Note: Descriptions are shown in the official language in which they were submitted.
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CLONED LYSOP~OSP~ATIDIC ACID RECEPTORS
FIELD OF THE INVENTION
The invention is in the field of molecular biology. It relates, more particularly, to cloned
Iysophosphatidic receptors and their use in drug screening and related applications.
BACKGROUND TO THE INVENTION
Lysophosphatidic acid, LPA, is a phospholipid sign~lling molecule that has a wide variety of
effects on many dilrerelll cell types (Moolenaar, 1995, Curr. Opin. Cell Biol. 7:203-210),
incl~l~ing neuronal cells. Possible functions of LPA in cortical neurogenesis, based on known
bioactivities of LPA and biological events occurring within the ventricular zone of the cerebral
cortex, include regulation of cytoskeletal events such as interkinetic nuclear movement, cell
rounding, and cleavage plane orientation, mitogenesis, gap junction regulation and influence on
the binding and assembly of fibronectin which is expressed in the embryonic cortex. Additionally,
regulation of apoptosis, recently shown to occur in the vz may also be infiuenced by LPA
sign~lling Further, recent evidence implicates LPA in the proliferation of certain cancer cells (Xu
et al. 1995, J. Cell. Physiol. 163:441-450).
Although LPA is believed to act through a G-protein coupled receptor(GPCR), a cDNA clone of
this receptor has not been identified, in part reflecting the chemical characteristics of LPA that
result in unacceptably high levels of non-specific binding, making techniques such as expression
cloning impractical for discovery of this receptor.
SI~RY OF THE INVENTION
The LPA receptor has now been cloned and characterized. Accordingly, the present invention
provides an isolated polynucleotide encoding a LPA receptor. In aspects of the invention, nucleic
acid coding for LPA receptor is utilised for expression to obtain functional receptor protein and
for further gene cloning to identify structurally related receptor proteins. In related aspects of the
O 2 1 9 9 4 5 5
invention, anti-sense versions of LPA receptor-encoding nucleic acids and fragments thereof are
obtained and utilised to regulate LPA receptor expression.
In another of its aspects, LPA receptor is provided as a product of recombinant production in a
cellular host. In related aspects, there are provided recombinant host cells that express LPA
receptor, as well as receptor-bearing membranes derived from such cells, and expression
constructs in which nucleic acid coding for the LPA receptor is linked to expression controls
functional in the selected host cell.
In another of its aspects, the LPA receptor is utilised in a chemical screening program to identif~
LPA receptor ligands. This method comprises the steps of incubating the candidate ligand with an
LPA receptor-producing cell of the present invention, or with a membrane preparation derived
thelerlolll, and then assessing the interaction by determining receptor/candidate ligand binding.
In another of its aspects, the invention provides antibodies directed to the LPA receptor, for use
for example in diagnosis of conditions wherein the levels of LPA receptor are altered.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS
The invention relates in one respect to polynucleotides that code for lysophosphatidic acid ~LPA)
receptors. Such polynucleotides may be in the form of RNAt or in the forrn of DNA including
cDNA, genomic DNA and synthetic DNA. The LPA receptors are characterized by structural
features common to the G-protein coupled receptor class, including seven transmembrane regions,
and by the functional properties of binding LPA and, when expressed functionally in a host cell,
responding to LPA binding by signal transduction.
The activity of a G-protein coupled receptor such as a LPA receptor may be measured using any
of a variety of appropriate functional assays in which activation of the receptor results in an
observable change in the level of some second messenger system, such as adenylate cyclase,
calcium mobilization, inositol phospholipid hydrolysis or guanylyl cyclase. Alternatively, cell
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proliferation, actin-based cytoskeletal changes, Rho/Rac/ Cdc 42 activation, serum response
stim~ tion or transcription of certain genes may be measured.
In one embodiment of the invention, the LPA receptor is encoded by the nucleic acid sequence of
SEQ ID NO. 1. This particular LPA receptor-encoding nucleic acid, also referred to as the vzg-1
gene, is a cDNA of murine origin and encodes an LPA receptor characterized structurally as a
single 364 amino acid (41kD) polypeptide chain of SEQ ID NO 2. Wlth respect to structural
domains of this LPA receptor, hydropalhy analysis reveals seven putative tr~n.sm~mbrane domains, one
~ in~ residues 47-70 inclusive (TM 1), another sp~nning residues 80-105 (I~M Il), a third sp~nnin~
residues 121-144 (TM m~, a fourth spanning residues 160-182(TM IV), a f~h spanning residues 205-
224 (TM V), a sixth sp~nning residues 256-280 ( TM VI) and a seventh spanning residues 290-310
(TM V~.
