Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-08
2004-12-28
Wax, Robert A. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S252330, C435S254200, C435S320100, C435S358000, C536S023500
Reexamination Certificate
active
06835539
ABSTRACT:
The present invention relates to nucleic sequences encoding a protein capable of interacting with the AT2 receptor, to oligonucleotides contained in the said sequences, to their applications as probes and for the expression of the said proteins, to the vectors useful for the said expression, to the host cells containing the said vectors and to a model for studying the AT2 receptor.
The present invention also relates to the said proteins and to their applications.
The octapeptide, angiotensin II, mainly known as a regulator of blood pressure, has also been described as an important modulator of cell growth. Interestingly, this peptide appears to exert opposite effects on cell growth according to whether it is bound to one or the other of its two subtypes of membrane receptors (AT1 or AT2).
The AT2 receptor subtype, which also belongs to the G protein-coupled receptor family, is still poorly characterized both from the point of view of its mechanisms of activation and its physiological role (C. Nahmias et al.,
Trends Pharmacol Sci,
1995, 16, 223-225). Several arguments suggest, however, a role for this receptor in the phenomena of cell proliferation, differentiation or adhesion.
The AT2 receptor is highly expressed during foetal life, disappears in adults in most tissues, but becomes reexpressed under pathophysiological conditions involving restructuring of the tissues.
Studies carried out in vivo have demonstrated the inhibitory role exerted by the AT2 subtype on the proliferation of the muscle cells of the tunica intima vasorum after vascular lesion (P. Janiak et al.,
Hypertension,
1992, 20, 737-745; M Nakajima et al.,
Proc. Natl. Acad. Sci. USA,
1995, 92, 10663-10667).
Moreover, the stimulati on of the AT2 receptor activates phosphatase SHP-1 (Bedecs K., et al;
Biochem. J.,
1997, 325, 449-454). The fact that the AT2 receptor activates a phosphatase is consistent with its antiproliferative effects.
In the light of the above, it has been shown that, on cells in culture, the AT2 receptor:
inhibits the synthesis of DNA and proliferation, which are induced by angiotensin II (Ang II) and bFGF (M. Stoll et al.,
J. Clin. Invest.,
1995, 95, 651-657),
induces apoptosis (T. Yamada et al.,
Proc. Natl. Acad. Sci. USA,
1996, 93, 156-160), and
induces neuronal differentiation (L. Laflamme et al.,
J. Biol. Chem.,
1996, 271, 22729-22735).
Studies of the signalling pathways associated with the AT2 receptor have been undertaken in cells of the N1E-115 line which are derived from a murine neuroblastoma and which express only the AT2 subtype. A first study has made it possible to demonstrate rapid and transient dephosphorylation of some proteins on the tyrosine residues following the treatment of N1E-115 cells with angiotensin II (C. Nahmias et al.,
Biochem. J.,
1995, 306, 87-92). It has also been shown that the AT2 receptor interferes with the pathways for activation of growth factor receptors and inhibits the activity of MAP kinases (ERK1 and ERK2). (mitoqen-activated protein), which play a key role in the phenomena of cell proliferation and differentiation. The inhibitory effect of AT2 on the activation of MAP kinases is rapid and transient, does not involve a regulatory protein sensitive to the pertussis toxin (of the Gi/Go type), but involves the activation of an orthovanadate-sensitive tyrosine phosphatase.
Taking into account the role of the AT2 receptor in cell proliferation, the inventors have sought to develop tools capable of regulating the action of the AT2 receptor. Indeed, the activation of the AT2 receptor may have repercussions in cancerology (inhibition of cell proliferation).
In general, the AT2 receptor has opposite effects to those of AT1 on the activation of MAP kinases and on cell proliferation; study of the communication which may exist between these two receptor subtypes, which bind the same ligand, is consequently of interest.
The study of the signalling pathways and of the regulation of the AT2 receptor also represents a major stake for human health knowing that antagonists of the AT1 receptor are currently administered to patients with hypertension. In this context, it is essential to know the biological effects associated with the AT2 receptor which remains activable by circulating Ang II in this type of treatment.
