The hypothalamic neuropeptide somatostatin (SRIF) acts on several organs and tissues widespread distributed in the organism, exerting mainly an inhibitory effect on hormone secretion, as well as on other biological processes (Moller et al., 2003).
These inhibitory, but sometimes stimulatory effects (Castaño et al., 1996) are exerted through a family of seven transmembrane domains receptors (7TMDs) coupled two G proteins (GPCRs), called somatostatin receptors or ssts. The ssts share a common structure consisting in an extracellular amino terminal domain, connected to seven hydrophobic domains inserted into the membrane, which are connected by eight hydrophilic segments ending in an intracellular carboxyl terminal domain, this latter important in the modulation of second messengers pathways.
To date, in mammals, there are five different sst subtypes, from sst1 to sst5, and additionally, in rat and mouse, two isoforms of the subtype 2 (sst2A and sst2B) produced by alternative splicing of the precursor mRNA which encode two proteins differing at their intracellular carboxy terminal region and that possess a different ability to regulate second messengers pathways. However, in fish, there have been described other isoforms of each sst subtype, but due to duplication events instead of alternative splicing.
GPCRs, and among them the ssts, are involved in many cellular processes of high clinical relevance, mediated by signal transduction pathways depending on G proteins coupling. More specifically, one of these sst subtypes, the human sst5 (WO 0177172, WO 0155319, WO 0136446, EP 1369698, WO 03104816) has been linked, in mammals, with many pathologies as hematological diseases, cardiovascular diseases, alterations of the central and peripheral nervous systems, cancer, inflammatory diseases, hepatic diseases, gastrointestinal and urinary diseases, etc. (WO03104816).
The human somatostatin receptor type 5, sst5, is deposited in public databases with accession numbers G139756975, G121954086, G113937340. These sequences contain the cDNA corresponding to the coding sequence as well as the complete genetic structure of the receptor, including the promoter sequence, introns and the 5′ and 3′ untranslated sequences. To date, there has not been described an alternative splicing of the mRNA that originates an alternative isoform different to the deposited in the databases and widely described in bibliography.
Recently, it has been cloned the sequence corresponding to the mRNA containing the CDS of the porcine sst5, as well as the 5′ and 3′ non coding regions (Duran et al., 2005; publication in preparation). During the cloning by RACE PCR, two partial and latter complete variants of the mRNA were obtained which, by alternative splicing, encode two new isoforms of the receptor, similar to that reported for the murine sst2, but in this instance, encoding receptors of six and three transmembrane domains, named porcine isoforms sst5B and sst5C (p-sst5B and psst5C) respectively.
There are data of truncated GPCRs originated by alternative splicing of the mRNA, that encode proteins with less than seven transmembrane domains, as described previously for the GHRH receptor (Rekasi et al., 2000), GnRHR (Pawson et al., 2005), prostaglandin receptor (Ishii et al., 2001), etc., being some of them functional and having possible relevance in tumor processes.
After the results obtained for the porcine sst5, it began the cloning by RACE PCR of the putative human homologues of the sst5B and sst5C isoforms, to further evaluate their presence and importance in endocrine tumors, using the PCR technique.
For the correct interpretation of the present text the following concepts are detailed:
A “somatostatin receptor” is a transmembrane protein coupled to a G protein, belonging to the family of seven transmembrane domains, which is activated by the hypothalamic peptide somatostatin.
“RACE PCR” is referred to Random Amplification of cDNA Ends. It is a PCR (Polymerase Chain Reaction) based technique that allow the introduction of known oligonucleotide sequences into unknown cDNA sequences, that are used as target to amplify by PCR the cDNA sequence comprised between the mentioned oligonucleotides and the region of interest.
“Pituitary Cushing” is referred to the “cushing” syndrome or hypercortisolism. It is a disease caused by an increase of cortisol synthesis or by the excessive use of this or other steroid hormones. A pituitary “cushing” is when the disease is due to an increase of the production of the pituitary adrenocorticotroph hormone.
