agonist. These phosphorylated GPCRs are internalized in the absence of an agonist. The present inventors have developed agonist-independent methods for screening for agonists of GPCR internalization using these modified GRKs. These methods do not require the GPCR modifications described in US patent application Ser. No. 10 / 054,616.
[0108]
Previously, certain GRKs have been shown to be constitutively localized at the plasma membrane. Inglese et al. Constructed GRK2-C20, which is constitutively isoprenylated and localized to the membrane.
[0109]
The present inventors have identified that GPCRs are vigorously desensitized by cellular expression of GRK constitutively located in the plasma membrane. These GRKs can be overexpressed, can induce expression, and the nucleic acids encoding them can be localized in a vector or integrated into the genome. The present inventors have constructed a host cell that expresses GRK constitutively located in the plasma membrane. These host cells can also express arrestin. The GPCRs of interest were transfected into these host cells. Using GRK-containing cells, the expression of the GPCR of interest at the plasma membrane was identified, the ability of the GPCR to bind arrestin was analyzed, and a method for detecting constitutively desensitized GPCRs was developed. These desensitization methods were constructed and a method for identifying agonist-independent compounds that alter GPCR desensitization was developed. These methods are useful for identifying compounds, known or unknown, that alter GPCR internalization, which are GPCR agonists.
[0110]
We have also identified whether increasing wild-type or modified GRK increases desensitization, whether or not GRK is constitutively localized in the plasma membrane.
[0111]
The present invention relates to a modified GPCR, a modified GPCR polypeptide, a nucleic acid molecule encoding the modified GPCR, a vector containing the modified GPCR-encoding nucleic acid molecule, a vector capable of constructing the modified GPCR nucleic acid, and a cell containing the modified GPCR. About. The present invention further provides an assay system using the modified GPCR, an assay system using cells containing the modified GPCR, a compound identified using the assay system, a therapeutic method using the identified compound, and a disease using the assay system. And a kit comprising the assay reagent of the present invention and the cell of the present invention.
[0112]
The GPCR or modified GPCR can be mutated to change a particular codon to a codon encoding a different amino acid. Such mutations are generally made by changing the fewest nucleotides possible. The resulting protein may be in a non-conservative manner (ie, by changing codons from an amino acid belonging to an amino acid group having one particle size or characteristic to an amino acid belonging to another group) or in a conservative manner (i. Alternatively, such substitution mutations can be made such that the amino acid is changed (by changing the codon from an amino acid belonging to the characteristic amino acid group to an amino acid belonging to the same group). Generally, such conservative changes do not significantly alter the structure and function of the resulting protein. Non-conservative changes will likely alter the structure, activity, or function of the resulting protein. The present invention should consider including conservatively altered sequences in which the activity and binding properties of the resulting protein are not significantly altered.
[0113]
In certain embodiments, the modified GPCRs of the present invention include GPCRs modified to have one or more phosphorylation sites, preferably a cluster of phosphorylation sites, appropriately positioned in the carboxy-terminal tail. These modified GPCRs recruit to endosomes within about 30 minutes of agonist stimulation. These modified GPCRs recruit to endosomes in the cells described herein, and the GPCRs are phosphorylated in an agonist-dependent manner.
[0114]
The modified GPCRs of the present invention comprise one or more phosphorylation sites, preferably a cluster of one or more phosphorylation sites, suitably located at the carboxyl-terminal tail. We believe that one or more phosphorylation sites, preferably one or more clusters of phosphorylation sites, appropriately located in the carboxyl terminal tail, after stimulation with an agonist described herein. Alternatively, it was discovered that the affinity for arrestin was increased and co-localized with arrestin in endosomes during GPCR phosphorylation, either in an agonist-independent manner. The present inventors have also discovered that one or more phosphorylation sites, preferably a cluster of phosphorylation sites, must be optimally present in the GPCR tail so that the GPCR's affinity for arrestin is increased. did. Thus, a modified GPCR can be constructed such that one or more phosphorylation sites, preferably a cluster of phosphorylation sites, is optimally located within the carboxy-terminal tail. The portions of the polypeptides that are fused together to form the modified GPCR are selected such that one or more phosphorylation sites, preferably a cluster of phosphorylation sites, is properly located within the carboxy-terminal tail. Alternatively, the location of individual point mutations to create a modified GPCR can be selected such that one or more phosphorylation sites, preferably a cluster of phosphorylation sites, is properly located within the carboxy-terminal tail. .
[0115]
The present inventors have discovered that the modified GPCRs of the present invention are useful in screening assays for compounds that can alter G protein-coupled receptor (GPCR) activity. Examples of assays in which the present invention can be used include U.S. Patent Nos. 5,891,646 and 6,110,693, which are incorporated herein by reference. Includes, but is not limited to, the described assays. Further examples of assays in which the present invention can be used include assays using fluorescence resonance energy transfer (FRET) and Angers, S. et al. Salahpour, A .; Jolly, E .; Hilairet, S .; , Chelsky, "β in living cells using bioluminescence resonance energy transfer (BRET). ₂ Dimerization of adrenergic receptors, "Proc. Natl. Acad. Sci. USA, 97, 7, 3684-3689, including but not limited to assays using the bioluminescence resonance energy transfer (BRET) technique.
[0116]
The present inventors have identified that these modified GPCRs are useful in agonist-independent methods for screening for compounds that can alter GPCR internalization. Further examples of assays in which the present invention can be used include, but are not limited to, the assays described herein.
[0117]
GPCR desensitization enhancement
The present invention provides a method for enhancing GPCR desensitization. One embodiment relates to the expression of a GRK that can be modified. GRK can be overexpressed or its expression can be induced. These methods can be used to analyze desensitization of GPCRs, including modified GPCRs, orphan GPCRs, taste receptors, mutant GPCRs, β2ARY326AGPCR mutants, or another GPCR. Certain GPCRs useful in the present invention are listed in FIG.
[0118]
In a preferred embodiment, a cell comprising the expression system and a nucleic acid encoding GRK is obtained. GRK can be modified so that GPCR is constitutively desensitized by expression of GRK. GRK can be overexpressed and its expression induced.
[0119]
Preferably, a host cell containing GRK and arrestin that can be modified is obtained. The GPCR is then added to these cells. Agonist independent GPCR desensitization is detected. FIGS. 4, 5, 6, and 7 are examples of this method. The detection method is described below.
[0120]
The present invention provides a method for identifying whether a GPCR of interest is expressed at the plasma membrane. GPCRs expressed at the plasma membrane are useful in the compound identification methods described above.
[0121]
A preferred method of identifying whether the GPCR of interest is expressed at the plasma membrane is (a) a cell comprising a GPCR, arrestin, and GRK, wherein the arrestin is detectably labeled. Obtaining cells; (b) determining the cell distribution of the arrestin; and (c) correlating the cell distribution of the arrestin with the ability of the GPCR to express at the plasma membrane.
[0122]
Preferred embodiments of the present invention are described in Examples 2, 3, 4, 5, 6, and 7, and are illustrated in Figures 4, 5, 6, and 7.
[0123]
Another method of identifying whether a GPCR of interest is expressed at the plasma membrane is (a) a GPCR, GRK, and a cell containing the GRK, wherein the GRK is detectably labeled. (B) determining the cell distribution of the GRK, and (c) correlating the cell distribution of the GRK with the ability of the GPCR to express on the plasma membrane.
[0124]
The present invention provides a method for analyzing the ability of a GPCR to bind to arrestin. GPCRs that bind to arrestin are useful in the methods for identifying compounds described above.
[0125]
A preferred method for analyzing the arrestin binding ability of a GPCR comprises: (a) obtaining a cell comprising a GPCR, arrestin, and GRK, wherein the arrestin is detectably labeled; b) determining the cell distribution of the arrestin; and (c) correlating the cell distribution of the arrestin with the arrestin binding ability of the GPCR.
[0126]
Preferred embodiments of the present invention are described in Examples 2, 3, 4, 5, 6, and 7, and are illustrated in Figures 4, 5, 6, and 7.
[0127]
Using the methods of the present invention, certain GPCRs bind and desensitize arrestin. However, certain GPCRs do not desensitize without altering the GPCR as described in US patent application Ser. No. 09 / 993,844. We varied several GPCRs, including the known and orphan GPCRs listed in FIG. Upon modification, these modified GPCRs were constitutively desensitized in the above system.
[0128]
[Modified GPCR]
The present invention relates to modified GPCRs. A modified GPCR of the invention may include one or more variables in its carboxy-terminal tail. These modifications may include one or more phosphorylations, preferably clusters of phosphorylation sites, within certain regions of the carboxyl-terminal tail. Thus, all or part of the carboxy terminal tail can be modified. The carboxy-terminal tail of many GPCRs begins immediately after the conserved NPXXXY motif, which marks the end of the seven transmembrane domain (ie, the sequence following the NPXXXY motif is the carboxy-terminal tail of the GPCR). The carboxy-terminal tail of many GPCRs contains a putative palmitoylation site about 10-25 amino acid residues, preferably 15-20 amino acid residues downstream of the NPXXXY motif. This site is typically one or more cysteine residues. The carboxy terminus of the GPCR may be relatively long, relatively short, or virtually absent. We have identified that the carboxy-terminal tail of the GPCR determines the affinity of arrestin binding.
