CA2253832A1 - Ob receptor isoforms and nucleic acids encoding them - Google Patents

Abstract

The ob receptor has numerous isoforms resulting from alternative splicing; three novel isoforms, designated c', f, and g are disclosed. The nucleic acids encoding these isoforms are taught. Also part of the invention are vectors containing the nucleic acid encoding the receptors, host cells transformed with these genes, and assays which use the genes or protein isoforms.

Description

CA 022~3832 1998-11-04 W 097/42340 PCTfUS97107521 TITLE OF THE INVENTION
OB RECEPTOR ISOFORMS AND NUCLEIC ACIDS ENCODING THEM

FIELD OF THE ~NVENTION
This invention relates to oh receptor protein isoforms, to DNA and RNA sequences encoding them, and to assays using the receptor isoform proteins.

BACKGROUND OF THE INVENTION
Recently the identification of mutations in ~everal genes involved in the onset of obesity in rodents have been identified. Of particular interest are mutations discovered in the peptide hormone, leptin, which is a component of a novel signal transduction pathway that regulates body weight (Zhang et al. 1994, Nature 372:425-432;
Chen et al. 1996, Cell ~4:491-495). Leptin was initially discovered by the positional cloning of the obesity gene, ob, in mice. Two different ob alleles have been identified: one mutation causes the premature terrnination of the leptin peptide resulting in a truncated protein, and the other mutation changes the transcriptional activity of the obesity (oh) gene, resulting in a reduced amount of circulating leptin.
There is a correlation between a decrease in the levels of biologically active leptin and the overt obese phenotype observed in ob/ob mice. Recombinant leptin has been shown to induce weight loss in the oblob mouse but not in the diabetic phenotype dbldb mouse (Campfield et al. 1995, Science 269: 546-549; Halaas et al.
1995, Science 269: 543-546; Pellymounter et (11. 1995, Science 269:540-543; Rentsch et al. 1995, Biochem. Biophys. Res. Comm.
214: 131 - 136; and Weigle et al. 1995, J. Clin . Invest. 96:2065-2070).
Although the synthesis of leptin occurs in the adipocyte, its ability to decrease food intake and increase metabolic rate appears to be mediated centrally by the hypoth~l~mus. Injection of recombinant leptin into~the third ventricle of the brain elicits a similar response as peripheral ~lmini,stration of leptin.

CA 022~3832 1998-11-04 W 097/42340 PCT~US97107521 Furthermore, the recent cloning of the human receptor for the leptin, the ob-receptor (OB-R), reveals that it is transcribed in the hypoth~l~mus (Tartaglia etal. 1995, Cell ~3:1263-1271; Stephens et al. 1995, Nature 377: 530-532). In addition, a mutation that 5 results in premature termination of the long-form of the mouse OB-R, which is preferentially expressed in the hypothalamus, appears to be responsible for the obese phenotype of the dbldb mouse (Lee et al. 1996, Nature 379:632-635; Chua et al. 1996, Science 271:994-996; and Chen etal. 1996, Cell 84:491-495).
The OB-R from wild type (lean) rats and from rats having thefat~y mutation (both heterozygous and homozygousfa ) have been isolated and sequenced. (Patent Application Serial No.s.
, Attorney Docket No.s. 19642PV and 19642PV2, filed February 22, 1996 and March 22, 1996, which are hereby 15 incorporated by reference.) Various isoforms of the OB-Rs have also been identified. These isoforms are due to alternative splicing. For example, in the mouse the a form has 5 amino acids following the Lysine at 8~9; the b form has 273 amino acid.s after Lysine ~9; the c 20 form has 3 amino acids after Lysine ~9; and the d form contains 11 amino acids after Lysine ~9.
It would be desirable to be able to further experiment with variou~s isoforms in order to better understand obesity, and to be able to clone and produce novel o~ receptor isoforms to use in 25 assays for the identification of ligands which may be useful in understanding obesity and for its prevention and treatment.

