IE83567B1 - G protein coupled glutamate receptors - Google Patents
G protein coupled glutamate receptorsInfo
- Publication number
- IE83567B1 IE83567B1 IE1991/4314A IE431491A IE83567B1 IE 83567 B1 IE83567 B1 IE 83567B1 IE 1991/4314 A IE1991/4314 A IE 1991/4314A IE 431491 A IE431491 A IE 431491A IE 83567 B1 IE83567 B1 IE 83567B1
- Authority
- IE
- Ireland
- Prior art keywords
- dna
- cdna
- cells
- rna
- receptor
- Prior art date
Links
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Description
PATENTS ACT, 1992
4314/91
G PROTEIN COUPLED GLUTAMATE RECEPTORS
ZYMOGENETICS, INC. and
THE BOARD OF REGENTS OF THE UNIVERSITY OF WASHINGTON
The majority of nerve cell connections are
chemical synapses. A neurotransmitter is released from
the presynaptic terminal, typically in response to the
arrival of an action potential in the neuron, and
diffuses across the synaptic space to bind to membrane
receptor proteins of the postsynaptic terminal. The
binding of neurotransmitters to membrane receptors is
coupled either to the generation of a permeability change
in the postsynaptic cell or to metabolic changes.
Neurotransmitters produce different effects
according to the type of receptor to which they bind. In
general, those which produce effects that are rapid in
onset and brief in duration bind receptors that act as
ligand—gated ion channels, where binding almost instantly
causes an ion flow across the membrane of the
postsynaptic cell. Those neurotransmitters which act
more like local chemical mediators bind to receptors that
are coupled to intracellular enzymes, thereby producing
effects that are slow in onset and more prolonged. These
neurotransmitters alter the concentration of
intracellular second messengers in the postsynaptic cell.
Four second messenger systems have been linked
to neurotransmitter or hormone receptors and have been
studied for their roles in the control of neuronal
excitability. They are the adenylate cyclase/cyclic AMP-
dependent protein kinase system, guanylate cyclase and
CGHP-dependent protein kinase, the inositol
trisphosphate/diacyl glycerol-protein kinase C system,
and systems which are activated by calcium ions, such as
the calcium/calmodulin—dependent protein kinase system.
Thus, binding of a transmitter to a receptor may
activate, for example, adenylate cyclase, thereby
increasing the intracellular concentration of CAMP, which
in turn activates protein kinases that phosphorylate
specific proteins in the cells, such as those which form
ion channels and thus alter the cells’ electrical
behavior. As with the ligand—gated ion channel
transmitters, the effects can be either excitatory or
inhibitory, and may affect the cell at many levels,
including the pattern of gene expression. It is also
believed that these chemical synapses, associated with
’second-messenger systems, may be involved in long-term
changes that comprise the cellular basis of learning and
memory.
The ligand—activated membrane receptors do not
activate the second messenger systems directly, however,
but via a membrane-bound protein, the GTP—binding protein
(G protein), which binds GTP on the cytoplasmic surface
of the cell membrane and thereby acts to couple adenylate
cyclase to the membrane receptor. Neurotransmitter
binding to the membrane receptor is believed to alter the
conformation of the receptor protein to enable it to
activate the G protein in the lipid bilayer, which then
binds GTP at the cytoplasmic surface and produces a
further change in the G protein to allow it to activate,
e.g., an adenylate cyclase molecule to synthesize CAMP.
When a ligand binds a receptor, an enzymatic cascade
results as each receptor activates several molecules of G
protein, which in turn activate more molecules of
adenylate cyclase which convert an even larger number of
ATPS to CAMP molecules, producing a substantial
amplification from the initial event.
Glutamate, aspartate and their endogenous
derivatives are believed to be the predominant excitatory
neurotransmitters in the vertebrate central nervous
system. (Krinjrvic, Phys. Rev. 54:4l8-540, 1974).