[0129]
The present inventors have discovered that specific amino acid motifs in the carboxy-terminal tail can promote the formation of a stable GPCR / arrestin complex, thereby ultimately promoting recruitment of arrestin to endosomes. These amino acid motifs include one or more amino acids, preferably clusters of amino acid residues, that are efficiently phosphorylated to readily function as phosphorylation sites. Amino acid clusters are contiguous amino acid positions such as two of two, two of three, three of three, four of four, four of four, five of four, five of five, etc. Can occupy. Therefore, the amino acid cluster that promotes stable GPCR / arrestin complex formation is the “phosphorylation site cluster”. These clusters of phosphorylation sites are preferably clusters of serine and threonine residues.
[0130]
A GPCR that forms a stable complex with arrestin contains one or more phosphorylation sites, preferably a cluster of phosphorylation sites. In addition to one or more phosphorylation sites, preferably a cluster of phosphorylation sites, this site must be properly present in the carboxy-terminal tail to promote stable GPCR / arrestin complex formation Was found. To promote the formation of a stable GPCR / arrestin complex, one or more phosphorylation sites, preferably a cluster of one or more phosphorylation sites, is about 15-35 of the putative palmitoylation site of the GPCR ( (Preferably 15 to 25) amino acid residues. Further, one or more phosphorylation sites, preferably a cluster of one or more phosphorylation sites, may be located about 20-55 (preferably 30-45) amino acid residues downstream of the NRXXY motif of the GPCR. GPCRs containing one or more appropriately positioned phosphorylation sites, preferably a cluster of phosphorylation sites, are typically class B receptors.
[0131]
As an example, the V2R receptor contains a cluster of phosphorylation sites (SSS) that promote the formation of a stable GPCR / arrestin complex 19 amino acid residues downstream of the putative palmitoylation site and 36 amino acid residues downstream of the NRXXY motif. It was discovered. The NTR-2 receptor contains a cluster of phosphorylation sites (STS) that promote the formation of a stable GPCR / arrestin complex 26 amino acid residues downstream of the putative palmitoylation site and 45 amino acid residues downstream of the NRXXY motif. The oxytocin receptor (OTR) donor is one 20 amino acid residues downstream of the putative palmitoylation site and the other 29 amino acid residues downstream of the NRXXY motif, and one 38 amino acid residues downstream of the NRXXY motif and the other NRXXY It contains a cluster of two phosphorylation sites (SSLST and STLS) that promote the formation of a stable GPCR / arrestin complex 47 amino acid residues downstream of the motif. The substrate P receptor (SPR, also known as the neurokinin 1 receptor) promotes the formation of a stable GPCR / arrestin complex 32 amino acid residues downstream of the putative palmitoylation site and 50 amino acid residues downstream of the NRXXY motif. Contains a cluster of phosphorylation sites (TTIST).
[0132]
We note that GPCRs lacking one or more phosphorylation sites, preferably clusters of phosphorylation sites, present in the carboxyl-terminal tail are less stable and dissociate at or near the plasma membrane. It was identified as forming a GPCR / arrestin complex. These GPCRs are typically class A receptors, orphan receptors, taste receptors, and the like. However, the present inventors have shown that modification of the carboxyl-terminal tail of these receptors, which originally lacks one or more phosphorylation sites, can yield stable GPCR / arrestin complexes with low affinity for arrestin. discovered. Preferably, the carboxyl terminal tail is modified to include one or more phosphorylation sites, preferably a cluster of one or more phosphorylation sites, appropriately located within the carboxyl terminal tail.
[0133]
The invention includes the polypeptide sequences of these modified GPCRs. The modified GPCRs of the present invention include GPCRs that have been modified to have one or more phosphorylation sites, preferably one or more clusters of phosphorylation sites, appropriately positioned in their carboxy-terminal tail. The polypeptide sequences of the modified GPCRs of the present invention also include sequences having one or more additions, deletions, substitutions, or mutations. These mutations are preferably substitution mutations made in a conservative fashion (ie, changing codons from amino acids belonging to an amino acid group having a particle size or characteristic to the same group). Such conservative changes generally do not significantly alter the structure and function of the resulting protein. The present invention should consider including conservatively altered sequences in which the activity or binding properties of the resulting protein are not significantly altered.
[0134]
The modified GPCRs of the present invention include a GPCR containing the NPXXXY motif, a putative palmitoyl site about 10-25 amino acid residues (preferably 15-20 amino acids) downstream of the NPXXXY motif, and a modified carboxy-terminal tail. The modified carboxy-terminal tail is one or more phosphorylation sites, preferably one or more, such that the phosphorylation site is about 15-35, preferably 15-25 amino acid residues downstream of the putative palmitoylation site of the modified GPCR. It has a cluster of multiple phosphorylation sites. The modified carboxyl-terminal tail may include one or more phosphorylation sites, preferably one or more phosphorylation sites, such that the phosphorylation site is about 20-55, preferably 30-45 amino acid residues downstream of NPXXXY of the modified GPCR. It has a cluster of oxidation sites.
[0135]
The invention further includes an isolated nucleic acid molecule encoding the modified GPCR. It should also be recognized that a DNA sequence encoding a modified GPCR that encodes a modified GPCR that has the same amino acid sequence as the modified GPCR but is degenerate is within the scope of the invention. "Degenerate to" means identifying a particular amino acid using different three letter codons.
[0136]
As the skilled artisan will readily appreciate, the carboxyl tail of many GPCRs can be identified by the NPXXXY motif, which marks the end of the seven transmembrane domain.
[0137]
To create a modified GPCR comprising a modified carboxyl-terminal region of the invention, a GPCR that lacks a phosphate site or cluster of phosphate sites, or has a low or unknown affinity for arrestin, has an amino acid in its carboxyl-terminal tail. Residues may include one or more additions, substitutions, deletions, or mutations. These carboxyl-terminal tails are added, substituted, deleted or mutated such that they are modified to include one or more phosphorylation sites, preferably a cluster of phosphorylation sites. As an example, amino acid residues can be individually point mutated to obtain a modified GPCR. By way of example, three consecutive amino acids can be mutated to a serine residue to obtain a modified GPCR. These mutations are made so that one or more phosphorylation sites, preferably a cluster of phosphorylation sites, are properly located within the carboxyl-terminal tail.
[0138]
In addition, to create a modified GPCR containing a modified carboxyl-terminal region, the nucleic acid sequence of a GPCR that lacks a phosphate site or cluster of phosphate sites or has a low or unknown affinity for arrestin differs from a specific codon by Mutations can be made to alter codons encoding amino acids, preferably serine or threonine. Generally, such mutations are made by the fewest nucleotide changes possible. Such substitution mutations that alter amino acids in the resulting protein can be made to create one or more phosphorylation sites, preferably clusters of phosphorylation sites. As an example, individual point mutations of a nucleic acid sequence can be made. The phosphorylation site is positioned to be located about 15-35 amino acid residues downstream of the putative palmitoylation site of the modified GPCR.
[0139]
Furthermore, to obtain the modified GPCRs of the present invention, GPCRs that lack properly positioned phosphorylation sites or have low or unknown affinity for arrestin will have all or part of their carboxyl-terminal tails properly positioned. Can be replaced with that of a GPCR having a cluster of phosphorylation sites. The exchange site may be after or include the conserved NPXXXY motif. Alternatively, a putative palmitoylation site of the GPCR can be identified about 10-25 (preferably 15-20) amino acid residues downstream of the conserved NPXXXY motif, wherein the exchange site is located after or after the palmitoylated cysteine. Including. Preferably, the carboxyl-terminal tail of the GPCR, which lacks a cluster of properly located phosphorylation sites or has low or unknown affinity for arrestin, is replaced with an amino acid residue in close proximity to the putative palmitoylation site. More preferably, the carboxyl-terminal tail of the GPCR, which lacks a cluster of properly located phosphorylation sites or has low or unknown affinity for arrestin, has about 10 ng of the NPXXXY motif such that palmitoylated cysteine residues are maintained. Exchange at the predicted palmitoylation site 〜25 (preferably 15-20) amino acid residues downstream. The preferred mode of exchange facilitates the positioning of the cluster of phosphorylation sites at the appropriate position within the carboxyl-terminal tail of the modified GPCR. Because the tails can be swapped, modified GPCRs can be constructed by manipulation of the nucleic acid sequence or the corresponding amino acid sequence.
