G protein-coupled receptors from the rat and human

Details G protein-coupled receptors from the rat and human G protein-coupled receptors from the rat and human

1999-03-03

2004-02-24

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S069100, C435S320100, C435S325000, C530S350000

Reexamination Certificate

active

06696257

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the general field of biological receptors and the various uses that can be made of such receptors. More specifically, the invention relates to nucleic acids encoding novel G protein-coupled receptors and to the receptors per se.
BACKGROUND AND PRIOR ART
G protein-coupled receptors (GPCRs) constitute a family of proteins sharing a common structural organization characterized by an extracellular N-terminal end, seven hydrophobic alpha helices putatively constituting transmembrane domains and an intracellular C-terminal domain. GPCRs bind a wide variety of ligands that trigger intracellular signals through the activation of transducing G proteins (Caron, et al.,
Rec. Prog. Horm. Res
. 48:277-290 (1993); Freedman et al.,
Rec. Prog. Horm. Res
. 51:319-353 (1996)).
More than 300 GPCRs have been cloned thus far and it is generally assumed that there exist well over 1000 such receptors. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Cudermann, et al.,
J. Mol. Med
. 73:51-63 (1995)). Of particular interest are receptors located in dorsal root ganglia. This region of the central nervous system is densely innervated with primary or afferent sensory neurons involved in the transmission, modulation and sensation of pain. Thus, receptors from this region may be used in assays for the identification of new agents for anesthesia and analgesia
SUMMARY OF THE INVENTION
The present invention is based upon the discovery of a novel G protein-coupled receptor which is distinct from previously reported receptors in terms of structure and in being expressed preferentially in dorsal root ganglia. One dorsal root receptor (DRR) has been isolated and sequenced from the rat and six from the human. The rat receptor was given the designation rDRR-1 and its amino acid sequence is shown as SEQ ID NO:1. The human receptors were designated as
hDRR-1 (SEQ ID NO:3);
hDRR-2 (SEQ ID NO:5);
hDRR-3 (SEQ ID NO:7);
hDRR-4 (SEQ ID NO:9);
hDRR-5 (SEQ ID NO:11); and
hDRR-6 (SEQ ID NO:13).
Unless otherwise specified, the term “DRR” as used herein refers to all of the receptors from both human and rat.
In its first aspect, the invention is directed to proteins, except as existing in nature, comprising the amino acid sequence consisting functionally of a rat or human DRR as shown in SEQ ID NO:1, 3, 5, 7, 9, 11, or 13. The term “consisting functionally of” is intended to include any receptor protein whose sequence has undergone additions, deletions or substitutions which do not substantially alter the functional characteristics of the receptor. Thus, the invention encompasses proteins having exactly the same amino acid sequence as shown in the sequence listing, as well as proteins with differences that are not substantial as evidenced by their retaining the basic, qualitative binding properties of the DRR receptor. The invention further encompasses substantially pure proteins consisting essentially of a DRR amino acid sequence, antibodies that bind specifically to a DRR (i.e. that have at least a 100 fold greater affinity for the DRR than any other naturally occurring non-DRR protein), and antibodies made by a process involving the injection of pharmaceutically acceptable preparations of such proteins into an animal capable of antibody production. In a preferred embodiment, monoclonal antibody to human or rat DRR is produced by injecting a pharmaceutically acceptable preparation of the receptor into a mouse and then fusing mouse spleen cells with myeloma cells.
The invention is also directed to a substantially pure polynucleotide encoding a protein comprising the amino acid sequence consisting functionally of the sequence of rat DRR (as shown in SEQ ID NO:1) or a human DRR (as shown in SEQ ID NOs 3, 5, 7, 9, 11 or 13). This aspect of the invention encompasses polynucleotides encoding proteins consisting essentially of the amino acid sequences shown in the sequence listing, expression vectors comprising such polynucleotides, and host cells transformed with such vectors. Also included are the recombinant rat and human DRR proteins produced by host cells made in this manner.
Preferably, the polynucleotide encoding rat DRR has the nucleotide sequence shown in SEQ ID NO:2 and the polynucleotide encoding a human DRR has the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, 9, 11 or 13. It is also preferred that the vectors and host cells used for the expression of DRR contain these particular polynucleotides.
In another aspect, the present invention is directed to a method for assaying a test compound for its ability to bind to a rat or human DRR. The method is performed by incubating a source of DRR with a ligand known to bind to the receptor and with the test compound. The source of the DRR should be substantially free of other types of G protein-coupled receptors, i.e. greater than 85% of such receptors present should correspond to the DRR. Upon completion of incubation, the ability of the test compound to bind to the DRR is determined by the extent to which ligand binding has been displaced. The rat DRR should, preferably correspond to rDRR-1 as shown in SEQ ID NO:1. The human receptor should preferably be HDRR-1 (SEQ ID NO:3); hDRR-2 (SEQ ID NO:5); hDRR-3 (SEQ ID NO:7); hDRR-4 (SEQ ID NO:9); hDRR-5 (SEQ ID NO: 11); or hDRR-6 (SEQ ID NO:13). Either transformed cells expressing recombinant DRR may be used in the assays or membranes can be prepared from the cells and used. Although not essential, the assay can be accompanied by the determination of the activation of a second messenger pathway such as the adenyl cyclase pathway. This should help to determine whether a compound that binds to DRR is acting as an agonist or antagonist.
An alternative method for determining if a test compound is an agonist of any of the DRRs disclosed herein is to use a cell signaling assay, e.g., an assay measuring either intracellular adenyl cyclase activity or intracellular calcium concentration. The test compound is incubated with cells expressing the DRR but substantially free of other G protein-coupled receptors, typically a cell transfected with an expression vector encoding the DRR. Test compounds that are agonists are identified by their causing a statistically significant change in the results obtained from the cell signaling assay when compared to control transfectants not exposed to test compound. For example, the cells exposed to the test compound may show a significant increase in adenyl cyclase activity or in intracellular calcium concentration.
The invention also encompasses a method for determining if a test compound is an antagonist of a DRR which relies upon the known activation of G protein-coupled receptors that occurs when such receptors are expressed in large amounts. This method requires that DNA encoding the receptor be incorporated into an expression vector so that it is operably linked to a promoter and that the vector then be used to transfect an appropriate host. In order to produce sufficient receptor to result in constitutive receptor activation (i.e., activation in the absence of natural ligand), expression systems capable of copious protein production are preferred, e.g., the DRR DNA may be operably linked to a CMV promoter and expressed in COS or HEK293 cells. After transfection, cells with activated receptors are selected based upon their showing increased activity in a cell signaling assay relative to comparable cells that have either not been transfected or that have been transfected with a vector that is incapable of expressing functional DRR. Typically, cells will be selected either because they show a statistically significant increase in intracellular adenyl cyclase activity or a statistically significant increase in intracellular calcium concentration. The selected cells are contacted with the test compound and the cell signaling assay is repeated to determine if this results in a decrease in activity relative to control cells not contacted with the test compound. For example, a stat

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