The present invention comprises the determination of the DNA sequence encoding two new isoforms of the somatostatin receptor type 5 (sst5), named sst5B and sst5C, of five and four transmembrane domains respectively, produced by alternative splicing of the mRNA contained into the genomic sequence deposited in the database with accession number GI13937340 (
With the procedure described in the way to carry out the invention, it is possible to obtain recombinant DNA molecules encoding polypeptides that show, at least in part of the sequence, structural motifs of somatostatin receptors.
The invention is also referred to polynucleotide DNA sequences that hybridize, under restrictive conditions, with those of the new isoforms, which implies a homology level of at least 60% between their nucleotide sequences, preferably a homology of 75% and more preferably a homology of 90%, or sequences derived from them by variation of the genetic code or by mutagenesis.
The procedure used in the invention allows the obtaining of recombinant functional polypeptides for their further study. The recombinant DNA molecules as the described in SEQ ID 1, SEQ ID 3, SEQ ID 5 and SEQ ID 7 or derived from those are inserted into expression vectors. Both polypeptides expressed into host cells offer a new screening system to test new drugs and ligands able to bind selectively in vivo and in vitro to the sst5B and sst5C isoforms, as well as systems to study the modulation of second messenger pathways by each isoform in response to drugs.
The invention object of this application is referred to a purified human nucleic acid that encodes an isoform of the human somatostatin receptor type 5 (sst5) chosen between: sst5B (SEQ ID 5), sst5C (SEQ ID 7), their complementary sequence, a sequence with a 90% homology, and fragments of them.
The invention is also referred to a purified human nucleic acid characterized because it comprises a partial coding sequence contained in SEQ ID 1 and SEQ ID 3.
In a specific consecution, the invention is referred to a human nucleic acid characterized because it contains the 3′ RACE fragment corresponding to the sst5B which sequence is SEQ ID 1, or its partial fragments. In another particular consecution, the invention is referred to a human nucleic acid characterized because it contains the 3′ RACE fragment corresponding to sst5C which sequence is SEQ ID 3, or partial fragments derived from it.
In a preferable consecution, the invention is referred to a purified polypeptide characterized because its amino acid sequence is defined in SEQ ID 2, SEQ ID 4, SEQ ID 6 and SEQ ID 8, and is encoded by one of the oligonucleotides described before in the text.
In second thoughts, the invention is referred to an expression vector characterized because it comprises a nucleotide sequence described before, transcriptionally coupled to an exogenous promoter. In a specific consecution, the mentioned expression vector is characterized because its nucleotide sequence encodes a polypeptide like the defined previously in the text.
In a specific consecution, the methods described previously are characterized because are performed in vitro. In a preferable consecution, the searching is performed in whole cells. In a preferable consecution, the mentioned method is characterized because the polypeptides detailed in SEQ ID 2, SEQ ID 4, SEQ ID 6 and SEQ ID 8, come from an expression vector defined previously in the text. In a more preferable consecution, the mentioned polypeptide corresponds to one of the encoded by SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, or their fragments.
On the other hand, the invention is also referred to new oligonucleotide pairs detailed in the sequences SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, and SEQ ID 14 or sequences homologues in at least 90%, that allow the amplification by PCR of the isoforms A, B and C of the human sst5. In a specific consecution, the invention is referred to the use of these oligonucleotides to amplify selectively the isoforms sst5A, sst5B and sst5C, with any PCR variant. In a preferable consecution, the invention is also referred to the use of the oligonucleotides to study the quantitative tissue distribution of sst5A, sst5B and sst5C in normal and tumor tissues.
In a specific consecution, the invention is referred to a cDNA characterized because it hybridizes with the total or partial sequences contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 or SEQ ID 7.