DETAILED DESCRIPTION OF THE INVENTION
This invention relates to novel ol~ receptor isoforms 30 designated c', f and g which are substantially free from associated membrane proteins. It also relates to substantially purified oh receptor isoform c', f and g proteins. These isoforms are present in various species, including rat, mouse and human.

CA 022~3832 1998-11-04 WO 97/42340 PCTrUS97/07521 Another aspect of this invention is to nucleic acids which encode OB receptor isoforms c', f or g. The nucleic acid may be any nucleic acid which can encode a protein, such as genomic DNA, cDNA, or any of the various forms of RNA. Preferably, the nucleic ~S acid is cDNA.
This invention also includes vectors containing a OB-R
isoform c', f or g gene, host cells cont~ining the vectors, and methods of making susbstantially pure OB-R isoform c', f or g protein comprising the steps of introducing a vector comprising a OB-R isoform c', f or g gene into a host cell, and cultivating the host cell under appropriate conditions such that OB-R isoform c', f or g is produced. The OB-R isoform c', f or g so produced may be harvested from the host cells in conventional ways.
Yet another aspect of this invention are assays which employ OB-R isoform c', f or g. In these assays, various molecules, suspected of being OB-R isoform c', f or g ligands are contacted with a OB-R isoform c', f or g, and their binding is detected. In this way agonists, antagonists, and ligand mimetics may be identified. A
further aspect of this invention are the ligands so indentified.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE I is the amino acid se4uence of wild type rat OB-R.
FIGURE 2 is the cDNA sequence of wild type rat OB-R.
FIGU~E 3 is the cDNA se4uence encoding rat isoform.
FIGURE 4 is the cDNA specific for Rat isoform c'.
A~s used througout the specification and claims, the following definitions apply:
"Substantially free from associated membrane proteins"
means that the receptor protein is not in physical contact with any membrane proteins.
"Substantially purified OB-receptor isoform c', f or g"
means that the protein isoform is at least 90% and preferably at lea~st 95% pure.

CA 022~3832 1998-11-04 WO 97/42340 PCTrUS97/07~21 "Wild type" means that the gene or protein is substantially the same as that found in an ~nim~l which is not considered to have a mutation for that gene or protein.
'~" means that the gene or protein is substantially the same as that found in a rat homologous for thefatt~ mutation.
"Substantially the same" when referring to a nucleic acid or amino acid sequence means either it is the same as the reference sequence, or if not exactly the same, contains changes which do not affect its biological activity or function.
It ha.s been suprisingly found, in accordance with this invention that the OB-R exi,sts in a large variety of isoforms, including three novel ones, form c', f and g. These isoforrns apply to all species, but for convenience, throughout the specification and claims, numberings of amino acids and nucleotides will use the rat wild type sequences (FIGURES 1 and 2) as a reference. However, it is to be understood that this invention is not limited to rat wild type proteins and nucleic acids and specifically include.s rat (wild type and fatty), mouse, and human OB-R isoform c', f and g proteins and nucleic acids.

OB-R isoform f differs from wild type protein in that after the Lysine at position 889 (referring to the rat se4uence in FIGURE 1), there are six amino acids, ending at an Asparagine residue at position 895. In the cDNA, the codons are then followed by a Stop codon. One cDNA for rat isoform f is shown in FIGURE
3; this invention specifically includes all various cDNAs encoding an isoform f protein. The .superscripted numbers refer to protein position numbers.
Lys889 Iso890 Met891 Pro892 Gly893 Arg894 Asn895 In the human isoform f, Lysine 89 3 corresponds to the rat Lysine 889, the same six amino acids follow Lysine 889.
In a particularly preferred embodiment of thi.s invention, the OB-R isoform f is from rat origin.

CA 022~3832 1998-11-04 OB-R isoform g differs from the wild type in that it is much shorter that the wild type sequence. The following eighteen amino acids are found at the beginning of the protein with the superscript numbers indicating their position. The Arginine at S position lP~ is spliced to a large fragment of the wild type molecule, beginning at the Proline at position 166 (in both mouse and human).
This isoform then extends for the remainder of the wild type molecule.