In one embodiment the invention provides LPA encoding nucleic acids,or fragments thereof, as a tools
useful to identify structurally related nucleic acids. At low stringency hybridization conditions, for
instance, nucleic acid libraries can be probed to identify genes that are at least about 40% homologous
to vzg-1. Obviously, if the homolog from a particular species is sought e.g. human, the applopliate
library should be probed. To f~filit~te isolation of LPA receptor encoding homologs of vzg-1,
homologs desirably have 80% sequence identity at the nucleic acid level to vzg-l. More desirably they
are 90% identical, and most desirably they have at least 95% sequence identity when compared to vzg-
1. It will be clear that increasing the stringency of the hybridization conditions will result increased
sequence identity ofthe homolog thereby isolated, with vzg-l. In order to isolate LPA receptor
encoding homologs of vzg-1 it is desirsble but not ess~nti~l to screen libraries of brain origin; fetal brain
libraries are paticularly suitable sources, in~ ling fetal cortical libraries. Therefore, the invention
infllldes not only vzg-l but structural homologs and particularly those that code for proteins having
LPA receptor properties. Thus, the invention provides nucleic acids that encode LPA receptors,
including murine LPA receptor and m~mm~ n homologs thereof as well as synthetic variants of
these. Synthetically derived variants of the LPA receptor includè LPA binding variants that
incorporate one or more, e.g. 1-10, amino acid substitutions, deletions or additions, relative to the
LPA receptor.
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It will be appal ell~ to the skilled worker that sequences of the LPA receptor of at least about 15
nucleotides, and preferably of at least about 17 nucleic acids, could be used to generate probes useful
to identify nucleic acid molecules encoding LPA receptor encoding vzg-l homologs. With l ~relence to
SEQ ID No. 1 and the nucleotide numbering appeali~lg thereon, such nucleotide fr~gment~ include
those collespondi.lg in sequence to the tr~n.cm~.mbrane regions. These sequences, and the intact gene
itsel~ may be used of course to clone vzg-l-related genes by standard hybridization techniques. For
example, DNA coding for other vzg-l l~c~Lol~, for example other mouse receptor or other
m~mm~ n receptors, can be obtained by applying selected techniques of gene isolation or gene
synthesis. It is very likely that other species, in particular other m~mm~ inclll~ing the human encode
within their genomes a LPA binding receptor homolog of vzg-l . Isolation of the vzg-l homolog
typically will entail extraction of total m.?.c.c~nger RNA from a fresh source of fetal brain tissue,
followed by conversion of message to cDNA and formation of a library in for example a bacterial
plasmid, more typically a bacteriophage. Such bacteriophage harbouring fragrnPnt~ ofthe human DNA
are typically grown by plating on a lawn of susceptible E. coli bacteria, such that individual phage
plaques or colonies can be isolated. The DNA carried by the phage colony is then typically immobilized
on a nitro-cellulose or nylon-based hybridization membrane, and then hybridized, under carefully
controlled conditions, to a radioactively (or otherwise) labelled probe sequence to identify the
particular phage colony carrying the fragment of DNA of particular interest, in this case a vzg-l
homolog. The phage carrying the particular gene of interest is then purified away from all other phages
from the library, in order that the foreign gene may be more easily characterized. Typically, the gene or
a portion thereof is subcloned into a plasmidic vector for convenience, especially with respect to the
full determination of its DNA sequence. Therefore, having herein provided the mouse LPA receptor, it
will be appreciated by one of ordinary skill in the art of molecular biology that the human homolog of
the mouse vzg-l gene is available by screening a human embryonic brain cDNA or genomic library by
using the mouse vzg-1 gene or fr~n.ont.~ thereof as a probe, using standard molecular biological
techniques.
In the altemative, nucleic acids homologous with vzg-l can be sourced via available databases that
store and allow for searching of sequences electronically. In this regard it has been found that the
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following genes, of unknown function, have the requisite homologies: the mouse clone Recl.3
(Genebank accession No. U48235, available 27 February 1996); the bovine clone Recl .3 (Genebank
accession No. U48236, available 27 February 1996) and the human clone Edg-2 (GPneb~nk accession
No. Y09479, available 18 November 1996).
As an alternative to obtaining LPA encoding DNA directly as a DNA insert from an available or a
constructed cDNA library, in light of the present disclosure it can be synthesized de novo using
established techniques of gene synthesis. Because ofthe length ofthe LPA receptor-encoding DNA
of SEQ ID NO 1, application of automated synthesis may require staged gene construction, in which
regions ofthe gene up to about 300 nucleotides in length are synth~ci7ed individually and then ligated
in correct s lcce.~cion for final assembly. Individually synth~si7ed gene regions can be amplified by PCR
The application of automated synthesis may typically be applied by synthesi7ing specific regions or
fragments of the gene and ligating them, usually via designed overlaps, in correct succession to form
the final gene sequence. In this case, the longer the oligonucleotide building blocks, the fewer wi~l be
the ligations needed, resulting in greater ease of assembly.
