Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-01-27
2002-08-27
Pak, Michael (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100
Reexamination Certificate
active
06441133
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of biological receptors and the various uses that can be made of such receptors. More specifically, it relates to nucleic acids encoding a novel receptor for thyrotropin-releasing hormone and to the receptor protein itself.
BACKGROUND OF THE INVENTION
Thyrotropin-releasing hormone (TRH) is a tripeptide (pyroglutamic acid-histidine-proline-amide) present in the central nervous system (thalamus, cerebral cortex, spinal cord) as well as in the periphery (pancreas, gastrointestinal tract, placenta). In the hypothalamus, TRH is synthesized by peptidergic neurons of supraoptic and paraventricular nuclei. It is then axonally transported to the median eminence where it is stored. Upon secretion into the bloodstream, TRH is transported to the pituitary where it stimulates the production of thyroid stimulating hormone (TSH) which, in turn, stimulates the production of thyroxin (T4) in the thyroid gland (Gaillard, in
Pharmacologie: Des Concepts Fondamentaux Aux Applications Thérapeutiques,
M. Shorderet ed., pp. 415-448 (1992)).
In addition to its role in regulating the synthesis and secretion of hormones from the anterior pituitary, there is evidence that TRH acts as a neurotransmitter (Wu, et al.,
Neurosci. Let.
142:143-146 (1992)). TRH is found abundantly in the central nervous system and exogenous administration of TRH elicits a variety of behavioral changes. It produces a rapid onset, neurotransmitter-like, excitation of spinal lower motor neurons and reduces neurological deficits observed after traumatic spinal cord injury in cats.
The distribution of TRH-containing cells, fibers or receptors suggests a potential role for TRH in the perception of noxious stimuli. Specifically, TRH is present in the periaqueducal gray (PAG), the nuclei raphe magnus (NMR), in the pallidus and dorsal horn of the spinal cord. TRH binding sites have been found in the brain, pituitary, dorsal and ventral horns of the spinal cord, and in peripheral tissues. When injected centrally (I.C.V. and I.C.), TRH induces a short lasting supraspinal antinociception. The analgesia induced by I.C.V. TRH injection is twice as great, on a molar basis, as that induced by morphine (Boschi, et al.,
Br. J. Pharmacol.
79:85-92 (1983)). This antinociceptive effect is detected in models of chemically and mechanically, but not thermally, induced pain.
The actions of TRH are mediated by the stimulation of specific cell surface receptors. There is evidence that TRH receptors found in the pituitary transmit their signal to the cell interior through a G protein to trigger the inositol phospholipid-calcium-protein kinase C transduction pathway (Straub, et al.,
Proc. Nat'l. Acad. Sci. U.S.A.
87:9514-9518 (1990); Duthie, et al.,
Mol. Cell. Endocrinol.
95:R11-R15 (1993)). A cDNA sequence encoding a G protein-coupled TRH receptor was first isolated from mouse pituitary cells using an expression cloning strategy (Straub, et al.,
Proc. Nat'l. Acad. Sci. U.S.A.
87:9514-9518 (1990)). Subsequently, several groups have described the cloning of rat TRH receptor cDNAs expressed in either a pituitary tumor cell line (GH3) or in pituitary gland (Duthie, et al.,
Mol. Cell. Endocrinol.
95:R11-R15 (1993); De La Pena, et al.,
J. Biol. Chem.
267:25703-25708 (1992)). In addition, two isoforms of the rat TRH receptor have been shown to be generated from a single gene by alternative splicing (De La Pena, et al.,
J. Biol. Chem.
267:25703-25708 (1992)).
In addition to receptors isolated from the mouse and rat, a human TRH receptor cDNA has been cloned by Matre et al. (
Biochem. Biophys. Res. Comm.
195:179-185 (1993)). With the exception of its C-terminal region, the predicted amino acid sequence of the human receptor was found to be more than 95% homologous to its counterparts from the rat and mouse.
Using synthetic TRH analogues, a dissociation of endocrine and CNS effects has been observed, suggesting that subtypes of TRH receptor may exist. Certain analogues were found to affect sleeping time and breathing frequency in test animals even though they failed to bind to pituitary or brain receptors and had no measurable TSH release activity (Alexandrova, et al.,
Gen. Physiol. Biophys.
10:287-297 (1991)). Other analogues, modified in the C-terminal region, have been identified which are ineffective in treating traumatic spinal cord injury but which maintain the same endocrine effects as normal TRH (Faden,
Brain Research
486:228-235 (1989)).
The existence of distinct TRH receptor subtypes has also been suggested by biochemical experiments. Specifically, TRH receptors isolated from the brain were found to have an isoelectric point of 5.5 whereas those isolated from the pituitary were found to have an isoelectric point of only 4.9. One possible explanation for this difference is that the receptors in the brain and those in the pituitary have different amino acid sequences (Burt,
Ann. NY Acad. Sci.
553:188 (1989)). In addition, electrophysiological experiments and measurements of intracellular calcium concentration have suggested that TRH and TRH metabolites present in the brain may act by binding to different subtypes of TRH binding sites (Toledo-Aral, et al.,
J. Physiol.
472:327-340 (1993)).
Therapeutically, it is clear that agonists and antagonists of TRH binding have potential value in regulating endocrine function, controlling pain, and in the treatment of spinal cord injury. The ability to identify such agents will depend upon the availability of purified TRH receptors suitable for binding assays. Such assays could be used to screen for TRH agonists and antagonists; to determine the extent to which a patient's plasma contains an appropriate level of binding activity; and to help monitor the purity and effectiveness of agents at all stages of drug development.
SUMMARY OF THE INVENTION
To date, the only TRH receptor which has been cloned has been designated TRHR-1. The present invention is based upon the discovery of a new receptor for TRH which differs from TRHR-1 in terms of structure, tissue distribution and binding characteristics. Thus, in its first aspect, the invention is directed to a protein, except as existing in nature, comprising the amino acid sequence consisting functionally of the sequence of SEQ ID NO:2. The term “consisting functionally of” refers to proteins in which the sequence of SEQ ID NO:2 has undergone additions, deletions or substitutions which do not substantially alter the functional characteristics of the receptor. The term is intended to encompass proteins having exactly the same amino acid sequence as that of SEQ ID NO:2, as well as proteins with sequence differences that are not substantial as evidenced by their retaining the basic, qualitative ligand binding properties of TRHR-2.
The invention also encompasses substantially pure proteins with sequences consisting essentially of that of SEQ ID NO:2; antibodies that bind preferentially to such proteins (i.e., antibodies having at least a 100-fold greater affinity for TRHR-2 than any other protein); and antibodies made by a process involving the injection of a pharmaceutically acceptable preparation of TRHR-2 into an animal capable of antibody production. In a preferred embodiment, monoclonal antibody to TRHR-2 is produced by injecting the pharmaceutically acceptable preparation of TRHR-2 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 SEQ ID NO:2. This aspect of the invention encompasses polynucleotides encoding proteins consisting essentially of the amino acid sequence of SEQ ID NO:2, expression vectors comprising such polynucleotides, and host cells transformed with such vectors. Also included is the recombinant TRHR-2 protein produced by host cells made in this manner. Preferably, the polynucleotide encoding TRHR-2 has the nucleotide sequence shown in SEQ ID NO:1, and the vectors and host cells used for exp
AstraZeneca Canada Inc.
Pak Michael
Sanzo Michael A.
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