The subject of the present invention is an isolated nucleic acid (DNA or RNA) fragment, encoding a protein capable of binding to the AT2 receptor, which fragment is selected from the group consisting of the sequences SEQ ID NO:1, 3, 5, 7 and 9, as represented in the sequence listing included in the present application.
These various sequences correspond to the complementary DNA (cDNA) encoding all or part of the protein called hereinafter ATIP (AT2 interacting protein).
The sequence SEQ ID NO:1 (1803 bp) corresponds to the complete nucleic sequence of mouse ATIP and includes both the parts encoding the AT2 receptor binding protein and the noncoding parts.
The sequence NO:3 (1323 bp) corresponds to the nucleic acid sequence of the coding part of the sequence SEQ ID NO:1, while the sequence SEQ ID NO:5 corresponds to the sequence NO:1 fragment obtained by the two-hybrid technique (A Plessis et al., M/S, 1994, 9, I-1K; J. Luban et al.,
Curr. Op. Biotechnol.,
1995, 6, 59-64).
The sequence SEQ ID NO:7 (3742 bp) corresponds to the complete nucleic sequence of the human cDNA and includes both the parts encoding the protein homologous to the mouse ATIP and the noncoding parts.
The sequence SEQ ID NO:9 (1308 bp) corresponds to the coding part of the sequence SEQ ID NO:7.
The subject of the present invention is also transcripts, characterized in that they are complementary to the sequences in accordance with the invention and are in particular generated from the said sequences.
The subject of the present invention is, in addition, fragments of the said sequences comprising between 20 and 400 bp, useful as probes or as primers, for the detection of the sequences SEQ ID NO:1, 3, 5, 7 or 9, or of homologous sequences.
Among the said fragments, there may be mentioned in particular a probe of 354 bp (SEQ ID NO:5) as well as any fragment of 20 bp to 400 bp included in the sequences SEQ ID NO:1, 3, 5, 7 or 9.
As primer, there will be used in particular the sequence SEQ ID NO:10 (antisense oligonucleotide) which makes it possible in particular to amplify the 5′ parts of the various mRNAs corresponding to ATIP (5′ RACE technique: Marathon cDNA amplification kit, Clontech).
It is also possible to use, as amplification primers, any pair of oligonucleotides of more than 20 bp and comprising part of the ATIP (human or mouse) nucleic sequence, in particular the pair SEQ ID NO:11-SEQ ID NO:12.
The preferred hybridization (prehybridization and hybridization) conditions are in particular the following: 45% formamide, 9% dextran sulphate, 0.2% BSA, 0.2% polyvinyl pyrrolidone, 0.2% Ficoll, 0.1% sodium pyrophosphate, 0.01% SDS, 0.05 mM Tris pH 7.5, 0.9 M NaCl and rinses to a stringency corresponding to the buffer: 1×SSC, 0.1% SDS.
The subject of the present invention is also a purified and isolated protein, called ATIP, which is capable of interacting with the AT2 receptor and which is selected from the group consisting of the sequences SEQ ID NO:2, 4, 6 or 8.
The murine and human sequences exhibit 85.6% homologies. The human sequence (human ATIP) possesses 5 amino acids less than the mouse sequence (mouse ATIP). The amino acids missing from the human sequence are situated at the level of amino acids: 162, 163, 164, 166 and 214 of the mouse ATIP sequence.
Comparisons (Blast) between the ATIP protein sequences according to the invention and the sequences contained in data banks indicate that human ATIP (like mouse ATIP) never exhibits more than 25% homology with a known sequence, and this being the case only over part of this sequence.
The subject of the present invention is also a translational product, characterized in that it is encoded by a nucleotide sequence in accordance with the invention.
The subject of the present invention is, in addition, a
Elbaz Nathalie
Nahmias Clara
Strosberg Arthur Donny
Alston & Bird LLP
Wax Robert A.
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