Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-06-07
2003-06-10
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S300000, C530S350000, C435S252300, C435S320100, C536S023100, C536S023500
Reexamination Certificate
active
06576442
ABSTRACT:
INTRODUCTION
The present invention relates generally to newly identified rdgB proteins and related products and methods.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention and references cited therein are not admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine phosphatases (TPs) and tyrosine kinases (TKs), including receptor tyrosine kinases and non-receptor tyrosine kinases.
A tyrosine protein kinase named PYK2, is described in U.S. patent application Ser. No. 08/460,626, filed Jun. 2, 1995, which is a continuation-in-part application of U.S. patent application Ser. No. 08/357,642, filed Dec. 15, 1994, both of which are hereby incorporated herein by reference in their entirety including any drawings. PYK2 contains an N-terminal domain, a catalytic domain, two proline-rich regions, potential Src homology 2 (SH2) binding regions, and a region homologous to the focal adhesion targeting domain.
A type of protein found in Drosophila, called Drosophila retinal degeneration B protein(rdgB)is described in Vihtelic et al.,
J. of Cell Biology
122, :1013-1022, 1993. The sequence described in this reference, however, contained a false stop codon sequencing error and thus the authors were not aware that the Drosophila rdgB contains a PYK-2 binding domain. In addition, this sequence was incorrectly identified as a member of the 6-transmembrane domain family of proteins. These rdgB proteins function in many sensory and neuronal cells of the fly and are directly associated with sight in the fly.
The sequence of a genomic clone of a portion of
C. elegans
has been placed on a computer database, and (although unappreciated), this sequence contains an rdgB sequence with introns. Thus, the GENEBANK database contains raw data of the nucleotide sequence of a series of genomic clones of
c. Elegans.
Using portions of the human rdgb sequence, the present invention identifies an open reading frame that has been to this point unrecognized. An rdgB was thus found segregated into 14 exons in two separate cosmids C54C6 (assc. #Z77131) and MO1F1 (assc. #Z46381).
SUMMARY OF THE INVENTION
The present invention relates to rdgB polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such polypeptides, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Such rdgB polypeptides are involved in various signal transduction pathways and thus the present invention provides several agents and methods useful for diagnosing, treating, and preventing various diseases or conditions associated with abnormalities in these pathways.
The present invention is based in part upon the identification and isolation of a series of novel non-receptor tyrosine kinase binding molecules, termed hrdgB1, hrdgB2, and hrdgB3. The full length nucleic acid sequences encoding these proteins are set forth respectively in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The full length amino acid sequences are set forth respectively in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. RDGBs are generally comprised of 3 structural domains. The N-terminal PIT domains described herein have approximately 45% amino acid identity to human PPI1 and PPI2. The PIT domains of RDGB2 and RDGB3 (RDGB1 lacks a PIT domain) have approximately 72% identity with each other and approximately 62-65% identity with the drosophila and
C elegans
rdgB's. The full length amino acid sequence for
c. Elagans
is set forth in SEQ ID NO:7 and the full length Drosophila nucleic acid sequence set forth in SEQ ID NO:8, and the full length Drosophila amino acid sequence is set forth in SEQ ID NO:9. The PIT domains of the rdgBs have a conserved putative ATP binding motif similar to that seen in protein kinases.
The second central domain is present in all human rdgbs described herein and has no sequence homology to any other known domain. The three human rgdbs share 43-47% identity over the 600 to 675 amino acid stretch and show 25-35% identity to the invertebrate rdgB's. This large domain contains three subdomains with much higher identity (66-88% in the human rdgbs and 35-75% with the invertebrate rdbgs.) This high level of conservation, especially across such a diverse set of species, suggests an important functional role for these stretches. The N-terminal portion of the central domain is a conserved acidic region of 10 to 15 amino acids comprised almost exclusively of glutamatic and aspartate residues that may function as a calcium binding motif.
The third rdgB domain is particularly unique to these proteins and consists of the C-terminal 343 to 384 residues of the proteins. There is 60-63% identity amongst the human rdgbs and 40-60% with the invertebrate rdgB's. The comparison with the drosophila rdgb is based on the unique knowledge of this domain and its functional significance as described herein. The published sequence contained a framseshift mutation such that the protein was previously thought to terminate less than halfway through this domain. By comparison with the human sequences, the present invention provides a sequence that extends beyond the end of the drosophila sequence to include amino acids 1054-1249.
Within the PYK2 binding domain is a distinct motif with primary sequence homology to the nucleotide binding region of the ras-related GTP-binding proteins. All members of this family (ras, rho, rac, rab, ran) contain a sequence characterized by the conserved hydrophobic-hydrophobic-G-X-K-X-D-hydrophobic amino acid sequence. The G-X-K motif in the rdgBs is at aa 614 (rdgb1), aa898 (rdgb2), aa 983 (rdgb3) and aa 987 (dm). Based on analysis of the three dimensional structure (by X-ray crystalography) of this region from ras and ran, this motif grasps the nucleotide ring of GDP/GTP as part of the molecular “on-off” switch in these proteins. The rdgbs however lack the upstream p-llop or A-box present in these small G-proteins.
RdgB proteins are involved in key signal transduction pathways related to neurotransmitter signaling. This is based in part on the recognition of existence and significance of domains found in rdgB proteins (see FIG.
1
). For example, the experiments described herein demonstrate that rdgB proteins contain a PYK2 binding domain. PYK2 is believed to be responsible for regulating neurotransmitter signaling. The rdgB proteins also contain a PIT domain, which in Drosophila is involved in PI transfer. PI transfer in humans is involved in the recycling of synaptic vesicles. Thus, in view of the roles of the PYK2 binding domain and the PIT domain, rdgB proteins may be useful in the treatment of conditions of nervous system by enhancing or inhibiting such signaling.
Thus, in a first aspect the invention features an isolated, purified, enriched or recombinant nucleic acid encoding a rdgB polypeptide. Preferably such nucleic acid encodes a mammalian rdgB polypeptide, more preferably it encodes a human rdgB polypeptide.
By “isolated” in reference to nucleic acid is meant a polymer of 2 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but does indicate that it is
Lev Sima
Plowman Gregory D.
Schlessinger Joseph
Burrous Beth A.
Foley & Lardner
Saidha Tekchand
Sugen Inc.
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