On the other hand, the invention is also referred to a cDNA contained in SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID or sequences with at least a 90% homology, characterized because it is able to silence independently or together, the genetic expression of the sst5B and sst5C isoforms.
A specific consecution of the present invention is referred to the use of sequences contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 or SEQ ID 7 to generate selective antibodies that distinguish between the sst5B and sst5C isoforms.
The present invention allows the developement of new drugs able to bind selectively to the new sst5 isoforms, sst5B and sst5C, acting as agonists, antagonists or inverse agonists, using second messenger measurement techniques as the microfluorimetric measurement of intracellular calcium (Landa et al., 2005).
More specifically, the insertion of the recombinant DNA, contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 and SEQ ID 7 or derived from them, in eukaryotic expression vectors pCDNA3 like (Invitrogen) allows the transfection of those constructs in tumor cell lines as CHO-K1 and HEK-293T, widely used in the study of other somatostatin receptor subtypes. The process, which methodology is detailed in Landa et al., (Landa et al., 2005) is outlined as follows:
The present invention makes posible the developement of new drugs that modify the basal state of the new somatostatin 5 isoforms, sst5B and sst5C. Therefore, the present invention will allow using FRET (Fluorescence Resonance Energy Transfer) technologies to measure the physical interaction of the sst5B and sst5C isoforms with themselves and with other proteins belonging to the GPCR family. With this technique it is possible to study, in a rapid and accurate way, changes in the basal state of the receptor, whether they are due to aggregation or dissociation of ternary protein complexes, in response to a drug. More specifically, the insertion of the recombinant DNA molecules contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 and SEQ ID 7, or derived from them, in eukaryotic expression vectors variants E-GFPN1 like (Clontech), as E-CFPN1 and E-YFPN1 will allow the cotransfection of these recombinant constructs in tumor cell lines like CHO-K1 or HEK-293T. The process, which methodology is detailed in Vilardaga et al., (Vilardaga et al., 2003) is outlined as follows:
Hereafter it is described an example for a better understanding of the invention.
To clone the partial sequences of the sst5B and sst5C isoforms, the subsequent steps described in
The amplification method described was carried out following the indications of the “GeneRacer® kit”, from Invitrogen® life technologies, with the exception of steps (5) and (6).
To clone the coding sequences and for the functional expression of the isoforms sst5B and sst5C, it was used the strategy outlined in
Also, they were designed oligonucleotide pairs able to discriminate among the isoforms sst5A, sst5B and sst5C (SEQ ID 9 and SEQ ID 14) and that can be used with quantitative aims, amplifying selectively each isoform with the PCR conditions detailed subsequently in the text.
Nucleic Acids Isolation.
RNA isolation. It was performed using the Trizol reagent Invitrogen®, according the suppliers recommendations. Tissues corresponding to two pituitary adenomas diagnosed as non functioning and “cushing” were used as starting material for total RNA extraction. The resulting RNA was resuspended in 12 μl DEPC treated H2O and 1 μl was used for spectrophotometric quantification. For the retrotranscription, 2 μg of ARN from each sample were used in a 20 μl final volume. In addition, RNA from HeLa cells was used, in this instance supplied with the “GeneRacer kit”, using the same amount of RNA for the retrotranscription reaction (
Genomic DNA isolation. DNA was isolated from 107 human lymphocytes as starting material, using the Trizol reagent. The genomic DNA obtained was quantified spectrophotometrically.
PCR Amplification and Obtaining of Partial Sequences of the sst5B and sst5C Isoforms.
As indicated previously, the amplification was carried out using the “GeneRacer® kit” from Invitrogen® combined with oligonucleotides specifically designed, specified in Table 1.