The application of automated gene synthesis techniques provides an opportunity for generating
sequence variants of the naturally occurring LPA receptor encoding genes. It will be appreciated, for
example, that polynucleotides coding for the LPA receptor herein described can be generated by
substituting synonymo=us codons for those repl esell~ed in the naturally occurring polynucleotide
sequences herein provided. In addition, polynucleotides coding for synthetic variants ofthe LPA
recèptor herein provided can be generated which for example incorporate single amino acid
substitutions, deletions or additions. Since it will be desirable typically to retain the natural ligand
binding profile of the receptor for screening purposes, it is desirable to limit amino acid substitutions,
for example to the so-called conservative replacements in which arnino acids of like charge are
substitute~l, and to limit substitutions to those sites less critical for receptor activity e.g. in the third
intMc.ellnl~r loop i.e. residues 225-255.
~ Having LPA receptor encoding nucleic acid in hand, LPA receptor can be produced in a number
of ways, including in vitro transcription and via incorporation of the DNA into a suitable
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eApression vector and expression in the appropriate host, for example a bacteria such as E.coli,
yeast or a m~mm~ n cell. A variety of gene expression systems have been adapted for use with these
hosts and are now conlmercially available, and any one ofthese systems can be selected to drive
expression of the LPA I eceplor-encoding DNA. Expression vectors may be selected to provide
ru,l,led cell lines that express the receptor-encoding DNA either transiently or in a stable manner.
For transient expression, host cells are typically ~ srulllled with an expression vector harbouring an
origin of replication functional in a m~mm~ n cell. For stable expression, such replication origins are
unneces~ry, but the vectors will typically harbour a gene coding for a product that confers on the
rollnall~s a survival advantage, to enable their selection such as a gene coding for neomycin
resistance in which case the ll~lsrùllll~ are plated in medium supplemented with neomycin.
These systems, available typically in the form of plasrnidic vectors, incorporate expression cassettes the
functional components of which include DNA conctil~lting expression controlling sequences, which are
host-recognized and enable expression of the 1 eceplor-encoding DNA when linked 5' thereof. The
systems further incorporate DNA sequences which termin~te expression when linked 3' ofthe
receptor-encoding region. Thus, for çxpression in the selected m~mm~ n cell host, there is generated
a recolllbinal~l DNA expression construct in which the receptor-encoding DNA is linked with
expression controlling DNA sequences recognized by the host, and which include a region 5' of the
receptor-encoding DNA to drive expression, and a 3' region to terrninate expression.
Tnr.lllded among the various recolllbill~l~ DNA expression systems that can be used to achieve
m~mm~ n cell expression ofthe receptor-encoding DNA are those that exploit promoters of viruses
that infect m~mm~ n cells, such as the promoter from the cytomegalovirus (CMV), the Rous sarcoma
virus (RSV), simian virus (SV4û), murine ,~ "~1 y tumor virus (MMTV) and others. Also useful to
drive expression are promoters such as the LTR of retroviruses, insect cell promoters such as those
re~ ted by temperature, and isolated from Drosophila, as well as m~mm~ n gene promoters such as
those re~ ted by heavy metals i.e. the metalothionein gene promoter, and other steroid-inducible
promoters.
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In another embodiment the invention provides cells or membranes derived therefrom expressing at
the cell surface an LPA receptor encoded by a heterologous DNA molecule. For incorporation
into cell plasma membranes the vector can be designed to provide a suitable heterologous signal
peptide sequence or the naturally occurring signal peptide encoding sequence can be incorporated
into the expression vector. Conceivably any may be useful in this regard, provided that the
endogenous response, if any, is accounted for. Suitable cells include the mouse cell lines TSM or
TR (Chun et al. 1996, Mol. Neurosci. 7, 304-321). Other cell lines which may be used for this purpose
and which are currently available include the Chinese hamster ovary (CHO) cells for example of K1
lineage (ATCC CCL 61) inc~ ing the ProS variant (ATCC CRL 1281); the fibroblast-like cells
derived from SV40-transformed African Green monkey kidney ofthe CV-1 lineage (ATCC CCL 70),
ofthe COS-1 lineage (ATCC CRL 1650) and ofthe COS-7 lineage (ATCC CRL 1651); murineL-
cells, murine 3T3 cells (ATCC CRL 1658), murine C127 cells, human embryonic kidney cells ofthe
293 lineage (ATCC CRL 1573), human c~lollla cells inr.ll-t1ing those ofthe HeLa lineage (ATCC
CCL 2), and neuroblastoma cells ofthe lines IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10)
and SK-N-SH (ATCC HTB 11). Such cells or membrane preparations are useful to screen LPA
receptor candidate ligands.
For use in screening assays, cell lines expressing the receptor-encoding DNA can be stored frozen for
later use. Such assays may be performed either with intact cells, or with membrane prel)al~lions
derived from such cells. The membrane preparations typically provide a more convenient substrate for
the ligand binding experiments, and are therefore preferred as binding substrates. To prepare
membrane ple~ ions for screening purpose, i.e., ligand binding exp~nm~nt.~, frozen intact cells are
homogenized while in cold water suspension and a membrane pellet is collected a~er centrifugation.
The pellet is then washed in cold water, and dialyzed to remove endogenous LPA receptor ligands that
would otherwise compete for binding in the assays. The dialyzed membranes may then be used as
such, or after storage in Iyophilized form, in the ligand binding assays.