Metl Phe2 Gln3 Thr4 Pro5 Arg6 Ile7 Val8 Pro9 Glyl0 Hisl 1 Lysl2 ASP13 Leul4 Ile15 serl6 Ly,S17 Argl~ prol66 After Pro 166, the remainder of the protein may be the same as wild type, or, alternatively it could also contain another isoform variation, such as isoform a, b, c, d, e, or f.
A particularly preferred embodiment is the rat isoform g.

OB-R isoform c' is similar to the OB-R isoform c which was previously described [Lee et al., Natu~ e 379: 632-635]. After 20 Lysine at position ~9, it only has three amino acids, Val~s90 Thr~s9 1 Phe~92 Stop. As can be seen, isoform c' differs from isoform c in that the final amino acid is phenylalanine rather than valine found in isoform c. Further, there are untranslated se~luences in the DNA
encoding isoform c' which do not appear to be pre~sent in isoform c.
25 The cDNA encoding the rat isoform c' is given in FIGURE 4. In humans, the Val, Thr, Phe follow Lysine ~s91.

One aspect of this invention is the molecular cloning of these variou.s isoforms of OB-R. The wild type and fà receptor 30 proteins contain an extracellular, a transmembrane domain. In the rat, the extracellular domain extends from amino acids 1-~30; the transmembrane domain is from amino acids X39-X60; and the cytoplasmic domain is from amino acids ~60-1162. Similar domains have bene identified for the mouse and human proteins. This CA 022~3832 1998-ll-04 W O 97/42340 PCTrUS97/07521 invention also includes isoform C~7 f and g proteins which lack one or more of these domains. Such deleted proteins are useful in assays for identifying ligands and their binding activity.
In the rat wild type protein, amino acids 1-28 form a 5 signal sequence; thus the mature proteins extend from amino acids 28-1162. The mature protein isoforms form yet another aspect of this invention. This differs somewhat from the signal sequence of 1-22 reported for mouse and human OB-R; the mature mouse and human isoforrns form yet another aspect of this invention.
The OB-R isoform c', f or g gene can be introduced into virtually any ho.st cell using known vectors. Preferred ho.st cells include ~. coli as well as m~mm~lian and yeast cell lines.
One of ordinary skill in the art is able to choose a known vector which is appropriate for a given host cell; generally 15 plasmids or viral vectors are preferred. The OB-R isoform c', f or g gene may be present in the vector in its native form, or it may be under the control of a heterologous promoter, and if desired, one or more enhancers, or other sequences known to regulate transcription or translation. The host cell containing the OB-R isoform c', f or g 20 gene is cultured, and the OB-R isoform c', f or g gene is expressed.
After a suitable period of time the OB-R c', f or g isoform protein may be harvested from the cell using conventional separation techniques.
A further aspect of this invention is the use of an OB-R
25 c'~ f or g isoforrn in assays to identify OB-R c', f or g isoform ligands. A ligand binds to the OB-R isoforrn receptor, and in vivo may or may not result in an activation of the receptor. Ligands may be agonists of the receptor (i.e. stimulate its activity), antagonists (inhibit its activity) or they may bind with little or no effect upon the 30 receptor activity.
In an assay for ligands, an OB-R isoform of this invention is exposed to a putative ligand, and the amount of binding is measured. The amount of binding may be measured in many ways; for example, a ligand or the OB-R isoform being investigated CA 02253832 1998-ll-04 may be labeled with a conventional label (such as a radioactive or fluorescent label) and then put in contact with the OB-R isoform under binding conditions. After a suitable time, the unbound ligand is separated from the OB-R isoform and the amount of ligand which 5 has bound can be measured. This can be performed with any of the OB-R isoforms of this invention; alternatively the amount of binding of the various isoforms can be compared. In a competitive assay, both the putative ligand and a known ligand are present, and the amount of binding of the putative ligand is compared to the amount 10 of binding to a known ligand. Alternatively, the putative ligand's ability to displace previously bound known ligand (or vice-versa) may be measured. In yet other embodiments, the assay may be a heterogeneous one, where the OB-R isoform may be bound to a surface, and contacted with putative ligands. Dectection of binding 15 may be by a variety of methods, including labelling, reaction with antibodies, and chomophores.
In another assay, the OB-R isoforms of this invention may be used in a "trans" activation a.ssay. Such assays are described in U.S. Application Serial No. , Attorney Docket No.
20 196P~6PV, which was filed on April 22, 1996 and which is hereby incorporated by reference. In this assay, a cell which expresses an OB-R isoform of this invention (either naturally or through recombinant means) is transfected with a reporter gene construct comprising a minimal promoter, a leptin activation element and a 25 reporter gene. Transcription of the reporter gene is dependant upon activation of the leptin activation element. Binding of a ligand to the receptor isoform activates the leptin activation element, which then allows transcription of the reporter gene.
The following non-limiting Example,s are presented to 30 better illustrate the invention.