[0140]
Further alternatively, the exchange site can be selected because the carboxyl tail of a GPCR (eg, a GPCR without the NPXY motif) can be inferred from a hydrophobicity plot. Based on the hydrophobicity plot, one skilled in the art can predict the sites where the GPCR can be immobilized in the membrane and where the putative palmitoylation site has been introduced. Using this technique, a GPCR that does not contain an NPXXXY motif or a putative palmitoylation site can be modified to create a reference point (eg, a putative palmitoyl site). The tail exchange can be located using the putative palmitoyl site introduced.
[0141]
The carboxyl-terminal tail used for exchange has one or more clusters of properly located phosphorylation sites and a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NXXY motif. It can be from a second GPCR. The identified tail can be exchanged for or include a conserved NPXXXY motif. Alternatively, the putative palmitoylation site of the GPCR can be identified about 10-25 (preferably 15-20) amino acid residues downstream of the conserved NPXXXY motif, and the tail is replaced or replaced after palmitoylated cysteine. Can be included. Preferably, the carboxyl terminal tail of a GPCR having a cluster of phosphorylation sites is replaced with an amino acid residue that is very close to the putative palmitoylation site. More preferably, the carboxyl-terminal tail of a GPCR having a cluster of phosphorylation sites is modified such that a portion of the carboxyl-terminal tail containing the cluster of phosphorylation sites begins at the amino acid residue immediately downstream of the palmitoylated cysteine residue. Exchange at a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream. With the exchange in a preferred manner, the cluster of phosphorylation sites is easily located at the appropriate position within the carboxyl-terminal tail of the modified GPCR. The carboxyl-terminal tail with the cluster of phosphorylation sites used for exchange may have a detectable molecule attached to the carboxyl terminus. Since the tail can be exchanged, a modified GPCR can be constructed by manipulation of the nucleic acid sequence or the corresponding amino acid sequence.
[0142]
In addition, the carboxyl-terminal tail used for the exchange is synthesized to have an amino acid sequence corresponding to the amino acid sequence from a GPCR having one or more phosphorylation sites, preferably a cluster of one or more phosphorylation sites. Can be derived from the polypeptide. Synthetic polypeptides may have a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NPXXXY motif. Synthetic polypeptides can have one or more amino acid residue additions, substitutions, deletions that do not affect or change the structure and function of the polypeptide.
[0143]
In addition, the carboxyl-terminal tail used for exchange may originate from a naturally occurring polypeptide that recognizes having an amino acid sequence corresponding to a GPCR-derived amino acid sequence having one or more clusters of phosphorylation sites. . The polypeptide may have a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NPXXXY motif. A polypeptide can have one or more amino acid residue additions, substitutions, deletions that do not affect or change the structure and function of the polypeptide.
[0144]
A modified GPCR containing a modified carboxyl-terminal region is prepared by combining a first carboxyl-terminal tail portion of a GPCR lacking a well-located cluster of phosphorylation sites or having a low or unknown affinity for arrestin with one or more phosphorylations It can be made by fusion with a GPCR having a cluster of sites or a second carboxyl-terminal tail of a polypeptide. The second GPCR or polypeptide may have a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NPXXXY motif. Thus, the modified carboxyl-terminal region of the modified GPCR lacks an appropriately located cluster of phosphorylation sites or has an affinity for arrestin fused to a portion of the carboxy-terminal tail of a GPCR or polypeptide that has a cluster of phosphorylation sites. Includes a portion of the low or unknown carboxyl terminal tail from the GPCR. The tail of the GPCR lacking a properly positioned phosphorylation site cluster can be replaced or include a conserved NPXXXY motif followed by a fusion to the carboxyl terminal tail containing the phosphorylation site cluster after the conserved NPXXY motif. Or include this. Alternatively, the tail of the GPCR lacking a properly positioned cluster of phosphorylation sites can be replaced or include after the palmitoylation cysteine, and the tail containing the cluster of phosphorylation sites after the palmitoylation motif. Can be fused or included. Since the tail can be exchanged, a modified GPCR can be constructed by manipulation of the nucleic acid sequence or the corresponding amino acid sequence.
[0145]
Further alternatively, the carboxyl tail of a GPCR (eg, a GPCR without the NPXXXY motif) can be inferred and replaced from a hydrophobicity plot. Exchange sites can be selected according to the hydrophobicity plot. Based on the hydrophobicity plot, one skilled in the art can predict the sites where the GPCR can be immobilized in the membrane and where the putative palmitoylation site has been introduced. Using this technique, a GPCR that does not contain an NPXXXY motif or a putative palmitoylation site can be modified to create a reference point (eg, a putative palmitoyl site). The tail exchange can be located using the putative palmitoyl site introduced. After introduction of the putative palmitoylation site, the resulting tail has one or more clusters of one or more phosphorylation sites and a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NPXXXY motif. The GPCR or the second carboxyl tail portion of the polypeptide can be fused.
[0146]
Preferably, the modified carboxyl-terminal region of the modified GPCR is replaced with an amino acid residue in close proximity to the putative palmitoylation site. More preferably, the portion from the first GPCR that has low affinity for arrestin has amino acid residues from the NPXXY motif due to a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NPXY motif. And a portion from the first GPCR that has a cluster of phosphorylation sites and a putative palmitoylation site about 10-25 (preferably 15-20) amino acid residues downstream of the NPXXXY motif, extending to the end of the carboxyl terminus. The modified carboxyl-terminal region of the modified GPCR is fused to include an amino acid residue starting from the amino acid residue immediately downstream of the putative palmitoylation site of the second GPCR. The cluster of phosphorylation sites is easily located at the appropriate position within the carboxyl-terminal tail, and the modified GPCR maintains its structure and ability to function.
[0147]
As an example, a class A or orphan receptor can have a portion of the carboxy terminal tail replaced with a portion of the carboxy terminal tail from a known class B receptor. In addition, receptors that are virtually free of a carboxy-terminal tail (eg, orphan and taste receptors) have a portion of the carboxy-terminal tail that has been replaced by a portion of the carboxy-terminal tail from known class B receptors. May be provided. The class B receptor tail used for these exchanges may have a detectable molecule fused to the carboxyl terminus.
[0148]
Modified GPCRs can be made by standard molecular biology techniques in the field of genetic engineering, including but not limited to polymerase chain reaction (PCR), restriction enzymes, expression vectors, plasmids, and the like. As an example, vectors such as pEArrB (with enhanced arrestin binding) can be designed to enhance the affinity of GPCRs lacking a cluster of phosphorylation sites for arrestin. To form a vector such as pEArrB, a PCR amplified GPCR carboxyl-terminal DNA fragment that forms a stable complex with arrestin can be digested with appropriate restriction enzymes and cloned into a plasmid. A schematic diagram of one such plasmid is shown in FIG. 4A. As shown in FIG. 4B, the DNA of the GPCR to be modified can also be PCR amplified, digested at the appropriate location with restriction enzymes, and subcloned into a vector such as pEArrB. When expressed, the modified GPCR comprises a polypeptide fused to a carboxyl terminus. The polypeptide contains a cluster of phosphorylation sites. Preferably, the polypeptide originates from the GPCR carboxyl terminus of the receptor forming a stable complex with arrestin.
[0149]
Such modified GPCRs can also occur naturally as a result of abnormal gene splicing or single nucleotide polymorphism. Such naturally occurring modified GPCRs are expected to have modified endocytosis targeting. These naturally occurring modified GPCRs can be associated with a number of GPCR-related diseases.
[0150]
As shown in FIG. 3, we modified some GPCRs. As described herein, β2 adrenergic receptor, dopamine D1A receptor, mu opioid receptor, orphan GPR3, orphan GPR6, orphan GPR12, orphan GPR7, orphan GPR8, orphan GPR55. , Orphan SREB2, and orphan SREB3 were modified. These modified GPCRs contain a V2R cluster (SSS) of phosphorylation sites properly located within the modified GPCR tail.
[0151]
As can be shown by standard receptor binding assays, the modified receptor is not a protein that has increased affinity for arrestin with increased affinity for arrestin, the ability to act on arrestin, and the ability to reuse and resensitize Is essentially indistinguishable from its wild-type counterpart. For example, the modified receptor is suitably expressed at the membrane and has a similar affinity for agonists or ligands. However, because of the increased affinity for arrestin, the modified GPCR may form a complex with arrestin that is more stable than the wild-type subject, and may remain bound to arrestin when transported to endosomes.
[0152]
These modified GPCRs are useful in GPCR agonist screening assays and agonist-independent assays for compounds that alter GPCR desensitization.
[0153]
[Assay of GPCR activity using modified GPCR]
The modified GPCRs of the present invention are useful in assays for GPCR activity. The modified GPCRs of the invention can be used in research assays for GPCRs that are weaker than the desired interaction and association with arrestin and GPCRs that have an unknown interaction or association with arrestin. The method of the present invention using a modified GPCR can provide a sensitive assay, for example, to improve the detection of arrestin / GPCR in endosomes. Assays using the modified GPCRs of the invention can be useful for screening compounds and sample solutions for ligands, agonists, inverse agonists, desensitizing active compounds, and the like. Once identified, these compounds can modulate GPCR activity and can be useful as drugs useful in the treatment of one or more diseases associated with GPCRs.