The binding of a c~n~ te ligand to a selected LPA receptor of the invention is performed typically
using a predetermined amount of cell-derived membrane (measured for example by protein
d~ellllill~lion), generally from about 25ug to 100ug. Generally, competitive binding assays will be
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useful to evaluate the affinity of a test compound relative to LPA. This competitive binding assay is
performed by incubating the membrane preparation with radiolabelled LPA, for example [3Hl-LPA, in
the presence of unlabelled test compound added at varying concentrations. Following incubation, either
displaced or bound radiolabelled LPA can be recovered and measured, to determine the relative
binding ~ffinities of the test compound and LPA for the LPA receptor used as substrate. In this way,
the affinities of various compounds for the LPA receptor can be measured.
Alternatively, intact, fresh cells, harvested about two days after transient transfection or after about the
same period following fresh plating of stably transfected cells, can be used for ligand binding assays by
the same methods as used for membrane pl ~ ~lions. In this case, the cells must be harvested by more
gentle centrifugation so as not to damage them~ and all washing must be done in a buffered medium.
As an alternative to using cells that express receptor-encoding DNA, ligand characterization may also
be perforrned using cells for exarnple Xenopus oocytes, that yield functional membrane-bound receptor
following introduction of messenger RNA coding for a LPA receptor. In this case, the LPA receptor
gene of the invention is typically subcloned into a plasmidic vector such that the introduced gene may
be easily transcribed into RNA via an ~ljac~nt RNA transcription promoter supplied by the plasmidic
vector, for example the T3 or T7 bacteriophage promoters. RNA is then transcribed from the inserted
gene in vitro, and can then be injected into Xenopus oocytes. Each oocyte is a single cell, but is large
enough to be penetrated by a fine-tipped microneedle without causing irreparable damage Following
the injection of nL volumes of an RNA solution, the oocytes are left to incubate for up to several days,
whereupon the oocytes are tested for the ability to respond to a particular ligand molecule supplied in a
bathing solution.
In addition to using the receptor-encoding DNA to construct cell lines useful for ligand screening,
expression of the DNA can according to another aspect of the invention be performed to produce
fragments of the receptor in soluble form, for structure investigation, to raise antibodies and for other
experimental uses. It is expected that the portion of the LPA receptor responsible for binding a ligand
molecule resides on the outside of the cell, i.e., is extracellular. It is therefore desirable in the first
instance to f~r.ilit~te the charactelization ofthe receptor-ligand interaction by providing this
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extr~c~ r ligand-binding domain in quantity and in isolated form, i.e., free from the remainder of the
receptor.
To accomplish this, the full-length LPA receptor-encoding DNA may be modified by site-directed
mutagenesis, so as to introduce a translational stop codon into the extracellular N-terminal region,
immediately before the sequence encoding the first tr~nsm~rnbrane domain (TMl), i.e., before residue
47 as shown in SEQ ID No 2. Since there will no longer be produced any transmembrane domain(s)
to "anchor" the leceplor into the membrane, expression ofthe modified gene will result in the
secretion, in soluble form, of only the extracellular ligand-binding domain. Standard ligand-binding
assays may then be performed to ascertain the degree of binding of a candidate compound to the
extr~c~ r domain so produced. It may of course be necessary, using site-directed mutagenesis, to
produce several diae~ versions of the extracellular regions, in order to op~ the degree of ligand
binding to the isolated domains.
It will be appreciated that the production of such extracellular ligand binding domains may be
accomplished in a variety of host cells. l~;~mm~ n cells such as CHO cells may be used for this
purpose, the expression typically being driven by an expression promoter capable of high-level
expression, for example the CMV (cytomegalovirus) promoter. Alternately, non-mammalian cells, such
as insect Sf 9 (Spodoptera frugiperda) cells may be used, with the expression typically being driven by
expression promoters of the baculovirus, for example the strong, late polyhedrin protein promoter.
Fil~m~ntous fungal expression systems may also be used to secrete large quantities of such extracellular
domains of the LPA receptor. Aspergillus nidulans, for example, with the expression being driven by
the alcA promoter, would constitute such an acceptable system. In addition to such expression hosts, it
will be further appreciated that any prokaryotic or other eukaryotic expression system capable of
~X~I es~ g heterologous genes or gene fr~gmPnt~, whether intracellularly or extracellularly would be
similarly acceptable.
The availability of isolated extracellular ligand-binding domains of the receptor protein makes it feasible
to determine the 3-dimensional structures of these ligand-binding regions, with or without a candidate
ligand complexed thereto, by a combination of X-ray crystallographic and advanced 2D-NMR
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techniques. In this way, additional new candidate compounds, predicted to have the required
interactions with the 3-dimensional receptor structure, can be specifically designed and tested.
With large domains, crystallography is the method of choice for structure determination of both the
domain in isolation, and ofthe co-complex with the natural ligand (or an applop~iate antagonist or
agonist molecule). If a particular domain can be made small enough, for example approx. 100-130
amino acids in length, then the powerful technique of 2-D NMR can also be applied to structure
determination. This enables not only the determination of the domain structure, but also provides
dynamic information about the drug-receptor interaction.
