Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-16
2002-09-10
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S242000, C435S320100, C530S333000, C530S350000
Reexamination Certificate
active
06448011
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Although adrenergic receptors (ARs) bind the same endogenous catecholamines (epinephrine and norepinephrine, NE) their physiological as well as pharmacological specificity is markedly diverse. This diversity is due primarily to the existence of at least nine different proteins encoding three distinct adrenergic receptors types (&agr;
1
, &agr;2, and &bgr;). These proteins belong to the super-family of G-protein coupled receptors, and are characterized by a single polypeptide chain which span the plasma membrane seven times, with an extracellular amino terminus, and a cytoplasmic carboxyl terminus. The molecular cloning of three genes encoding &agr;
1
-ARs supports the existence of pharmacologically and anatomically distinct &agr;
1
-receptor subtypes. The &agr;
1b
-receptor was originally cloned from a hamster smooth muscle cell line cDNA library, and encodes a 515 a.a. peptide that shows 42-47% homology with other ARs. The message for the &agr;
1b
-receptor is abundant in rat liver, heart, cerebral cortex and kidney, and its gene was localized to human chromosome 5 (4). A second cDNA clone from a bovine brain library was found which encoded a 466-residue polypeptide with 72% homology to the &agr;
1b
-AR gene. It was further distinguished from &agr;
1b
by the finding that its expression was restricted to human hippocampus, and by its localization to human chromosome 8 and it has been designated as the &agr;
1c
-AR (20). The cloning of an &agr;
1a
-AR has been reported recently. This gene, isolated from a rat brain cDNA library, encodes a 560-residue polypeptide that shows 73% homology with the hamster &agr;
1b
-receptor. The message for this subtype is abundant in rat vas deferens, aorta, cerebral cortex and hippocampus, and its gene has been localized to human chromosome 5 (12).
Pharmacological studies have demonstrated the existence of two &agr;
1
-adrenergic receptor subtypes. The studies of &agr;
1
-AR-mediated responses in vascular tissue suggested the possible existence of receptor subtypes, based on the potency and efficacy of adrenergic agonists, as well as differential sensitivity of &agr;
1
receptor-mediated responses to extracellular calcium and calcium channel blockers (6, 24). Although radioligand binding studies of brain &agr;
1
-ARs with either [
3
H]WB4101 and [
3
H]prazosin showed good agreement with the potency of &agr;-adrenergic antagonists on vascular responses (23, 10), subsequent binding studies of rat brain &agr;
1
-ARs provided strong evidence for the existence of receptor heterogeneity, based on the relative affinities for prazosin and WB4101 (15). These observations were supported by the finding that chloroethylclonidine (CEC) inactivated 50% of the &agr;
1
sites from rat cerebral cortex and 80% of the binding sites from liver or spleen (&agr;
1b
), but did not inactivate &agr;
1
-receptors from the hippocampus or vas deferens (&agr;
1a
) (14). Taken together, these results suggested a classification of the &agr;
1a
-subtype as high affinity for WB4101 and insensitive to alkylation by CEC, and &agr;
1b
-subtype as 10 to 20 fold lower affinity for WB4101, but sensitive to inactivation by CEC. Consistent with this evidence the transfection of the hamster &agr;
1b
gene into COS-7 cells induced the expression of an &agr;1-receptor with high affinity for WB4101, 95% of which could be inactivated by CEC. Conversely, upon expression of the rat &agr;
1a
receptor gene in COS-7 cells, it showed a 10-fold higher affinity for WB4101 than the &agr;
1b
-receptor, and the binding site was resistant to inactivation by CEC. The existence of the &agr;
1c
receptor was not predicted from pharmacological data and upon expression it showed 16 and 30 fold higher affinity for WB4101 and phentolamine respectively, than the &agr;
1b
-receptor and was partially inactivated (65%) by CEC.
Molecular cloning and pharmacological studies have demonstrated the existence of at least three &agr;
1
-adrenergic receptor subtypes. However, it is not clear whether the pharmacological properties of these three cognates might be due also to species differences. This caveat is particularly relevant in the case of the bovine &agr;
1c
receptor, due to its restricted species and tissue expression. The cloning and expression of the human &agr;
1
adrenergic receptors will allow the further characterization of the pharmacology of the individual human &agr;
1
receptor subtypes.
SUMMARY OF THE INVENTION
This invention provides and isolated nucleic acid molecule encoding a human &agr;
1
adrenergic receptor. This invention further provides an isolated nucleic acid molecule encoding a human &agr;
1a
receptor. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid pcEXV-&agr;
1a
. This invention also provides an isolated nucleic acid molecule encoding a human &agr;
1b
receptor. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid pcEXV-&agr;
1b
. This invention further provides an isolated nucleic acid molecule encoding a human &agr;
1c
receptor. In one embodiment of this invention, the nucleic acid molecule comprises a plasmid pcEXV-&agr;
1c
.
This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human &agr;
1a
receptor, adapted for expression in a bacterial, a yeast cell, or a mammalian cell which additionally comprise regulatory elements necessary for expression of the DNA in the bacteria, yeast or mammalian cells so located relative to the DNA encoding the human &agr;
1a
receptor as to permit expression thereof. This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human &agr;
1b
receptor, adapted for expression in a bacterial, a yeast cell, or a mammalian cell which additionally comprise regulatory elements necessary for expression of the DNA in the bacteria, yeast or mammalian cells so located relative to the DNA encoding the human &agr;
1b
receptor as to permit expression thereof. This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human &agr;
1c
receptor, adapted for expression in a bacterial, a yeast cell, or a mammalian cell which additionally comprise regulatory elements necessary for expression of the DNA in the bacteria, yeast or mammalian cells so located relative to the DNA encoding the human &agr;
1c
receptor as to permit expression thereof.
This invention provides a mammalian cell comprising a DNA molecule encoding a human &agr;
1a
receptor. This invention also provides a mammalian cell comprising a DNA molecule encoding a human &agr;
1b
receptor. This invention also provides a mammalian cell comprising a DNA molecule encoding a human &agr;
1c
receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human &agr;
1a
receptor. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human &agr;
1b
receptor. This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human &agr;
1c
receptor.
This invention provides an antisense oligonucleotide having a sequence capable of specifically binding to any sequences of an mRNA molecu
Bard Jonathan A.
Forray Carlos C.
Weinshank Richard L.
Cooper & Dunham LLP
Dunham Christopher C.
Synaptic Pharmaceutical Corporation
Wang Andrew
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