[0154]
In a preferred assay of the invention, cells expressing a modified GPCR of the invention are obtained, and these cells may further comprise a conjugate of arrestin with a detectable molecule.
[0155]
Arrestin bound to a detectable molecule can be detected and its function in the GPCR pathway monitored. The location of arrestin (eg, uniformly dispersed in the cytoplasm, densely in cell membranes, densely in clathrin-coated pits, localized in endosomes, etc.) can be detected. In response to agonist stimulation, access of arrestin to GPCRs and access to any other cellular structure can be monitored. For example, in response to agonist stimulation, arrestin can be detected by access to GPCRs at cell membranes, clustering with GPCRs in clathrin-coated pits, co-localization with GPCRs on endosomes, and the like.
[0156]
The modified GPCR of the present invention has an increased affinity for arrestin, and a stable complex of the GPCR and arrestin can be obtained. Therefore, the GPCR and arrestin are co-localized to endosomes. In the assay method of the present invention, arrestin is detected, for example, in the cytoplasm, near the GPCR at the cell membrane, near the GPCR in clathrin-coated pits, and as co-localized with the GPCR on endosomes. be able to. Preferably, arrestin can be detected as co-localized with the GPCR on endosomes.
[0157]
The association of arrestin with the GPCR at the cell membrane can be rapidly detected after addition of the agonist (eg, about 1 second to 2 minutes). Co-localization of arrestin with GPCR on endosomes can be detected within minutes (eg, about 3-15 minutes) of agonist addition and retained for a long time (eg, 1 hour). The association of arrestin with GPCRs on endosomes can yield strong and easily recognizable signals. With a 40 × objective, the signal may show a donut-like appearance. Signals obtained from arrestin and GPCR compartmentalization co-localized in endosomal vesicles are typically easy to detect and can be retained for long periods of time.
[0158]
A preferred assay for the GPCR pathway of the invention comprises: (a) expressing at least one modified GPCR of the invention to obtain cells further comprising a conjugate of arrestin and a detectable molecule; And c) detecting the interaction of arrestin with the modified GPCR along the translocation pathway.
[0159]
The interaction of arrestin with the modified GPCR can be detected, for example, as enrichment in endosomes, clathrin-coated pits, and near cell membranes. Preferably, the interaction of arrestin with the modified GPCR is detected in endosomes. The interaction of arrestin with the GPCR in the endosome can be detected within minutes (eg, about 3-15 minutes) of agonist addition and retained for a long time (eg, 1 hour). The interaction of arrestin with GPCRs on endosomes can result in strong and easily recognizable signals that are maintained for long periods of time.
[0160]
The method for screening a compound for GPCR activity of the present invention results in cells expressing at least one modified GPCR. The cell contains arrestin that binds to a detectable molecule. The cells are exposed to a test compound. Detect the location of arrestin in cells. The position of arrestin in the cell in the presence of the compound is compared to the position of arrestin in the cell in the absence of the compound, and (1) the position of arrestin in the cell in the presence of the compound and (2) Correlate differences in the location of arrestin within the cell in the absence of compound.
[0161]
As an example, compounds and sample solutions can be screened for GPCR agonist activity using the modified GPCRs of the present invention. In this way, cells are obtained which express at least one modified GPCR of the invention and further comprise a conjugate of an arrestin and a detectable molecule. The cells are exposed to the compound to be tested or a sample solution. It is detected whether the interaction of the arrestin with the modified GPCR increases after exposure to the test compound and the solution, and this increased interaction is indicative of the compound or solution having GPCR agonist activity. The interaction of arrestin with a GPCR can be detected in endosomes, clathrin-coated pits, near cell membranes, and the like. The modified GPCR can also be linked to a molecule, preferably at the carboxyl terminus. As explained above, modifications to the GPCR in the present invention should not affect the natural affinity of the GPCR for agonists or ligands.
[0162]
As an example, compounds and sample solutions can be screened for GPCR antagonist or inverse agonist activity using the modified GPCRs of the invention. Cells are obtained which express at least one modified GPCR of the invention and further comprise a conjugate of arrestin and a detectable molecule. The cells are exposed to the compound or sample solution to be tested and a known agonist of the GPCR. It is detected whether the interaction of the arrestin with the modified GPCR is reduced after exposure to the test compound and the solution, and a decrease in this interaction is indicative of the compound or solution having GPCR antagonist or inverse agonist activity. The interaction of arrestin with a GPCR can be detected in endosomes, clathrin-coated pits, near cell membranes, and the like. The modified GPCR can also be linked to a molecule, preferably at the carboxyl terminus. As explained above, modifications to the GPCR in the present invention should not affect the natural affinity of the GPCR for agonists or ligands.
[0163]
As a further example, compounds and sample solutions can be screened for GPCR desensitization activity using the modified GPCRs of the present invention. Cells are obtained that express at least one first modified GPCR of the invention and further comprise a conjugate of arrestin and a detectable molecule. The first cell is exposed to the compound or sample solution to be tested and a known agonist of the first GPCR. It is detected whether the interaction of the arrestin with the first modified GPCR decreases or does not increase after exposure to the test compound and the solution, wherein a decrease or lack of this interaction indicates that the compound or solution has desensitizing activity An indicator of that. The interaction of arrestin with a GPCR can be detected in endosomes, clathrin-coated pits, near cell membranes, and the like. Cells are obtained which express at least one second modified GPCR of the invention and further comprise a conjugate of arrestin and a detectable molecule. The second modified GPCR is not related to the first modified GPCR. The second cell is exposed to the compound or sample solution to be tested and a known agonist of the second GPCR. It was detected whether the interaction of the arrestin with the second modified GPCR was reduced or not increased after exposure to the test compound and the solution, and a decrease or lack of this interaction was independent of the GPCR in which the compound or solution was expressed. It is an indicator of having the GPCR desensitizing activity. The interaction of arrestin with a GPCR can be detected in endosomes, clathrin-coated pits, near cell membranes, and the like.
[0164]
The methods of assessing GPCR pathway activity of the present invention also include cell-free assays. In the cell-free assay of the present invention, a precipitated substrate (the modified GPCR of the present invention) is obtained. A fluid comprising a conjugate of arrestin and a detectable molecule is also obtained. Induces arrestin translocation and detects the interaction of arrestin with GPCRs. GPCRs and arrestins are obtained from whole cells and can be used for cell-free assays after purification. The modified GPCR has an arrestin binding site and an agonist binding site and can be supported in a multilayer or bilayer lipid vesicle. The vesicles supporting the modified GPCR precipitate on the substrate, supporting the modified GPCR in lipid vesicles on the substrate such that the arrestin binding site is exposed to the arrestin and the receptor binding site is accessible to the agonist. Can be precipitated. Substrates include any artificial substrate on which the GPCR can be precipitated (glass, plastic, ceramic, semiconductor, silica, fiber optics, diamond, biocompatible monomers, biocompatible polymers, polymer beads (organic and inorganic polymers), etc. But not limited thereto).
[0165]
The invention relates to compounds identified as ligands, agonists, antagonists, inverse agonists or DACs by the assay method of the invention. These compounds can be used to treat any one of the diseases involving GPCRs. The identified compounds can be administered to a human or non-human in a therapeutically effective amount for treating or ameliorating a condition, disorder, or disease involving a GPCR. A therapeutically effective amount refers to that amount of the compound that is sufficient to ameliorate the symptoms of such a condition, disorder, or disease.
[0166]
[Method of identifying compound that regulates GPCR desensitization]
The present invention relates to a method for screening a compound that regulates GPCR desensitization. The method uses a modified GRK that is constitutively dephosphorylated by constitutively phosphorylating the GPCR. Even though GPCR agonists are unknown, they can be used to identify compounds that alter GPCR desensitization. Once identified, these compounds can modulate GPCR activity and can be useful as drugs useful in the treatment of one or more diseases associated with GPCRs.
[0167]
In a preferred method of the present invention, cells are obtained that comprise an expression system and a nucleic acid encoding a modified GPCR and that are constitutively desensitized to GPCRs expressed in the cells. These cells may further contain arrestin that binds to GFP.
[0168]
Preferred methods for identifying compounds that inhibit GPCR internalization include: (a) obtaining cells containing a GPCR, arrestin, and modified GRK; (b) exposing the cells to the compound; and (c) exposing the cells to the GPCR. Or determining the cellular distribution of arrestin; (d) (1) the position of the labeled molecule in the cell in the presence of the compound and (2) the position of the labeled molecule in the cell in the absence of the compound Correlating the difference with the regulation of GPCR internalization. Non-limiting examples of this method are described in FIGS. 4, 5, 6, and 7 and Examples 2, 3, 4, 5, 6, and 7.
[0169]
The GRK of step (a) above can be GRK 1, 2, 3, 4, 5, 6, or any other GRK (including splice variants, biologically active fragments, or modified GRKs). GRK can be overexpressed and / or expression can be inducible. GRKs can include a CAAX motif.