For use particularly in detecting the presence and/ or location, for example in brain tissue, the present
invention also provides, in another of its aspects, labelled antibody to a LPA receptor. To raise such
antibodies, there may be used as immunogen either the intact, soluble receptor or an immunogenic
fragment thereof, produced in a microbial or m~mm~ n cell host as described above or by standard
peptide synthesis techniques. Regions of the LPA receptor particularly suitable for use as
irnmunogenic fragments include those corresponding in sequence to an extracellular region ofthe
receptor, or a portion of the extracellular region, such as peptides consisting of amino acids 1-46 of
SEQ ID No. land peptides corresponding to regions between transmembrane domains thereof such as
a peptide consisting of peptides corresponding to amino acids: 105-121; 182-205; 280-290 of SEQ ID
No. 2
The raising of antibodies to the desired LPA receptor or fragrnent imrnunogen can be achieved, for
polyclonal antibody production as described above or as in Example 4, from the blood of an animal
that has been immuni7ed with the immunogen. Alternatively, for monoclonal antibody production,
immunocytes such as splenocytes can be recovered from the immllni7ed animal and fused, using
hybridoma technology, to a myeloma cells. The fusion products are then screened by culturing in a
selection mediurn, and cells producing antibody are recovered for continuous growth, and antibody
recovery. Recovered antibody can then be coupled covalently to a detectable label, such as a
radiolabel, enzyme label, luminescent label or the like, using link:er technology established for this
purpose.
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Animal model systems which elucidate the physiological and behavioural roles of the LPA
receptor are produced by creating transgenic animals in which the activity of the LPA receptor is
either increased or decreased, or the amino acid sequence of the expressed LPA receptor is
altered, by a variety of techniques. Examples of these techniques include, but are not limited to:
1) Insertion of normal or mutant versions of DNA encoding a LPA receptor, by microinjection,
electroporation, retroviral transfection or other means well known to those skilled in the art, into
appropriate fertilized embryos in order to produce a transgenic animal or 2) Homologous
recombination of mutant or normal, human or animal versions of these genes with the native gene
locus in transgenic animals to alter the regulation of expression or the structure of these LPA
receptor sequences. The technique of homologous recombination is well known in the art. It
replaces the native gene with the inserted gene and so is useful for producing an animal that
cannot express native LPA receptor but does express, for example, an inserted mutant LPA
receptor, which has replaced the native LPA receptor in the animal's genome by recombination,
resulting in underexpression of the transporter. Microinjection adds genes to the genome, but
does not remove them, and so is useful for producing an animal which expresses its own and
added LPA receptorS, resulting in overexpression of the LPA receptors.
One means available for producing a transgenic animal, with a mouse as an example, is as follows:
Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The
eggs are stored in an appropriate medium such as M2 medium. DNA or cDNA encoding a LPA
receptor is cesiumchloride purified from a vector by methods well known in the art. Inducible
promoters may be fused with the coding region of the DNA to provide an experimental means to
regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory
elements may be fused with the coding region to permit tissue-specific expression of the trans-
gene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which
may be made from capillary tubing using a piper puller) and the egg to be injected is put in a
depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is
injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse ( a
mouse stim~ ted by the appropriate hormones to m~int~in pregnancy but which is not actually
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pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above,
microinjection is not the only methods for inserting DNA into the egg cell, and is used here only
for exemplification purposes.
Example 1 Isolation of the LPA receptor ( vz~
Poly-A+ RNA was isolated from the neocortical murine cell lines TR and TSM cells (Chun and
J~enicch, 1996, Mol. Cell Neurosci. 7:304-321 ). The RNA was twice selected for poly A+ on
oligo-dT cellulose (Pharmacia, Piscataway, NJ) and 10.5 llg of RNA was reverse transcribed
using oligo-dT or random hexamer primers in 50 mM Tris, pH 8.3, 6 mM MgCl2, 40 mM KCl, 1
mM DTT, 1 mM each dNTPs, and 10 U/~LI Moloney murine leukemia virus reverse transcriptase
(Gibco, Gaithersburg, MD). RNA and primers were heated to 65~C (5 min), cooled to RT,
additional reagents added, then heated to 37~ (2 h). This cDNA was PCR amplified using a
degenerate primer set derived from the conserved regions of transmembrane (TM) domain II and
VII of the GPCR farnily. as follows:
PCR reactions used 40 ng of cDNA in 10 mM Tris, pH 8.3, 50 mM KCI, 2 ~lM of each primer,
1.5 rnM MgCl2, 0.2 ~,IM each dNTPs, and 2.5 U Taq DNA polymerase. All 30 pairwise
combinations of primers were used. Reactions were placed in Perkin-Elmer 480 thermal cycler
(Applied Biosystems, Foster City, CA) at 94~C (3 min), then cycled 25-40 times at 96~C (45 sec),
47~C (144 sec) or 53~C (216 sec), and 72~C (3 min, 6 sec extension/cycle). Products were T/A
cloned~ screened by in si~u hybridization and sequenced. One product "513" was localized to the
vz. Northern blot analysis of embryonic brain detected a single 3.8kb transcript.