CA 022~3832 1998-ll-04 Preparation of mRNA and cDNA from rat tissues Tissues were collected from lean and fa/fa Zucker rats 5 and snap frozen in liquid nitrogen. The tissues collected included:
hypothalamus, pituitary, lung, liver, kidney, heart, adrenal glands, smooth muscle, skeletal muscle, and adipose tissue. The tissues were homogenized with a Brinkm~nn Polytron homogenizer in the presence of guanadinium isothiocyanate. mRNA was prepared from lO hypothalamus, lung, and kidney according to the instructions provided with the messenger RNA isolation kit (Stratagene, La Jolla, CA). cDNA was prepared from approximately 2 ,ug of mRNA with the SuperScriptTM choice system (Gibco/BRL Gaithersburg, MD).
The first strand cDNA synthesis was primed using l ,ug of 15 oligo(dT)l2 18 primer and 25 ng of random hexamers per reaction.
Second strand cDNA sythesis was performed according to the manufacturer's instructions. The quality of the cDNA was assessed by labeling an aliqout (l/lOth) of the second strand reaction with approximately 1 ,uCi of [oc-32P]dCTP (3000 Ci/mmol). The labeled 20 product~s were separated on an agarose gel and detected by autoradiography.

25 Preparation of a hypothalamic cDNA library Approximately 3.6 ~g of phosphorylatedBstXI adapters (Invitrogen, San Diego, CA) were ligated to approximately 3 ~g of cI~NA prepared as described in Example 1. The ligation mix was then diluted and size-fractionated on a cDNA sizing column 30 (Gibco/BRL Gaithersburg, MD). Drops from the column were collected and the eluted volume from the column was determined.
An aliqout from each fraction was analyzed on an agarose gel.
Fractions containing cDNA of greater than or equal to 1 kb were pooled and precipitated. The size-fractionated cDNA with the Bst Xl 35 adapters was ligated into the prokaryotic vector pcDNA II

CA 022~3832 1998-11-04 (Invitrogen, San Diego, CA). The vector (4 ~g) was prepared for ligation by first cutting with the restriction endonuclease Bst XI, gel purifying the linearized vector, and then dephosphorylating the ends with calf intestinal phosphatase (Gibco/BRL, Gaithersburg, MD) 5 according to the manufacturers instructions. The ligation contained approximately 10-20 ng of cDNA and approximately 100 ng of vector and was incubated overnight at 14~C. The ligation was transformed into 1 ml of XL-2 Blue Ultracompetent cells (Stratagene, La Jolla, CA) according to the manufacture's 10 intructions. The transformed cells were spread on 133 mm Colony/Plaque Screen filters (Dupont/NEN, Boston, MA), plated at a density of 30,000 to 60,000 colonies per plate on Luria Broth agar plates containing 100 !lg/ml Ampicillin (Sigma, St. Louis, MO).

