Note: Descriptions are shown in the official language in which they were submitted.
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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.
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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.
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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.
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"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.
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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
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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
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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.
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EXAMPLE 1
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.
EXAMPLE 2
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
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(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).
EXAMPLE 3
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
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- 10 -
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).
EXAMPLE 4
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
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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.
EXAMPLE 5
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
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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.