The product used to clone vzg-1 ("513") was independently isolated using
TM II primer 5'AA(C/T)T(A/G)(C/G)ATI(A/C)TI(C/G)TIAA(C/T)(C/T)TIGCIGTIGCIGA and
TM VII primers
5'CTGI(C/T)(G/T)(A/G)TTCATIA(A/T)I(A/C)(A/C)(A/G)TAIA(C/T)IA(C/T)IGG(A/G)TT,
021 99 455
5'TCIAT(A/G)TT(A/G)AAIGTIGT(A/G)TAIATIATIGG(A/G)TT, and
5 'AA(A/G)TCIGG(A/G)(C/G)(A/T)ICGI(C/G)A(A/G)TAIAT(C/G)AIIGG(A/G)TT .
Clone "513" was used to screen 500,000 phage at high stringency from a postnatal day 20 Balb/c
mouse brain library (Stratagene, La Jolla, CA). Clone "pSt3 ", cont~ining a 2249 bp insert, was
sequenced completely in both directions by the dideoxy chain termination method. This cDNA
contains an open reading frame encoding a 41kD protein with seven hydrophobic membrane
spanning domains, as well as other features of the GPCR family. These sequence data were made
available 30 November 1996 inGenBank/EMBL/DDBJ under the accession number U70622.
Example 2 Morpholo~ical Assay
Vectors for transfection contained the 1131 bp Ear I-Nae I vzg-l open reading frame fragment
from SEQ ID No. 2 in the sense or antisense orientation, blunt-end cloned into the EcoRV site of
pcDNAI/Amp (Invitrogen, San Diego) by standard protocols.
Neuronal cell line TSM, which has a low expression of endogenous vzg-l transcript compared to
thr TR cell line, was transfected both trasiently and stably with expression vectors cont~ininp;
vzg-1 in the sense or antisense orientation. Transient transfection used calcium phosphate
precipitation with a 10:1 molar ratio of vzg-l expression plasmid to ~-galactosidase expression
plasmid pCMV~ (Clonetech, Palo Alto, CA). After 18 h, cells were refed, grown for 24 h, then
fixed in 4% paraformaldehyde in PBS for 10 min, and stained for ~-galactosidase activity .
Positive cells (200/plate) were counted "blind." The statistical program Instat (Graphpad
Software, San Diego, CA) was used for one way ANOVA and the Student-Newman-Keulspairwise t-test. Stable transfection used a 10:1 molar ratio of vzg-1 expression plasmid to pSV2-
puro.(Vara et al. 1986, Nuc. Acid Res. 14, 4167-4624) and selection in medium cont~ining 10
~lg/ml puromycin. After 2 weeks of selection, single colonies of cells were picked using cloning
cylinders, expanded, then stored or processed for RNA isolation and northern analyses as
previously described.(Chun et al. Supra,~.
021 99 455
Stable cell lines (5000/well in 24 well plates) were serum-starved for 24 h, then media cont~ining
the desired agents was added to the required final concentration. Cells were fixed in 4%
paraformaldehyde in PBS to terrninate incubation and examined. Experiments were performed in
duplicate (200 cells counted/well) and representative samples were evaluated by multiple
investigators. Statistical methods used were identical to transient experiments.
Transfection with the vzg- 1 sense expression vector induced neurite retraction and cell rounding,
which was m~int~ined for at least 24 h "sustained cell rounding". This morphological change
required the presence of serum. Sense transfected cells exposed to serum had 48~3.6% round
morphology, compared to 22~5.0% without serum.
The reproducibility of cell rourding allowed its use as a bioassay to identify putative ligands for
vzg-1. Boiling the serum did not abolish its ability to mediate cell rounding, indicating that the
ligand was a heat stable molecule that might be associated with 1) cytoskeletal changes and 2) cell
proliferation, since vzg- 1 expression was restricted to the vz. A molecule present in serum that
met these criteria was LPA. Since endogenous vzg-1 should be active in the cell lines from which
it was identified, untransfected TS~I cells were first assayed for their ability to respond to LPA
(Avanti Polar Lipids (Alabaster, AL), all lipids were synthetic, 98-99% pure). TSM cells
responded with a rapid increase in the percentage of round cells. However, this response was
reversible such that hy 3 h the percentages of round cells returned to their baseline values.
Transfection of TSM cells with vzg-1 in the sense orientation sustained the rounding response to
LPA such that at 3 h 49~4.9% oftreated transfected cells still displayed a round morphology,
compared to 29~1.9% of untreated transfected cells. Importantly, thrombin, a serum component
which also induces cell rounding in some neural cell lines did not induce sustained cell rounding in
cells transfected with vzg-1, although it did induce sustained cell rounding in cells transfected with
the thrombin receptor. Thus, transient overexpression of vzg- 1 specifically alters LPA-mediated
changes in cell morphology.