[0170]
In the above method, an agonist may or may not be obtained.
[0171]
Methods for detecting labeled molecules and identifying cellular distribution of GPCRs or arrestins are described below.
[0172]
GPCRs useful in the invention include GPCRs with known agonists, GPCRs with no known agonists, GPCRs listed in FIG. 1, GPCRs shown in FIGS. 3, 4, 5, 6, and 7, Class A GPCRs, Class B GPCR, taste receptor, odorant receptor, orphan GPCR, modified GPCR, U.S. Patent Application Nos. 10 / 054,616, 09 / 993,844, 10 / 095,620, 10 / 101,235, 09 / 631,468, 10 / 141,725, 10 / 161,916, 09 / 469,554, 09 / 772,644, 60 / 393,789, and 60 / 379,986 (hereby incorporated by reference). Or a biologically active fragment of the GPCR described above. Not a constant.
[0173]
[Vector, nucleic acid, host cell for protein expression]
The present invention relates to modified GRKs, including GRKs that are overexpressed and whose expression is inducible.
[0174]
A nucleic acid encoding the modified GRK is obtained. The present invention relates to the expression, overexpression, and inducible expression of these proteins. As described below, it can be expressed by an appropriate expression system contained in the vector.
[0175]
One aspect of the present invention relates to a combination of (1) a nucleic acid encoding a modified GRK and (2) a modified GRK expression system in which a GPCR is constitutively desensitized. The modified GRK expression system can include a promoter or origin of replication.
[0176]
Another aspect of the invention relates to a modified GPCR, a nucleic acid encoding the modified GPCR, and a host cell for expressing the modified GPCR.
[0177]
A nucleic acid encoding the modified GPCR is obtained. The present invention relates to the expression, overexpression, and inducible expression of these proteins. As described below, expression can be achieved by an appropriate expression system contained in the vector.
[0178]
A feature of the invention is the expression of the DNA sequences disclosed herein. As is well known in the art, a DNA sequence is expressed in a suitable expression vector by operable linkage to an expression control sequence and use of the expression vector to transform a suitable single-cell host. be able to. The transforming DNA may or may not be integrated (covalently linked) into the chromosomal DNA making up the cell genome.
[0179]
Such an operable linkage of a DNA sequence of the invention to an expression control sequence, of course, includes an initiation codon (ATG) in the correct reading frame upstream of the DNA sequence, if not already part of the DNA sequence. It is.
[0180]
A wide variety of host / expression vector combinations can be used to express the DNA sequences of the present invention. Useful expression vectors can consist of, for example, segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include SV40 derivatives, known bacterial plasmids (such as E. coli plasmids colEI, pCR1, pBR322, pMB9, and derivatives thereof), plasmids such as RP4, phage DNAS (eg, a number of derivatives of λ phage). (Eg, NM989), and other phage DNAs (eg, M13 and filamentous single-stranded phage DNA), yeast plasmids (2μ plasmid or derivative thereof), vectors useful in eukaryotic cells (useful in insect or mammalian cells) And vectors derived from a combination of plasmid and phage DNA (such as a plasmid modified to use phage DNA or other expression control sequences).
[0181]
Any of a wide variety of expression control sequences (sequences that control the expression of an operably linked DNA sequence) can be used in these vectors to express a DNA sequence of the present invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma, or adenovirus, lac, trp, TAC, TRC, LTR, LTR, λ phage major. Operator and promoter regions, regulatory regions of coat proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatase (eg, Pho5), promoters of yeast alpha mating factor, and prokaryotic or eukaryotic cells Or other sequences known to regulate gene expression of the virus, as well as combinations thereof.
[0182]
A wide variety of unicellular host cells are also useful for expressing the DNA sequences of the present invention. These hosts include the well-known eukaryotic and prokaryotic hosts E. coli. E. coli, Pseudomonas, Bacillus, Streptomyces, and other strains, fungi such as yeast, plant cells, nematode cells, and HEK-293, U2OS, CHO, RI. 1, BW, and LM cells Animal cells, such as African green monkey kidney cells (eg, COS1, COS7, BSC1, BSC40, and BMT10), insect cells (eg, Sf9), and human cells and plants in tissue culture May include cells. In one aspect of the invention, the host cells include GRK-C20 and arrestin. In a further aspect of the invention, host cells include GRK-C20, arrestin, and GPCR.
[0183]
It is understood that, but not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of the present invention. Not all hosts will function equally well with the same expression system. However, one of ordinary skill in the art can select appropriate vectors, expression control sequences, and hosts without undue experimentation to achieve the desired expression without departing from the scope of the present invention. For example, in selecting a vector, the host must be considered because the vector must function in the host. The copy number of the vector, the ability to control copy number, and the expression of any other proteins encoded by the vector (such as antibiotic markers) are also considered.
[0184]
In selecting an expression control sequence, various factors are usually considered. These include, for example, the relative strength of the system, its modulatability, and its compatibility with the preferred DNA sequences that express, among other things, potential secondary structure. Suitable single-cell hosts include, for example, compatibility with selected vectors, secretion properties, ability to specifically fold the protein and fermentation requirements, and toxicity of the sample encoded by the expressed DNA sequence to the host and ease of purification of the expressed product. To choose.
[0185]
In view of these and other factors, one skilled in the art can construct various vector / expression control sequence / host combinations that express the DNA sequences of the present invention in fermentation or large animal culture.
[0186]
It is further contemplated that the modified GRK analog can be tailored from the nucleotide sequence of the protein complex / subunit derived within the scope of the present invention. Analogs such as fragments can be produced, for example, by pepsin digestion of GRK material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of the GRK coding sequence. Analogs that exhibit "GRK activity", such as small molecules that function as either promoters or inhibitors, can be identified by known in vivo and / or in vitro assays.
[0187]
As described above, a DNA sequence encoding a modified GRK can be prepared by synthesis rather than cloning. DNA sequences can be designed using the appropriate codons of the GRK amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is constructed from duplications of the oligonucleotides prepared by standard methods and assembled into the complete coding sequence. See, eg, Edge, Nature, 292, 756, 1981; Nambair et al., Science, 223, 1299, 1984; Jay et al. Biol. Chem. , 259, 6311, 1984.
[0188]
Synthetic DNA sequences allow genes that express GRK analogs or "variants" to be conveniently constructed. Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of a native or modified GRK gene or cDNA, and muteins can be made directly using conventional polypeptide synthesis.
[0189]
General methods for site-specific incorporation of unnatural amino acids into proteins are described in ChristopherJ. Noren, Spencer J. et al. Anthony-Cahill, Michel C .; Griffith, Peter G. Schultz, Science, 244, 182-188, April 1984. Using this method, analogs can be made with unnatural amino acids.
[0190]
Additional motifs, such as epitope tags or sequences, to aid in purification can be incorporated into the nucleic acid encoding the modified GRK or GPCR. Preferably, the nucleic acid encoding the motif may be at the 5 'or 3' end of the nucleic acid, causing the motif to be at the N or C terminus of the protein.
[0191]
[Joints (The conjugates)]
The cells used in the assays of the invention can contain a conjugate of an arrestin protein and a detectable molecule. In the cells and methods of the invention, the cells may also include a conjugate of the modified GPCR of the invention with a detectable molecule.
[0192]
All types of arrestins (naturally occurring arrestins, including visual arrestin, cone arrestin, β arrestin 1, and β arrestin 2, and engineered variants) can be used in the present invention. The modified GPCRs of the invention are capable of interacting with all types of arrestin at detectable levels.
[0193]
Detectable molecules that can be used to bind arrestin include spectroscopy, photochemistry, biochemistry, immunochemistry, electrical, radioactive, and optical means (including bioluminescence, phosphorescence, and fluorescence. , But not limited thereto). Detectable molecules include, but are not limited to, GFP, luciferase, β-galactosidase, rhodamine binding antibodies, and the like. Detectable molecules include radioisotopes, epitope tags, affinity labels, enzymes, fluorophores, chemiluminescers, and the like. Detectable molecules include those molecules that are detected indirectly or directly as a function of interaction with another molecule. These detectable molecules should be biocompatible molecules and should not interfere with the ability of arrestin to interact with the GPCR system, and the interaction of arrestin with the GPC system will increase the ability of the detectable molecule to detect Do not interfere. Preferred detectable molecules include optically detectable proteins that can be excited chemically, mechanically, electrically, or radioactively to emit fluorescence, phosphorescence, or bioluminescence. Is a molecule that can be detected. More preferred detectable molecules are molecules that are intrinsically fluorescent, such as fluorescent proteins, including green fluorescent protein (GFP). Detectable molecules can be conjugated to arrestin proteins by the methods described by Barak et al. (US Pat. Nos. 5,891,646 and 6,110,693). The detectable molecule can be conjugated at the arrestin front end, back end, or in the middle.