TTAGGATCCTCAGAGCAAGGCCAAGTTGCC-2457
ACTGGATCCTCAGCCTGGGCCTTTCTCCTG-2637
After the retrotranscription reaction, 100 ng of cDNA were used for each PCR, using the oligonucleotides Ra_hum_sst5—3′ (SEQ ID 16) and GeneRacer 3′ (
One μl of each PCR product was reamplified with the nested oligonucleotides Ra_hum_sst5—3′N (SEQ ID 17) and GeneRacer 3′N (
Both amplifications were performed with the Certamp (BioTools, Spain) polimerase mixture supplied with a specific buffer for complex amplifications. The different PCR reactions were carried out with all the cDNA in parallel. PCR products were visualized in a 1% agarose gels and the bands of interest were purified with the QuiaQuick Mini Elute kit (Quiagen). The purified blunted ends PCR products were cloned into the EcoRV site of the pBluescript KSII+ plasmid and then sequenced, resulting in the sequence of 609 base pairs (SEQ ID 1) obtained from cDNA coming from HeLa RNA and another sequence of 257 base pairs (SEQ ID 3), obtained from the cDNA coming from the pituitary “cushing”. Both cDNA obtained from HeLa and the pituitary “cushing” were further amplified with the oligonucleotides Hum_sst5_ATG (SEQ ID 15) and GeneRacer 3′ (
Obtaining of the Coding Sequence Corresponding to the sst5B and sst5C Isoforms.
For the cloning and functional expression of the human sst5B and sst5C isoforms, it was used a strategy based in the independent amplification of the two exons that constitute each isoform (
Selective Amplification with Quantitative Aims of Partial Sequences Corresponding to the sst5A, sst5B and sst5C Isoforms.
Oligonucleotide pairs with a diagnostic aim were developed allowing the selective discrimination by PCR of each of the human sst5 isoforms, sst5A (GI39756975), sst5B and sst5C. The oligonucleotide pair Hum_sst5A_cuant_U/Hum_sst5A_cuant_L, SEQ ID 9 and SEQ ID 10, respectively, amplifies a PCR product of 154 base pairs, using an annealing temperature of 68° C. The oligonucleotide pair Hum_sst5B_cuant_U/Hum_sst5B_cuant_L, SEQ ID 11 and SEQ ID 12, respectively, at an annealing temperature of 68° C., amplifies a PCR product of 142 base pairs contained into the sequence corresponding to the sst5B (SEQ ID 5) and does not amplify the isoforms sst5C or sst5A (GI39756975) while it amplifies a 1643 PCR fragment contained into the human genomic sequence GI13937340 which includes the intron located between the exons E1 and E2 of the sst5B isoform. The oligonucleotide pair Hum_sst5C_cuant_U/Hum_sst5C_cuant_L, SEQ ID 13 and SEQ ID 14, respectively, at an annealing temperature of 68° C., amplifies a PCR fragment of 137 base pairs contained into the sequence corresponding to the sst5C (SEQ ID 6), and also amplifies a 488 base pair fragment contained into the sequence corresponding to the sst5B, and additionally amplifies a 1989 base pairs fragment contained into the human genomic sequence GI13937340 which includes the intron located between the exons E1 and E2 of the sst5C isoform. The three PCR reactions were carried out in parallel using a PCR program consisting in an initial denaturation of 2 minutes at 94° C., followed by thirty seven cycles of a 10 seconds denaturation at 94° C., 10 seconds of annealing at 68° C. and 10 seconds of extension at 72° C. With these PCR settings, each oligonucleotide pair only amplifies selectively a specific isoform, avoiding the additional PCR fragments mentioned above. In all instances, the PCR reactions were supplemented with 1M betaine (Sigma). This methodology allowed screening the presence of the sst5B and sst5C isoforms in several pituitary tumors of different etiology.
This application is a divisional application of U.S. patent application Ser. No. 12/738,131, filed May 31, 2011, now allowed, based upon the United States national stage filing under 35 U.S.C. §371 of International (PCT) Application Number PCT/ES07/00627 filed Oct. 27 2007, and designating the US.
Number | Date | Country | |
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Parent | 12738131 | May 2011 | US |
Child | 13713058 | US |