Example 3 Membrane Isolation and Li~and Bindin~ Assay
14
0 2 1 9 9 4 5 5
Cell lines were grown to 90% confluence, washed with PBS, scraped from the plate, centrifuged
for 5 min (110 x g), washed with PBS, re-centrifuged, and the pellet then resuspended 10 ml of
ice cold 20 mM Tris, pH 7.5, disrupted by twenty strokes in a glass homogenizer, and sonicated
on ice with three 10 s bursts using a micro-ultrasonic cell disrupter (Kontes Glass, Hayward, CA).
After low speed centrifugation (1000 xg, 15 min, 4~C), membranes were pelleted from the
supernatant (16000 x g, 30 min, 4~C) and resuspended at 3 ~ l protein in ice cold 20 mM Tris,
pH 7.5. Just prior to analysis, membranes were re-sonicated for 5 s on ice until the solution
became transparent, then LPA binding assays carried out as previously described .(Thompson et
al. Mol. Pharmacol. 45, 718-723,~. Briefly, binding reactions (500 ~11 of 50 llg/ml membrane
protein in an assay buffer of 20 rnM Tris, pH 7.5 and 0.5 mM CuS04) were initiated by addition
of 1-oleoyl-(9,10-3H)-LPA (56.2 Ci/mmol) (DupontNEN, Boston, MA) to a final concentration of
8.5 nM (230,000 CPM) and incubated (30~C, 30 min). To assess nonspecific binding, 2 ~M
unlabeled 1-oleoyl-LPA was added to a set of parallel reactions. Reactions were run through PD-
10 Sephadex G25M columns (Pharmacia, Piscataway, NJ) to separate bound from free ligand.
The eluent (1.5 ml) was mixed with scintillation fluid (Ultima Gold; Packard Instrument Co.,
Meriden, CT) and counted at RT. About 5-10% of the total input CPM were collected in the
eluent. Uncorrected total binding ranged from 13,000 to 20,000 CPM. Because LPA forms
aggregates that are excluded from the column bed and thus collected in the eluent, each
experiment was performed in parallel using reactions without membranes, in the presence or
absence of 2 ',IM LPA, to determine eluted background CPM. Total and non-specific CPM were
corrected by subtraction of the appropriate background CPM. Specific CPM were calculated by
subtracting corrected non-specific CPM from corrected total CPM. Overall, with correction,
non-specific CPM were 61% of total CPM. Experiments were done in triplicate and the results
statistically analyzed using Student's t-test.
Example 4 Production of polyclonal antisera to the LPA receptor
A rabbit polyclonal antiserum was raised against Vzg- 1 by cDNA vaccination according to the
method of Donnelly et al., J. Infect. Dis. 1996, 173:314-320 and Raz et al., PNAS USA 1994
0 2 1 9 9 4 5 5
,91:9519-9523. Briefly, Twice weekly intradermal injections of vzg-1 expression construct (20
llg) in PBS for 6 weeks were followed by a week hiatus, then injections repeated for 4 weeks and
serum collected. Antiserum was collected, diluted in appropriate buffers and screened by
Westeryl blot anaysis. The antiserum recognizes an approximately 41kd protein in brain and cell
line extracts that is absent from pre-imrnune controls.
16
0 21 99 4 5 5
GAATTCGGCA CGAGGCACAG TGCTGCCCTC CGTAGGCTCC GGGTTGTGCT GGGGTGAGGC
6 I TTGGGTTGGG TTGGCCCGGC GGCTGCGTGA ACTGCGGAGC TGGACCTAGC AGGCTTACAG