Recently, glutamate has been described as playing a
major, widespread role in the control of neuroendocrine
neurons, possibly controlling not only the neuroendocrine
system but other hypothalamic regions as well. Four
major subclasses of glutamate receptors have been
described but their characterization has until recently
been limited to pharmacological and electrophysiological
functional analyses. See generally, Hollman et al.,
Nature 342:643—648 (1989) and Sommer et al., Science
249:1580—l585 (1990). Three of the receptors, the
quisqualate (QA/AMPA), kainate (KA), and N—methyl—D—
aspartate (NMDA) receptors, are believed to be directly
coupled to cation—specific ion channels and thus are
classified as ligand-gated ionotropic receptors. The
fourth glutamate receptor binds some of the agonists of
the ionotropic receptors (quisqualate and glutamate, but
not AMPA) but has no shared antagonists, and is coupled
to G protein. Thus, this receptor, referred to as the G
protein-coupled glutamate receptor, or Glugi, is
pharmacologically and functionally distinct from the
other major glutamate receptors. This receptor has also
been termed the metabotropic receptor.
Agonist binding to GlunR has been shown to
result in the activation of a number of second messenger
systems, depending on the system studied. One of the
best characterized is the quisqualate activation of
phospholipase C through a G protein coupled interaction
that leads to the stimulation of inositol phospholipid
metabolism. This activity has been studied in systems
that measure the accumulation of radiolabeled inositol
monophosphate in response to stimulation by glutamate.
The systems typically use brain slices from regions such
as the hippocampus, striatum, cerebral cortex and
hypothalamus (Nicoletti, et al., Proc. Natl. Acad. Sci.
fig s3:1931-1935 (1986), and Nicoletti, et al., J_.
Neurochem. 46:40-46 (1986)) neuronal cultures derived
from embryonic mouse and rat cerebellum, corpus striatum
and cerebral cortex (Nicoletti et al., J. Neurosci.
:1905—1911 (1986), Sladeczek et al., Nature 3l7:717—719
(1985), Dumuis, et al., Nature 347:182—l84 (1990), and
Drejer et al., J. Neurosci. 7:2910-2916 (1987)) and rat
brain synaptosomes (Recasens et al., Eur. J. Pharm. 141:
87-93 (1987), and Recasens et al., Neurochem. Int.
13:463-467 (1988)). A major disadvantage of each of
these model systems is the difficulty in analyzing the
pharmacological and functional activities of GIUGR in an
environment where other glutamate receptors and G
protein-coupled receptors such as muscarinic and
serotonin receptors are also present.
The Xenopus oocyte system has been used to
identify GlumR as a member of the family of G protein-
coupled receptors. An endogenous inositol triphosphate
second messenger-mediated pathway in the oocyte allows
the detection of GluGR after injection of total rat brain
mRNA, in that the oocyte responds to ligand via the
oocyte G protein-coupled PLC-mediated activation of a
chloride channel that can be detected as a delayed,
oscillatory current by voltage-clamp recording (Houamed
et al., Nature 310:318-321 (1984); Gunderson et al.,
Proc. Royal Soc. B221:127-143 (1984); Dascal et al., Mol.
Brain Res. 1:301—309 (1986); Verdoorn et al., Science
238:1114-1116 (1987); Sugiyama et al., Nature 325:531-
533 (1987); Hirono et al., Neuros. Res. 6:106-114 (1988);
Verdoorn and Dingledine, Mol. Pharmacol. 34:298-307
(1988); and Sugiyama et al., Neuron 32129-132 (1989)).
Injection of region-specific brain mRNA and of size
fractionated mRNA have suggested that Gh%R may be a
large mRNA (6-7 kb) and that it is enriched in the
cerebellum (Fong et al., Synapse 2:657-665 (1988) and
Horikoshi et al., Neurosci. Lett. 105:340-343 (1989)).
There remains considerable need in the art for
isolated and purified GlunR, as well as systems capable
of expressing Gh%R separate from other neurotransmitter
receptors. Further, it would be desirable to
specifically identify the presence of Gh%R in cells and
tissues, thereby avoiding the time-consuming, complex and
nonspecific functional electrophysiological and
pharmacological assays. It would also be desirable to
screen and develop new agonists and/or antagonists
specific for Glugh but to date this has not been
practical. Quite surprisingly, the present invention
fulfills these and other related needs.
