DE19930285A1 - Human P2YLi gene coding for a putative G protein coupled receptor used to develop new drugs and technologies and to evaluate existing drugs - Google Patents

Abstract

The P2YLi gene, having a 3180 base pair sequence, fully defined in the specification, including 5'- and 3'-untranslated regions, and which overlaps with the gene for acrosomal protein SP32, is new. Independent claims are also included for the following: (1) transcription factors, RNA polymerases, drugs and chemicals that positively or negatively modulate expression of the P2YLi gene; (2) mRNA transcripts of the P2YLi gene, including splice variants and isoforms; (3) cDNA derived from the mRNA or from the intronless P2YLi gene; (4) a protein derived or produced from the mRNA, gene or cDNA; (5) antibodies and antisera raised against one or more epitopes of the proteins or against the protein as a whole; (6) expression systems that express the protein of (4); (7) ligand binding studies and screening assays on native or recombinant receptors or cells or cell membranes containing the protein of (4); (8) transgenic animals and knockout animals which express the protein of (4) at an altered density or not at all; (9) gene therapy methods that concern the protein of (4) or its gene or its mRNA (cDNA) and their development and use; (10) sense and antisense oligonucleotides derived from the P2YLi gene and their use; and (11) diagnosis and treatment of diseases that are directly or indirectly associated with the protein of (4).

Description

With the help of the amino acid sequence of a G protein-coupled receptor Homology search in a publicly accessible database a potential gene for one identified a new G protein-coupled receptor on human chromosome 12 p13.3. The gene sequence became oligonucleotides for the amplification of the potential receptor gene and its derived cDNA (mRNA) sequence prepared and for the PCR amplification of the gene and the cDNA used. Using this primer, the intronless gene could human genomic DNA are cloned and sequenced.

The gene to be patented extends over 3180 bases. The coding area of the gene (Pos. 909 to 2024) consists of an open reading frame of 1116 bases and thus codes a protein of 372 amino acids. Hydrophobicity analysis of the amino acid sequence reveals that it is a G protein coupled receptor with seven transmembrane domains acts. The amino acid sequence is new and previously unknown and shows the best homology (37% identity; 58% homology) to the human P2Y5 purinoceptor and it also has a relatively high homology to human somatostatin-4 and human interleukin-8 Receptor (type B).

Furthermore, the sequence to be patented includes 908 bases of the 5 'untranslated Area of the gene which is believed to be the promoter (with typical TATA box and CAP Signal in the area around base 500) and 1156 bases of the 3 'untranslated Region of the gene which exhibits a typical polyadenylation signal in position 2997 to 3002 contain.

The expression of this gene was determined by means of polymerase chain reaction (PCR) and primers (sense: 5 'CAATCCCACTTTGGCACGATG 3'; antisense: 5 ' AGCGCAATGGCATGTGTGTTC 3 '), which flank the coding region of the gene proven. The following temperature program was used for the PCR: 94 ° C 1 min, 60 ° C 1 min, 72 ° C 3 min, 35 cycles. We were able to use the full coding cDNA (open Reproduce the reading frame) of this receptor from a human hippocampal cDNA bank. This is the expression of the mRNA of this receptor in this central nervous tissue clearly proven. PCR with genomic DNA and these primers resulted in a band of of identical size and by sequencing, it was clearly demonstrated that the gene was intronless is. Furthermore, by homology search in databases of expressed Sequence pieces of human genes (EST = expressed sequence tags) an expressed sequence of 449 base pairs with 100% identity to a sequence piece of the untranslated 3 ' Area (pos. 2127 to 2576) of the gene can be identified. This EST (AA769338) comes from a human B lymphocyte cDNA bank. This further proves that this sequence region (2127-2576) is contained on the mRNA of the receptor, and that the Gene is expressed in B lymphocytes.  

The amino acid sequence of the receptor derived from the cDNA sequence is 372 Amino acids long. Hydrophobicity analysis of the amino acid sequence gives a putative Secondary structure of the protein as an integral membrane protein with probably seven transmembrane domains. The protein contains two potential N-glycosylation sites in the N-terminal region (amino acid positions 4 and 9). Furthermore are in the amino acid sequence six potential protein kinase C phosphorylation sites (amino acid positions 21, 211; 226, 232, 307 and 332) contain, their optional phosphorylation (if they are located intracellularly) can be involved in the modulation of the receptor function. In Position 342 of the amino acid sequence is a potential casein kinase II Phosphorylation site. Potential N-myristoylation sites are in amino acid positions 36, 258 and 324.