121 TTCCTCCTAG CATGACCGAG ATCTGATCAG CCAACCCGCG CATTGCTTTT TGTGCCTGGC
181 ACTGCAGTGC AGGGGGCCTC TTCATCGCCC CAAACTACAG CACTGTCATG GCAGCTGCCT
241 CTACTTCCAG CCCTGTAATT TCACAGCCCC AGTTCACAGC CATGAACGAA CAACAGTGCT
301 TCTACAATGA GTCTATCGCC ~ l l lATA ACCGGAGTGG GAAATATCTA GCCACAGAAT
361 GGAACACAGT GAGCAAGCTG GTGATGGGAC TGGGCATCAC TGmGCGTG TTCATCATGT
421 TGGCCAATCT CCTGGTCATG GTGGCAATCT ACGTCAACCG CCGCTTCCAT TTCCCTAm
481 ATTACTTGAT GGCCAACCTG GCTGCTGCAG A(~ 1 1 (: 1 1 ( :GC TGGATTGGCC TACTTCTACC
541 TGATGTTCAA TACAGGACCT AATACCCGGA GACTGACTGT TAGCACGTGG CTCCTCCGGC
601 AGGGCCTCAT TGACACCAGC CTGACAGCTT CTGTGGCCAA CCTGCTGGCT ATTGCTATCG
661 AGAGGCACAT CACGGTTTTC CGCATGCAGC TCCATACACG AATGAGCAAC CGGCGCGTGG
721 TGGTGGTGAT TGTAGTCATC TGGACTATGG CCATTGTGAT GGGTGCTATA CCCAGTGTGG
781 GCTGGAACTG CATCTGTGAT ATCGATCACT GTTCCAACAT GGCACCCCTC TACAGTGACT
841 CCTACTTAGT CTTCTGGGCC ATTTTCAACC TGGTGACCTT TGTGGTCATG GTGGTTCTCT
901 ACGCTCACAT CmGGCTAT GTTCGCCAGA GGACTATGAG GATGTCTCGG CATAGTTCTG
961 GACCCAGGAG GAATCGGGAC ACCATGATGA GCCTTCTGAA GACTGTGGTC ATTGTGCTTG
1021 GTGCCmAT TGTCTGCTGG ACTCCGGGAT TGGTCTTGTT ATTGCTGGAT GTGTGCTGCC
1081 CGCAGTGCGA TGTCCTGGCC TATGAGAAGT TCTTCCTCCT CCTGGCCGAG TTCAACTCTG
1141 CTATGAACCC CATCATCTAC TCCTACCGCG ACAAAGAGAT GAGCGCCACC TTCAGGCAGA
1201 Tc(~l~lGl lG CCAGCGCAAC GAGAACCCTA ATGGCCCCAC GGAAGGCTCT GACCGCTCTG
1261 CCTCCTCCCT CAACCACACC ATTCTGGCTG GAGTTCACAG CAACGACCAC TCTGTGGTTT
1321 AGAAGGAAGC CAGCCGGCCT CTGTGGATCT GTGAACCCCA CCCTACCCCC CATTGCCAGG
1381 GCAAGGTGGG GAGCCAGAGG AGATGAGGAC ACTCCTGTAC TTAACACTAA CCAATGGCAG
1441 TAmGTCCC TAGACCCAAG AGACTTGAGG ATGAAmAT TTGGCAGGCC CCATCTTCTC
1501 CTTTGGAAAA CAGAAGGGGA CCGTCTTGTG GTGGAATTGA GAAATGGACT CTGGGGTGAC
1561 CGTGTAGCAT TCACTAACTA GACTTAAAAG ATTTTATGTG GmGGCTTA AGCCAGGAAA
1621 AAAAAATCTG CTGAATTGAG TATACAATCG AGTATACACA GGCTTCCCCT TTAAAGAACA
1681 AACAATACAT TGCAmATT AATGAGTATG mATGCCTG ACAGCATGTT TGTGATCGAA
1741 AAGACTGCTA AACTGACATA GATGAGTTGT 1 1 1 1 1 1 1 11 1 TT(~ 1 1 lG 1 11 1 1 1 1 1 lA
1801 CATGATGGAG GAAAAGTATA AATTAGAATG ATTTTTGTGT TTGmAGAA AGCAAGCATG
1861 TG~lc~l (i 1~1 ATTCAGTATG CCmCmA AAGATAAAAG GCCACTAm TAAATCTTCT
1921 AGGGAATAGA AGAATCTAGT AAAAACCAGT ATTCAmAG GCTACAGGAA AAACCATATC
1981 CTAATCAATT ACCTTTTAAT TAAAGTAATG AAATATACAT GAAAGGCAAA GTAATGTGAG
2041 CTTGTCACCC AAAGAGTGTG TGCTCTCCAA ACGCTGGAGG AGATGAAGCT GTAGCGTTGT
2101 CCCTGCATAG TGAAGATACC CACGTGCGTT CTCAGTGCCA GACCCTCAGT GGGACTTGTT
2161 TTAAAGCCTG TGGTTTTCCA AGTTAGAAAA TAATACCTAC TTACTATAGA AAACTTGAAA
2221 ATTGCAGAAC TGTGTGAAAA AAAAAAA~A
SEQ ID No. 1
02199 455
MAAASTS SPVISQPQFTAMNEQQCFYNESIAFF
TMI
34 YNRSGKYLATEWN ¦TVSKLVMGLGITVCVFIMLA¦
TM II
67 I NLLV¦MVAIYVNR R IFHFPIYYLMANLAAADFFAG I
TM III
100 I LAYFYLIMFNTGPNTRRLTVST~LLRQGLIDTSLI
133 ¦ TASVANLLAIAI¦ERHITVFRMQLHTR~SNRRVV¦
166 ¦ VVIVVIWTMAIVMGAI~ S VGWNCICDIDHC SNM
TM V
199 APLYSD¦SYLVFWAIFNLVTFVVMVVI~YAHIFGY
TM VI
232 VRQRTMRMSRHSSGPRRNRDTMMSLILKTVVIVL I
TM VII
265 IGAFIVCWTPGLVLLLL!DVCCPQCDVLAYEKFFLI
298 ¦ LLAEFNSAMNPII~SYRDKEMSATFRQILCCQR
331 NENPNGPTEGSDRSASSLNHTILAGVHSNDHSVV
SEQ I:D NO 2