[0194]
GPCRs and biologically active fragments thereof can also be conjugated to a detectable molecule. Preferably, the carboxyl terminus of the GPCR is conjugated to a detectable molecule. A carboxyl terminal tail conjugated or linked to a detectable molecule can be used in a carboxyl terminal tail exchange to obtain a detectably labeled GPCR.
[0195]
When the GPCR binds to a detectable molecule, the proximity of the GPCR to arrestin can be easily detected. Furthermore, in the case of a conjugate with a molecule capable of detecting a GPCR, compartmentalization of the GPCR with arrestin can be easily confirmed. Detectable molecules used for binding to GPCRs include, for example, optics that can be chemically, mechanically, electrically, or radioactively excited to emit fluorescence, phosphorescence, or bioluminescence. It may include the molecules described above, including those that are specifically detectable. Preferred optically detectable molecules can be detected by immunofluorescence, luminescence, fluorescence, and phosphorescence.
[0196]
For example, a GPCR can be an antibody labeled with an antibody coupled to an immunofluorescent molecule, or a GPCR can be coupled to a fluorescent donor. In particular, a GPCR can be conjugated, for example, to a luciferase (eg, Renilla luciferase) or a rhodamine-binding antibody (eg, a rhodamine-conjugated anti-HA mouse monoclonal antibody). Preferably, the carboxy-terminal tail of the GPCR can be conjugated to a fluorescent donor (eg, luciferase). L. S. Barak et al., “Functionally Intact β ₂ Internal transport and surface mobility of adrenergic receptor-green fluorescent protein conjugates, "Mol. Pharm. , 1997, 51, 177-184, a GPCR, preferably a carboxy-terminal tail, can also be attached to GFP.
[0197]
[Cell type and substrate]
The cells of the invention are capable of expressing at least one modified GPCR of the invention. The cell may further comprise a conjugate of the arrestin protein and a detectable molecule. Useful cells include prokaryotic and eukaryotic cells, including but not limited to bacterial cells, yeast cells, fungal cells, insect cells, nematode cells, plant cells, and animal cells. Suitable animal cells include, but are not limited to, HEK-293 cells, U2OS cells, HeLa cells, COS cells, and various primary mammalian cells. Animal models expressing conjugates of arrestin with a detectable molecule throughout the tissue or in a particular type of organ or tissue can also be used.
[0198]
The cells of the present invention are capable of expressing one variant protein in which the GPCR is localized in endocytic vesicles or endosomes in an agonist-independent manner.
[0199]
The substrate could accumulate on multiple cells of the invention. The substrate can be any biomatrix, including, but not limited to, glass, plastic, ceramic, semiconductor, silica, fiber optics, diamond, biocompatible monomers, or biocompatible polymeric materials.
[0200]
[Detection method]
The subcellular location of the detectably labeled arrestin, the subcellular location of the detectably labeled GPCR, the interaction of the detectably labeled arrestin, the GPCR or any other cellular structure (eg, arrestin or Methods for detecting other members of a GPCR / arrestin complex having GPCR clustering, co-localization of arrestin and GPCR in endosomes, arrestin or GPCR clustering in good b clathrin-coated pits, etc. Will vary depending on the detectable molecule used.
[0201]
One skilled in the art can devise an appropriate detection method for the detectable molecule used. For optically detectable molecules, any optical method can be used that allows a change in fluorescence, bioluminescence, or phosphorescence to be measured by redistribution or redirection of emitted light. Such methods include, for example, polarizing microscopy, BRET, FRET, evanescent wave excitation microscopy, as well as standard or mirror focus microscopy.
[0202]
In a preferred embodiment, arrestin can be conjugated to GFP and the arrestin-GFP conjugate can be detected with a mirror-focus microscope. In another preferred embodiment, arrestin can be conjugated to GFP, GPCR can be conjugated to an immunofluorescent molecule, and the conjugate can be detected with a microscope. In a further preferred embodiment, arrestin is linked to GFP, the carboxy terminus of the GPCR is linked to luciferase, and the conjugate can be detected by bioluminescence resonance emission techniques. In a further preferred embodiment, arrestin is conjugated to luciferase, GPCR is linked to GFP, and the conjugate can be detected in the organism by anti-resonance release technology. The method of the invention indicates detection of GPCR activity. The method of the invention improves the simultaneous monitoring of GPCR pathways.
[0203]
In a preferred embodiment, the localization pattern of the detectable molecule is identified. In a preferred embodiment, changes in the localization pattern of the detectable molecule can be identified. The localization pattern may indicate the cellular distribution of the detectable molecule. Certain detection methods are described in the United States and Provisional Application No. 10 / 095,620, filed on March 12, 2002, claiming priority of US Provisional Application No. 60 / 275,339, filed on March 13, 2001. (Herein incorporated by reference).
[0204]
The molecule can also be detected by interaction with another detectably labeled molecule, such as an antibody.
[0205]
[Test kit]
In a further embodiment of the present invention, a commercially available test kit can be provided that includes a screening assay system for potential drugs effective in modulating GPCR activity. A test kit can include a cell, nucleic acid, or protein described herein. The test kit can be used to perform any of the methods described herein. The GPCR of interest can be introduced into the host cells of the test kit. The test kit may be useful for identifying whether the GPCR is expressed at the plasma membrane, the phosphorylated or non-phosphorylated GPCR binds to arrestin, or the phosphorylated or non-phosphorylated GPCR is internalized. The test kit may be useful for identifying compounds that alter the desensitization of a GPCR of interest.
[0206]
The GPCR can be introduced into a test system, and the expected drug can be introduced into the resulting cell culture, and the culture can then be cultured for GPCR activity (eg, signal transduction, recycling, affinity for arrestin). (E.g., gender).
[0207]
The following examples are provided to more fully illustrate the preferred embodiments of the present invention. However, the examples should not be construed as limiting the scope of the invention.
[0208]
(Example)
[Example 1]
[Experimental procedure]
The present inventors sub-screened the bovine GRK-C20 cDNA (Inglese et al., Nature, 1992) on the expression vector pcDNA3.1zeo + (Invtrogen). Expression of this cDNA produces GRK2 with a CAAX motif (C is cysteine, A is a small aliphatic residue and X is an uncharged amino acid) attached to the carboxyl terminus. GRK2, a specific CAAX motif bound to the end of CVLL, directs geranylgeranylation (C20 isoprenylation) of this protein. It is localized to the plasma membrane by an enzyme-directed covalent attachment of a 20-carbon geranylgeranyl lipid group to the carboxyl terminus of GRK2 (Inglese et al., Nature, 1992).
[0209]
[Cell culture]
Human fetal kidney (HEK-293) cells were purchased from the American Type Culture Collection (ATCC) and supplemented with 10% (v / v) heat-inactivated fetal bovine serum and gentamicin (100 μg / ml) Eagle's minimum essential medium (EMEM). HEK-293 cells stably expressing arrestin-GFP (HEK293-ArrGFP) were generated by standard procedures using G418 selection (0.4 mg / ml). HEK-293 cells were transiently transfected with arrestin-GFP, the GPCR of interest, and GRK2-20. For control experiments performed in equilibrium, HEK-293 cells were transiently transfected using Arrestin-GFP, using the desired GPCR, and without GRK2-20. HEK-293 cells were transiently transfected with GPCR of interest and GRK2-20 using arrestin-GFP. For parallel control experiments, HEK293 cells transfected with arrestin-GFP were transfected using the GPCR of interest and without GRK2-C20. All transfections were performed by the calcium phosphate precipitation method described above (Oakley et al., 1999). After transfection, cells were maintained in culture medium (EMEM supplemented with 10% FCS and 10 μg / ml gentamicin) for about 24 hours. The cells were then plated in 35 mm glass bottom dishes (MatTek) and incubated for an additional 16-24 hours. Transformed GPCRs were incubated with known GPCRs (receptors whose natural ligands are known) and orphan GPCRs (receptors whose natural ligands have not yet been identified).
[0210]
[Mirror focus microscope]
Transfected HEK-293 cells were plated in 35 mm glass bottom dishes (MatTek) and incubated overnight. The next day, the medium was removed, replaced with serum-free medium supplemented with 10 mM HEPES, and further incubated at 37 ° C. for 1 hour. The distribution of arrestin-GFP was then evaluated using a Zeiss laser scanning confocal microscope (LSM 5 Pascal). Using singleline excitation (488 nm) and an LP505 emission filter, images containing 63 times the oil object were obtained from living cells.
[0211]
[Example 2]
[Agonist-independent desensitization of known GPCR upon expression of modified GRK]
The present invention provides that the overexpression of GRK2-C20 (Inglese et al., Nature, 192) expressed at the plasma membrane in cells expressing arrestin-GFP promotes arrestin-GFP binding to GPCRs in the absence of ligand. To be identified.
[0212]
HEK293 cells transfected with arrestin-GFP were transiently transfected with the desired GPCR with or without GRK2-C20. Arrestin-GFP distribution was determined using confocal microscopy. Desensitization of arrestin-GFP at clathrin-coated pits, endocytic vesicles, endosomes, or other stages of the desensitization pathway indicated arrestin-GFP binding to GPCRs. Therefore, desensitization of GPCRs visualized by arrestin-GFP binding to GPCRs was analyzed.