Screening a hypothalamic cDNA library Colonies on filters were replica plated onto a second filter set. The master filter was stored at 4~C for subsequent 20 isolation of regions containing colonies that gave a positive hybridization signal. The replica filters were grown for several hours at 37~C until colonies were visible and then processed for in situ hybridization of colonies according to established procedures (Maniatis, et al. Molecular Cloning: A Lahorato1y Manual, Cold 25 Spring Harbor Laboratory Publications, Cold Spring Harbor, NY, which is hereby incorporated by reference). A Stratalinker (Stratagene, La Jolla, CA) was used to crosslink the DNA to the filt~r. The filters were washed at 55~C for 2 hours in 2x SSC and 0.5% SDS to remove bacterial debris. Eight to ten filters were then 30 placed in a heat sealable bag (Kapak, Minneapolis, MN) containing 15-20 ml of lx hybridization solution (Gibco/BRL, Gaithersburg, MD) containing 50% formamide and incubated for 1 hour at 42~C.
The filters were hybridized overnight with greater than 1,000,000 cpm/ml of the radiolabeled probe described below in lx CA 022~3832 1998-11-04 WO 97t42340 PCT/US97/07521 hybridization buffer (Gibco/BRL, Gaithersburg, MD) containing 50% formamide at 42~C. The probe, a 2.2 kb fragment encoding the extracellular portion of the Ob-R was labeled by random priming with [alpha 32P]dCTP (3000 Ci/mmole, Amersham, Arlington 5 Heights, IL) using redi-prime (Amersham, Arlington Heights, IL).
The probe was purified from unincorporated nucleotides using a Probequant G-50 spin column (Pharmacia Biotech, Piscataway, NJ).
Filters were washed two times with 0. lx SSC 0.1% SDS at 60~C for 30 min and then subjected to autoradiography. Individual region.s 10 containing hybridization positive colonies were lined up with the autoradiogram of the hybridized filter. These were excised from the master filter, and placed into 0.5 ml Luria broth plus 20% glycerol.
Each positive was replated at a density of approximate 50-200 colonies per 100 by 15 mm plate and screened by hybridization as 15 previously described. Individual positive colonies were picked and plasmid DNA was prepared from an overnight culture using a Wizard kit (Promega, Madison, WI).

Amplification of Lean Rat OB-receptor cDNA u,sin~ PCR
To provide for a probe to screen the hypothalamic cDNA library, the rat OB receptor was initially obtained by PCR
usin~ degenerate primers based on the mouse and human OB-25 receptor amino acid sequences. A set of oligonucleotide primers,were designed to regions with low codon degeneracy. The pairing of the forward primers ROBR 2 (5'-CAY TGG GAR TTY CTI TAY
GT-3') and ROBR 3 (5'-GAR TGY TGG ATG AAY GG-3') corresponding to mouse amino acid sequences HWEFLYV and 30 ECWMKG, with reverse primers ROBR 6 (5 '-ATC CAC ATI GTR
TAI CC-3'), ROBR 7 (5'-CTC CAR TTR CTC CAR TAI CC-3'), ROBR P~ (5'-ACY TTR CTC ATI GGC CA-3') and ROBR 9 (5'-CCA YTT CAT ICC RTC RTC-3') representing mouse amino acids, GYTMWI, VYWSNWS, WPMSKV, and DDGMKW provided good 35 yields of the appropriately sized products. The fragments of interest CA 022~3832 1998-11-04 were amplified as long polymerase chain reaction (PCR) products by a modifying the method of Barnes (1994, Proc. Natl. Acad. Sci.
91:2216-2220, which is hereby incorporated by reference). In order to obtain the required long PCR fragments, Taq Extender 5 (Stratagene, La Jolla CA) and the Expand Long Template PCR
System (Boehringer Mannheim, Indianapolis, IN) were used in combination. The standard PCR reaction mix, in a final volume of 20 ,ul, contained 5 ng of template (lean rat cDNA), 100 ng of primers, 500 ~M dNTPs, 1 X Buffer 3 from the Expand kit, 0.1 ,ul 10 each of Taq Polymerase and Taq Expander. Reactants were assembled in thin walled reaction tube.s.
The amplification protocol was: 1 cycle of 92~C for 30 sec., followed by 32 cycles at 92~C for 30 sec., 45~C for 1 min. and 6~~C
for 3 min. using a Perkin-Elmer (Norwalk, CT~ 9600 Thermal 1 5 Cycler.
This strategy produced a series of PCR products with the largest being approximately 2.2 Kbp amplified from primers ROBR 2 and ROBR 9. These products were subcloned for DNA
~equence analysis as described below. The insert was excised from 20 the cloning vector with the restriction endonuclease Eco Rl, and fragments were separated from the vector by agarose gel electrophoresis. The fragments were eluted from the gel using a Prep-A-Gene kit (BioRad, Richmond CA) according to the manufacturer's instructions and radiolabeled as described above.

