Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Utility Patent
1993-03-10
2001-01-02
Ulm, John (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S252300, C435S320100
Utility Patent
active
06169173
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the purification of cholecystokinin (CCK) receptor protein to sequenceable-grade homogeneity. The present invention further relates to obtaining and expressing DNAs that code for CCK receptor protein.
A family of cholecystokinin peptides was originally isolated from the mammalian gastrointestinal tract and was one of the first gastrointestinal peptides to be discovered in the brain. The predominant molecular form of CCK peptide is cholecystokinin-octapeptide (CCK-8) which exists in a sulfated and non-sulfated form.
The cholecystokinin family of receptors is widely distributed throughout the gastrointestinal and central nervous systems where they regulate pancreatic and gastric secretion, smooth muscle motility, growth, anxiety and satiety, analgesia and neuroleptic activity. The receptor family includes CCK
A
and CCK
B
/gastrin receptors by virtue of their affinity for a structurally and functionally related family of peptides, including CCK and CCK analogues having identical COOH terminal pentapeptide sequence and varying sulfation at the sixth (gastrin) and seventh (CCK) tyrosyl residues. CCK
B
receptors more recently have been designated CCK
B
/gastrin receptors because of the suspected homology, perhaps even identity, between CCK
8
and gastrin receptors. Kopin et al.,
PNAS
USA 89: 3605 (1992).
Recently, nonpeptide agonists highly selective for each of the CCK receptor subtypes have been developed and further support the subtype classification. The most potent and selective antagonists are L-364,718 for CCK
A
and L-365,260 and PD134408 for CCK
B
/gastrin receptors. The CCK
A
receptor differs from CCK
B
/gastrin receptor particularly in its selectivity for CCK peptide analogues with a sulfate at the seventh position from the COOH terminus.
The CCK
A
receptor mediates physiologic gallbladder contraction, pancreatic growth and enzyme secretion, delayed gastric emptying, relaxation of the sphincter of oddi and potentiation of insulin secretion. The CCK
A
receptor also appears in the anterior pituitary, in the myenteric plexus, and in areas of the CNS (midbrain) where CCK interaction with dopaminergic neurons has been implicated in the pathogenesis of a schizophrenia, Parkinson's disease, drug addiction and feeding disorders. Experimental rat pancreatic carcinogenesis is promoted by CCK through the CCK
A
receptor.
CCK
A
receptors in pancreatic acinar cells have been most well characterized because of the ability to prepare a homogeneous preparation of a hormonally responsive effector system in dispersed acini. In pancreatic acinar cells, CCK peptide interacts specifically with its cell surface receptor, which is coupled to a guanine nucleotide regulatory protein (G protein) which activates phospholipase C, the breakdown of phosphoinositides, the mobilization of intracellular calcium, and the activation of protein kinase C.
CCK
A
receptors have been functionally expressed in the plasma membrane of oocytes after injection of rat brain total RNA, and of mRNA from rat pancreatic acinar carcinoma cell line, AR42J. Affinity labeling studies of CCK
A
receptors from rat pancreas and partial purification demonstrate an 85-95 kDa, heavily glycosylated, binding subunit with a deglycosylated core protein of 42 kDa.
The CCK
B
/gastrin receptor is found predominantly throughout the CNS, where it is thought to modulate anxiety and neuroleptic activity. Interaction between CCK peptide and CCK
B
/gastrin receptors on mesocorticolimbic, dopaminergic neurons influences the physiological states of stress and anxiety. The presence of CCK
B
/gastrin receptors on peripheral monocytes and monocyte-derived splenic cells suggests that CCK plays a role in the long suspected neuroendocrine modulation of the immune system.
CCK
B
/gastrin receptors, found on gastric parietal and chief cells, and gastrointestinal smooth muscle cells, regulate acid and pepsinogen secretion, and gastrointestinal motility, respectively. They are also present on some human gastric and colon cancer cells where they may regulate growth. CCK peptide, acting at peripheral CCK
A
receptors and at central CCK
A
and CCK
B
/gastrin receptors plays a significant role in the nervous system control of appetite.
Attempts have been made to purify CCK receptor protein to homogeneity, but these efforts were unsuccessful. Duong et al.,
J. Biol. Chem
. 264: 17990-96 (1989), used digitonin-solubilized rat pancreatic receptor to obtain a receptor preparation estimated to be of 80% purity. The Duong purification scheme included a three-step purification process utilizing cation exchange, Ulex europaeus agglutinin-I-agarose, and Sephacryl S-300. Szecowka et al.,
Regulatory Peptides
24: 215-24 (1989), employed a two-step purification scheme to partially purify digitonin-solubilized rat pancreatic receptor that included lectin and CCK affinity chromatography.
Researchers labored unsuccessfully for years to illuminate the molecular -structure of CCK receptor protein, but were limited by the inability to purify receptor protein to a homogeneity sufficient for sequencing purposes. Instead, attempts to obtain a purified preparation yielded a partially purified CCK receptor, along with non-CCK receptor proteins. As a result, the accuracy in studies relating, for example, to binding affinities and electrophysiology, was compromised by the inability to study a particular subtype without contamination by another type. Further, the inability to purify CCK receptor to sequenceable homogeneity prohibited cloning of receptor-encoding DNAs and the recombinant expression of a particular CCK receptor in a transformed cell line.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a method for obtaining CCK receptor protein, in a homogeneous form suitable for amino acid sequencing.
It is also an object of the present invention to provide DNA molecules that encode CCK receptor protein, as well as host cells that are transformed with such a DNA and that expresses functional CCK receptor protein.
In accomplishing these and other objects, there has been provided, in accordance with one aspect of the present invention, an isolated DNA molecule encoding a CCK receptor protein. In a preferred embodiment, the DNA molecule comprises a first nucleotide sequence that consists of nucleotides 199 to 1485 of FIGS.
1
A-E (SEQ ID NO:13), or a DNA sequence which hybridizes under high stringency conditions to an oligonucleotide probe consisting of nucleotides 199 to 1485. In another preferred embodiment, a DNA molecule can comprise, in addition to the aforementioned first nucleotide sequence, a second nucleotide sequence consisting of nucleotides 154 to 198 of FIGS.
1
A-E (SEQ ID NO:13), which second nucleotide sequence is positioned immediately upstream of nucleotide 199 of the first nucleotide sequence.
In another preferred embodiment, an isolated DNA molecule encoding a CCK receptor protein comprises a nucleotide sequence consisting of nucleotides 136 to 1491 of FIGS.
2
A-E (SEQ ID NO:15) or a DNA sequence which hybridizes under high stringency conditions to an oligonucleotide probe consisting of nucleotides 136 to 1491 of FIGS.
2
A-F (SEQ ID NO:15).
In another preferred embodiment of the invention, an isolated DNA molecule encoding a human CCK receptor protein comprises a nucleotide sequence consisting of nucleotides 1 to 1341 of FIGS.
12
A-E (SEQ ID NO:28) or a DNA sequence which hybridizes under high stringency conditions to an oligonucleotide probe consisting of nucleotides 1 to 1341 of FIGS.
12
A-E (SEQ ID NO:28).
In another preferred embodiment of the invention, an isolated DNA molecule encoding a human CCK receptor protein comprises a nucleotide sequence consisting of nucleotides 154 to 1437 of FIGS.
13
A-D (SEQ ID NO:30)or a DNA sequence which hybridizes under high stringency conditions to an oligonucleotide probe consisting of nucleotides 154 to 1437 of FIGS.
13
A-D (SEQ ID NO:30).
In accordance with another aspect of the present invention, an isolat
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The United States of America as represented by the Department of
Ulm John
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