[0213]
In the absence of agonist, arrestin-GFP is localized to small pits (probably clastin-coated pits) at the plasma membrane in cells expressing either GRK2-20C and cannabinoid type 2 receptor (CB2R) (FIG. 4). In addition, in the absence of agonist, arrestin-GFP can bind to GRK2-C20 and angiotensin II type IA receptor (AT1AR), vasopressin V2 receptor (V2R) (FIG. 4), or neurokinin1 / substrate P receptor ( NK-1 or SPR) were localized in endocytic vesicles in cells expressing either. In control cells that express each receptor (CB2R, AT1AR, V2R, or SPR), but lack GRK2-C20, arrestin-GFP is scattered and expressed in the cytoplasm and pits or small intracellular cells at the plasma membrane. It was not localized to any significant extent in the vesicle (FIG. 4).
[0214]
[Example 3]
[Agonist-independent desensitization of orphan GPCR upon expression of modified GRK]
The present invention provides that the overexpression of GRK2-C20 (Inglese et al., Nature, 192) expressed at the plasma membrane in cells expressing arrestin-GFP promotes arrestin-GFP binding to GPCRs in the absence of ligand. To be identified.
[0215]
As described above, HEK293 cells transiently transfected with arrestin-GFP were transiently transfected with the GPCRs of interest and with or without GRK2-C20. Arrestin-GFP distribution was determined using confocal microscopy. Desensitization of arrestin-GFP at clathrin-coated pits, endocytic vesicles, endosomes, or other stages of the desensitization pathway indicated arrestin-GFP binding to GPCRs. Therefore, desensitization of GPCRs visualized by arrestin-GFP binding to GPCRs was analyzed.
[0216]
In the absence of agonist, arrestin-GFP localized to small pits (probably crustin-coated pits) at the plasma membrane in cells expressing the orphan receptor GPR55 (FIG. 7). In control cells that express GPR55 but lack GRK2-C20, arrestin-GFP is scattered in the cytoplasm and is not localized to any significant extent in pits or intracellular vesicles at the plasma membrane. (FIG. 7). Other orphan GPCRs are described below.
[0219]
[Example 4]
[Method for analyzing arrestin binding ability of GPCR]
The present inventors have developed a method for identifying whether a GPCR of interest is expressed at the plasma membrane. Preferably, the expression of the orphan GPCR can be analyzed in a host cell where the GPCR is desensitized in an agonist-independent manner as described herein.
[0218]
As described above, HEK293 cells transiently transfected with arrestin GFP were transiently transfected with the GPCRs of interest and with or without GRK2-C20. Arrestin-GFP distribution was determined using confocal microscopy. Desensitization of arrestin-GFP at clathrin-coated pits, endocytic vesicles, endosomes, or other stages of the desensitization pathway indicated arrestin-GFP binding to GPCRs. Therefore, desensitization of GPCRs visualized by arrestin-GFP binding to GPCRs was analyzed.
[0219]
Certain GPCRs have been localized in clathrin-coated pits, endocytic vesicles, endosomes, or other stages of the desensitization pathway, as described above. This localization indicates that the GPCR has the ability to bind arrestin, since arrestin binding needs to be subsequently localized in the desensitization pathway. GPCRs that do not bind arrestin do not enter or localize the desensitization pathway. GPCRs that do not bind to arrestin can be altered to bind to arrestin. We have modified certain GPCRs to enhance arrestin affinity, as described below.
[0220]
[Example 5]
[Method for increasing the arrestin binding ability of GPCR]
We modified the GPCR to enhance its binding to arrestin. These modifications are described in US patent application Ser. No. 09 / 93,844. The GPCR was modified to be better phosphorylated by GRK at its C-terminal tail. These modified and phosphorylated GPCRs enhanced binding to arrestin. Demonstrated increased internalization. The letter E (enhanced phosphorylation) is added to the end of the name of the GPCR modified in this manner.
[0221]
A modified GPCR construct was created by the polymerase chain reaction according to standard protocols, which contains the HA epitope. A chimeric receptor was constructed in which the carboxy-terminal tail of the GPCR and V2R was exchanged one for the other after the putative palmitoylation site (Figure 3). The sequence of the DNA construct was confirmed by DNA sequencing.
[0222]
A nucleic acid of a target GPCR is prepared by adding a NotI restriction enzyme site (gcggccgc) immediately after a codon of a cysteine residue (presumed palmitoyl site) 10 to 25 amino acids (preferably 15 to 20) downstream of NRXY fused to the V2R carboxyl terminus. PCR amplification was performed with the transferred primers. The amplified receptor DNA fragment was then subcloned into the pEArrB-1 vector (US patent application Ser. No. 09 / 993,844) using a NotI restriction site and an additional restriction site upstream of the receptor atg start codon. When expressed, the modified GPCR contains a 31 amino acid peptide fused to the receptor carboxyl terminus. The first two amino acids are Ala residues due to the NotI site and the last 29 amino acids are from the V2R carboxyl terminus.
[0223]
We have modified the carboxyl-terminal tail of the following receptors described above and in US patent application Ser. No. 09 / 993,844. β2 adrenergic receptor (β2ARE), dopamine D1A receptor (D1ARE), mu opioid receptor (MORE), orphan GRK3 (GPR3E), orphan GRK6 (GPR6E), orphan GRK12 (GPR12E), orphan GRK7 (GPR7E), orphan GRK8 (GPR8E), orphan GRK55 (GPR55E), orphan GRKB2 (GPRB2E), and orphan GRKB3 (GPRB3E). “E” indicates “enhanced arrestin binding”. In the absence of an agonist, arrestin GFP localized to the endocytotic Commerce for each of the modified GPCRs described above when co-expressed with GRK2-C20 (FIGS. 4, 5, 6, and 7). For some of these modified receptors (orphan GPR6E), a small but significant amount of arrestin-GFP was localized in intracellular vesicles in regulatory cells lacking GRK2-C20 (FIG. 5). ). However, overexpression of GRK2-C20 at these receptors promoted a marked increase in this response (FIG. 5). The amino acid and nucleic acid sequences of these modified GPCRs, wild-type sequences, and HA-tagged modified GPCRs are shown in FIG. 3 and SEQ ID NOs: 35-90.
[0224]
[Example 6]
[Method for discriminating whether or not target GPCR is expressed in plasma membrane]
The present inventors have developed a method for identifying whether a GPCR of interest is expressed at the plasma membrane.
[0225]
HEK293 cells transiently transfected with arrestin-GFP were transiently transfected with the GPCRs of interest and with or without GRK2-C20. Arrestin-GFP distribution was determined using confocal microscopy. Desensitization of arrestin-GFP at clathrin-coated pits, endocytic vesicles, endosomes, or other stages of the desensitization pathway indicated arrestin-GFP binding to GPCRs. Therefore, desensitization of GPCRs visualized by arrestin-GFP binding to GPCRs was analyzed.
[0226]
Certain GPCRs have been localized in clathrin-coated pits, endocytic vesicles, endosomes, or other stages of the desensitization pathway, as described above. Since plasma membrane expression needs to be subsequently localized in the desensitization pathway, this localization indicates that the GPCR is expressed at the plasma membrane. GPCRs that are not expressed at the plasma membrane do not localize to the desensitization pathway. GPCRs that are not expressed at the plasma membrane can be altered to be expressed at the plasma membrane. For example, the expression of the GPCR can be altered, the amino acid sequence of the GPCR can be altered, or the GPCR can be transferred to another host cell.
[0227]
[Example 7]
[Monitoring of desensitization of GPCR mutant]
Desensitization in cells containing GPCR variants can be monitored. Desensitization of GPCR variants may depend on GRK overexpression.
[0228]
Human β containing point mutation ₂ The vector containing AR-EY-326A (in which tyrosine residue 326 has been converted to alanine) is transfected into cells expressing arrestin-GFP and GRK that can be modified. "E" indicates that the GPCR has been modified as described above. The Y326 mutant makes the GPCR dependent on overexpressed GRK for phosphorylation and subsequent desensitization. β ₂ Desensitizes upon expression of GRK-C20 in the absence of AR-Y326A agonist. GRK expression can be altered, including methods of altering the amount of GRK nucleic acid in a cell using an inducible promoter, a replication regulatory mechanism such as an origin of replication, and manually altering the amount of a vector in a cell. Including methods.
[0229]
Cells are seeded in 96-well or higher density plates and incubated overnight. The next morning, only the activator of the induction system or excipient is added to the wells to induce overexpression of GRK or modified GRK. An agonist is added to cells expressing GRK.
[0230]
The compound of interest is then added to the wells to see if they alter arrestin-GFP internalization. The cells are then fixed with 2% paraformamide and the amount of arrestin-GFP translocation is measured using an image analysis system.