Subclonin~ of PCR products PCR products of the appropriate size were prepared for 30 subcloning by separation on an agarose gel, excising the band, and extracting the DNA using Prep-A-Gene (BioRad, Richmond, CA).
PCR products were ligated into pCRTMII (Invitrogen, San Diego, CA) according to the instructions provided by the manufacturer. The ligation was transforrned into INVaF' cells and plated on Luria-35 Bertani plates containing 100 ,ug/ml ampicillin and X-Gal (32 ,ul of CA 022~3832 1998-11-04 W 0 97/42340 PCT~US97/07521 50 mg/ml X-Gal (Promega, Madison, WI). White colonies were picked and grown overnight in Luria-Bertani broth plus 100 ~g/ml ampicillin. Plasmid DNAs were prepared using the Wizard miniprep kit (Promega, Madison, WI). Inserts were analyzed by digesting the 5 plasmid DNA with EcoRI and separating the restriction endonulease digestion products on an agarose gel.
Plasmid DNA was prepared for DNA sequencing by ethanol precipitation of Wizard miniprep plasmid DNA and resuspending in water to achieve a final DNA concentration of 100 10 ,ug/ml. DNA sequence analysis was performed using the ABI
PRISMTM dye terminator cycle se4uencing ready reaction kit with AmpliTaq DNA polymerase, FS. The initial DNA sequence analysis was performed with M13 forward and reverse primers, subsequently primers based on the rat OB-R sequence were utilized. Following 15 amplification in a Perkin-Elmer 9600, the extension products were purified and analyzed on an ABI PRISM 377 automated sequencer (Perkin Elmer, Norwalk, CT). DNA sequence data was analyzed with the Sequencher program.

Claims (23)

WHAT IS CLAIMED IS:

1. Ob-receptor (OB-R) isoform c', f or g, sustantially free from associated proteins.

2. An OB-R isoform according to Claim 1 which is substantially pure.

3. An OB-R isoform according to Claim 1 which is a c' isoform.

4. An OB-R isoform according to Claim 1 which is an f isoform.

5. An OB-R isoform according to Claim 1 which is a g isoform.

6. An OB-R isoform according to Claim 1 which is from a rat.

7. An OB-R isoform according to Claim 6 which is from a wild-type rat.

8. An OB-R isoform according to Claim 6 which is from a fatty rat.

9. An OB-R isoform according to Claim 3 which is human.

10. An OB-R isoform according to Claim 4 which is human.

11. An OB-R isoform according to Claim 5 which is human.

12. An OB-R isoform according to Claim 3 which is from a mouse.

13. An OB-R isoform according to Claim 4 which is from a mouse.

14. An OB-R isoform according to Claim 5 which is from a mouse.

15. A nucleic acid encoding an OB-R of Claim 1.

16. A nucleic acid according to Claim 15 which is a cDNA.

17. A vector comprising a nucleic acid which encodes an OB-R of Claim 1.

18. A vector according to Claim 17 which is a plasmid.

19. A host cell containing a vector according to Claim 17.

20. A host cell according to Claim 19 which is E. coli, a mammalian cell, or a yeast cell.

21. An assay to determine if a putative ligand binds to an OB-R isoform c', f or g comprising: contacting the putative ligand with an OB-R isoform c', f or g, and determining if binding has occurred.

22. An assay according to Claim 17 wherein the ligand is labeled.

23. An assay to determine if a putative ligand binds to an OB-R isoforrn c', f or g which is a trans-activation assay.