Classification and potential functions of the receptor to be patented and its Gene (or eDNA; mRNA)

The receptor most likely belongs to the large gene amile of G protein-coupled receptors (GPCRs). Within this extended family, he belongs to the sub-family of "class A rhodopsin-like" receptors. It has high homology to purinoreceptors (in particular P2Y type 5), to somatostatin receptors (in particular type 4) and to interleukin receptors (in particular type 8).
Expression of the P2YLi receptor in human tissues:
The gene product is expressed in human B lymphocytes (sequence regions can be found in EST AA769338, 100% homology). Furthermore, a 100% homologous EST (AA993247) from human testis was found which is said to encode an acrosomal protein (SP32). This sequence overlaps with the 5 'untranslated region including start codon and the following 25 bases of the coding region. This finding shows that apparently two genes (the gene for the acrosomal protein SP32 and the gene for the P2YLi receptor) use common regions of the chromosome, that is to say the genes overlap.

Since all mammalian P2Y receptors cloned their cell response via activation of protein kinase C (PKC), this should also apply to the putative "P2YLi" - Apply receptor; d. H. the putative "P2YLi" receptor could also play a role in growth and differentiation processes. P2Y receptors are mostly smooth Muscle cells (intestines, blood vessels) before and mediate a release of Nitric oxide (NO) a relaxation (vasodilation).

The putative "P2YLi" receptor should therefore be physiological and pathophysiological Role in the regulation of bowel function, blood pressure, blood flow to organs or play body regions (e.g. heart, kidney, corpus cavernosum). The occurrence of P2Y receptors on dentritic cells, T cells and platelets and hematopoietic Cells suggest that the putative "P2YLi" receptor also functions in these cells play a role (e.g. in blood clotting, hematopoiesis, immune reactions). Also on Nervous system cells (e.g. Schwann cells) and CNS became P2Y receptors demonstrated why the putative "P2YLi" receptor has not yet been tested here either recognized role. After all, P2Y receptors are (and therefore possibly also the "P2YLi" receptor) is involved in the release of interleukins (e.g. IL-6), i.e. H. the "P2YLi" receptor could also be involved in inflammatory processes.  

The "P2YLi" receptor has a 33% identity with the somatostatin receptor type 4, which can also mean that the gene product of "P2YLi" is a previously unknown subtype of a somatostatin receptor. If so, the "P2yLi" receptor on the Inhibiting the release of growth hormones, inhibiting acid secretion and at the control of the neuronal activity of different CNS core areas (e.g. locus coeruleus).

The 31% identity (51% homology) of the "P2YLi" receptor with a human interleukin-8 receptor (hIL-8R) also includes the possibility that the gene product of the "P2YLi" is a previously unknown hIL subtype -8 receptor. In this case, the "P2YLi" receptor would be involved in inflammatory processes, the modulation of blood formation (hematopoiesis), tissue repair processes and the regulation of immune functions.

Claims (16)

1. The gene shown including the 5 'and 3' untranslated region and the finding, that the P2YLi gene overlaps with the gene for the acrosomal protein SP32. 2. Transcription factors, RNA polymerases and pharmaceuticals as well as chemicals that Affect gene expression in a positive or negative manner. 3. The messenger RNA, including that derived from the gene, transcribed Splice variants and isoforms. 4. The cDNA derived from the mRNA or from the intronless gene. 5. The protein derived or produced from the mRNA (or gene or cDNA). 6. Antibodies or antisera which act against one or more epitopes of the protein or against the whole protein. 7. Monoclonal antibodies or antisera against single or multiple epitopes of the Protein or against the whole protein. 8. Expression systems (eukaryotic cell lines, yeast cells, Xenopus oocytes, Baculovirus systems, bacterial expression systems) which contain the protein mentioned express (native or recombinant). 9. Ligand binding studies and screening assays on native or recombinant receptors or cells or cell membranes that contain this receptor. 10. Transgenic animals and knock out animals that have this receptor in modified density or do not express at all. 11. Methods of gene therapy which relate to this receptor or its gene or its extend mRNA (cDNA) and their development and application. 12. Sense and antisense oligonucleotides derived from this gene as well their application. 13. The diagnosis and treatment of diseases with this receptor in direct or communicating indirectly. 14. Methods for diagnosing diseases associated with this receptor (or its gene, mRNA) are connected in a direct or indirect manner such. B. Hybridization techniques, sequencing, SSCP, RFLP, Northern blot, Southern blot, Western blot, expression arrays, antibodies, mutation analyzes. 15. Use of information to develop new drugs, compounds and Chemicals and the evaluation of existing pharmaceuticals, compounds and chemicals as well as to develop new technologies or evaluate existing technologies. 16. The patent should also extend to points 1 to 15 for modified proteins, and gene, cDNA and mRNA sequences (amino acid changes, base changes).