[0231]
Although the invention is illustrated herein with reference to various specific materials, procedures, and examples, the invention is not limited to the specific material combinations and procedures selected for this purpose. It is understood that. As will be appreciated by those skilled in the art, many variations on such items may be contemplated.
[0232]
The following is a list of documents relating to the above disclosure as well as experimental procedures and considerations. The following documents and any documents referenced in the above specification should be considered to be incorporated herein by reference.
[Table 1]

[Table 1 (continued)]

[Brief description of the drawings]
FIG. 1A is a list of GPCRs that can be used in the present invention.
FIG. 1B is a list of GPCRs that can be used in the present invention.
FIG. 1C is a list of GPCRs that can be used in the present invention.
FIG. 2 is a list of GRKs that can be used in the present invention. 1 shows the amino acid and nucleic acid sequences of certain GRKs. 1 shows the amino acid sequence and nucleic acid sequence of GRK2-C20 (modified GRK).
FIG. 3 is a list of GPCRs with enhanced affinity for arrestin. 1 shows an amino acid sequence and a nucleic acid sequence.
FIG. 4 shows agonist-independent translocation of arrestin GFP to GPCR in the presence of GRK2-C20.
FIG. 5 shows agonist-independent translocation of arrestin GFP to GPCR in the presence of GRK2-C20.
FIG. 6 shows agonist-independent translocation of arrestin GFP to GPCR in the presence of GRK2-C20.
FIG. 7 shows agonist-independent translocation of arrestin GFP to GPCR in the presence of GRK2-C20.

Claims (49)

A method for identifying a compound that alters the internalization of a GPCR, comprising:
(A) obtaining a cell comprising a GPCR, an arrestin, and a modified GRK, wherein said GPCR is at least partially internalized in an agonist-independent manner upon expression of said GRK. Process and
(B) exposing the cells to the compound;
(C) determining the cell distribution of the GPCR, arrestin, or modified GRK;
(D) (1) the cellular distribution of the GPCR, arrestin, or modified GRK in cells in the presence of the compound; and (2) the GPCR, arrestin, or modified in cells in the absence of the compound. Monitoring the difference of GRK from the cell distribution. 2. The method of claim 1, wherein said GRK of step (a) is overexpressed. 2. The method of claim 1, wherein said GRK expression in step (a) is inducible. 2. The method of claim 1, wherein said GRK comprises a CAAX motif. 2. The method of claim 1, wherein the GPCR has been modified by GRK to enhance phosphorylation. _2. The method of claim 1, wherein said GPCR is β2AR (Y326A). 2. The GPCR of claim 1, wherein the GPCR is a GPCR, an orphan GPCR, a modified GPCR, a taste receptor, a class A GPCR, a class B GPCR, a mutant GPCR, or a biologically active fragment thereof as listed in FIG. the method of. 2. The method of claim 1, wherein said GRK is GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, or a biologically active fragment thereof. 2. The method of claim 1, wherein said GPCR, GRK, and arrestin are detectably labeled. 2. The method of claim 1, wherein the molecule associated with desensitization is detectably labeled, or a molecule that interacts with the molecule associated with desensitization is detectably labeled. 2. The method of claim 1, wherein the arrestin is visual arrestin, cone arrestin, beta arrestin 1, beta arrestin 2, or a biologically active fragment thereof. 2. The method according to claim 1, wherein no agonist is obtained. The method of claim 1, wherein the difference between (1) and (2) in step (d) indicates that GPCR internalization is regulated. A method for identifying a compound that alters phosphorylation of a GPCR, comprising:
(A) obtaining a cell containing a GPCR and GRK;
(B) exposing the cells to the compound;
(C) identifying whether GRK phosphorylation of a GPCR is altered in the presence of said compound. 15. The method of claim 14, wherein the cellular distribution of the GPCR or GRK is determined. Monitoring the difference between (1) the cellular distribution of the GPCR or GRK in the cell in the presence of the compound and (2) the cellular distribution of the GPCR or GRK in the cell in the absence of the compound. The method according to claim 14. 17. The method of claim 16, wherein the difference between (1) and (2) is correlated with phosphorylation of the GPCR. 15. The method of claim 14, wherein the GRK is absent from the plasma membrane, indicating that GRK phosphorylation of the GPCR has been altered. 15. The method of claim 14, wherein the phosphorylation status of the GPCR is determined. 15. The method of claim 14, wherein said GRK activity is determined. 18. The method of claim 17, wherein the ability of the GPCR to internalize is determined. A method for identifying whether a GPCR of interest is expressed at the plasma membrane,
(A) obtaining a cell comprising a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled;
(B) determining the cell distribution of the arrestin;
(C) correlating the cellular distribution of the arrestin with the ability of the GPCR to express at the plasma membrane. 23. The method of claim 22, wherein the arrestin is localized in vesicles, pits, endosomes, or elsewhere in the desensitization pathway. A method for determining whether a GPCR of interest is expressed at the plasma membrane, comprising:
(A) obtaining a GPCR, a GRK, and a cell containing the GRK, wherein the GRK is detectably labeled;
(B) determining the cell distribution of the GRK;
(C) correlating the cell distribution of the GRK with the ability of the GPCR to express at the plasma membrane. 25. The method of claim 24, wherein said GRK is located at the plasma membrane. A method for analyzing the arrestin binding ability of a GPCR, comprising: (a) obtaining a cell comprising a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled;
(B) determining the cell distribution of the arrestin;
(C) correlating the cell distribution of the arrestin with the arrestin binding ability of the GPCR. 27. The method of claim 26, wherein the arrestin or CPGR is located in vesicles, pits, or endosomes. A compound identified by claim 1. A method of treating a disease by modulating desensitization of a GPCR in a host cell, comprising:
(A) obtaining the compound identified in claim 1;
(B) administering the compound to a host. A host cell containing a GPCR and a modified GRK. 31. The host cell of claim 30, wherein said GRK is inducible or overexpressed. 31. The host cell of claim 30, wherein the host cell further comprises arrestin, wherein the arrestin can be detectably labeled. 31. The host cell of claim 30, wherein at least one GPCR, GRK, another molecule associated with desensitization, or a molecule that interacts with a molecule associated with desensitization is detectably labeled. A method for modifying a nucleic acid encoding a GRK in which a GPCR is constitutively internalized, comprising:
(A) obtaining a nucleic acid encoding GRK;
(B) modifying the nucleic acid encoding the GRK so that the encoded GRK contains a CAAX motif, wherein the modified GRK phosphorylates a GPCR in the presence of an agonist;
(C) expressing the modified GRK in the cells. 35. The nucleic acid encoding GRK comprises SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The method described in. A kit for identifying a compound that regulates internalization of the GPCR, comprising the host cell according to claim 30. A modified GPCR comprising an NPXXXY motif and a carboxyl terminal tail,
The carboxyl-terminal tail comprises a putative palmitoylation site and one or more phosphorylation clusters;
The carboxyl-terminal tail comprises a retaining portion of the carboxyl-terminal region of the first GPCR portion fused to a portion of the carboxyl terminus from the second GPCR;
Wherein said second GPCR comprises one or more phosphorylation clusters and further comprises a second putative palmitoylation site about 10 to 25 amino acid residues downstream of the second NPXXXY motif. . 38. The modified GPCR of claim 37, wherein said first GPCR is a class A receptor. 38. The modified GPCR of claim 37, wherein said first GPCR is hGPR3, hGPR6, hGPR12, hSREB2, hSREB3, hGPR8, or hGPR22. 38. The modified GPCR of claim 37, wherein said second GPCR is a class B receptor. 38. The modified GPCR of claim 37, wherein said class B receptor is selected from the group consisting of a vasopressin V2 receptor, a neurotensin 1 receptor, a substrate P receptor, and an oxytocin receptor. A nucleic acid encoding the modified GPCR of claim 37. SEQ ID NOs: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, A nucleic acid selected from the group consisting of 84, 86, 88, and 90. An expression vector comprising the nucleic acid according to claim 42. A host cell comprising the expression vector according to claim 44. A host cell comprising the nucleic acid of claim 42. A method of screening a compound for GPCR activity, comprising:
(A) obtaining a cell that expresses at least one modified GPCR of claim 37, wherein the cell further comprises arrestin conjugated to a detectable molecule;
(B) exposing the cells to the compound;
(C) detecting the position of arrestin in the cell;
(D) comparing the position of arrestin in the cell in the presence of the compound with the position of arrestin in the cell in the absence of the compound;
(E) correlating the difference between (1) the position of arrestin in the cell in the presence of the compound and (2) the position of arrestin in the cell in the absence of the compound. 50. The method of claim 47, wherein said arrestin is detected in endosomes, endocytic vesicles, or pits. 38. To identify a molecule that modulates the activity of a GPCR, comprising a cell that expresses at least one modified GPCR of claim 37, wherein said cell further comprises a molecule associated with desensitization conjugated to a detectable molecule. Kit.

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