Summarv of the Invention
The present invention provides isolated and
substantially pure preparations of mammalian G protein-
coupled glutamate receptors and fragments thereof. In
preferred embodiments the receptors are coupled to a G
protein in vertebrate cells, bind glutamate and
quisqualate and thereby activate phospholipase C, and are
capable of stimulating inositol phospholipid metabolism.
Having provided such receptors in isolated and purified
form, the invention also provides antibodies to the
receptors, in the form of antisera and/or monoclonal
antibodies.
In another aspect the invention provides the
ability to produce the mammalian G protein-coupled
glutamate receptors and polypeptides or fragments thereof
by recombinant means, preferably in cultured eukaryotic
cells. The expressed receptors or fragments may or may
not have the biological activity of corresponding native
receptors, and may or may not be coupled to a G protein
in the cell used for expression. Accordingly, isolated
and purified polynucleotides are described which code for
the receptors and fragments thereof, where the
polynucleotides may be in the form of DNA, such as CDNA,
or RNA. Based on these sequences probes may be used to
hybridize and identify these and related genes which
encode mammalian G protein-coupled glutamate receptors.
The probes may be full length CDNA or as small as from 14
to 25 nucleotide, more often though from about 40 to
about 50 or more nucleotides.
In related embodiments the invention concerns
DNA constructs which comprise a transcriptional promoter,
a DNA sequence which encodes the receptor or fragment,
and a transcriptional terminator, each operably linked
for expression of the receptor. For expression the
construct may also contain at least one signal sequence.
The constructs are preferably used to transform or
transfect eukaryotic cells, more preferably mammalian
cells which do not express endogenous G protein-coupled
glutamate receptors. when bound by an appropriate ligand
such as glutamate or quisqualate, the receptor may
activate phospholipase C in the host cell via coupling to
G protein. Further, for large scale production the
expressed receptor may also be isolated from the cells
by, for example, immunoaffinity purification.
Cells which express the G protein-coupled
glutamate receptors may also be used to identify
compounds which can alter the receptor—mediated
metabolism of a eukaryotic cell. Compounds may be
screened for binding to the receptor, and/or for
effecting a change in receptor-mediated metabolism in the
host cell. Agonists and/or antagonists of the G protein-
coupled glutamate receptors may also be screened in cell-
free systems using purified receptors or binding
fragments thereof for the effect on ligand—receptor
interaction, or using reconstituted systems such as
micelles which also provide the ability to assess
metabolic changes.
In yet other embodiments the invention relates
to methods for diagnosis, where the presence of a
mammalian G protein-coupled glutamate receptor in a
biological sample may be determined. For example, a
monospecific antibody which specifically binds a G
protein-coupled glutamate receptor is incubated with the
sample under conditions conducive to immune complex
formation, which complexes are then detected, typically
by means of a label such as an enzyme, fluorophore,
radionuclide, chemiluminescer, particle, or a second
labeled antibody. Thus, means are provided for
immunohistochemical staining of tissues, including brain
tissues, for the subject receptors.
Brief Description of the Figures
Figure 1 illustrates the construction of
plasmid pVEGT, where Fig. 1A shows the construction of
pVEG, Fig. 1B shows the construction of pVEG' and Fig. 1C
shows pVEGT'. Symbols used are T7 pro, the T7 promoter;
T1 and T2, synthetic and native T7 terminators,
respectively; M13, M13 intergenic region; the parentheses
indicate a restriction site destroyed in vector
construction; and pA is the Aspergillus gigg;
polyadenylate sequence.
Figure 2 illustrates representative responses
from voltage-clamp assays of oocytes injected with RNA
from positive pools.
Figure 3 illustrates a partial restriction map
of clone 45-A.
Figure 4 illustrates the cloning of the
receptor CDNA present in clone 45-A into Zem228R.
Figure 5 illustrates the DNA sequence and
deduced amino acid sequence of clone 45—A. Numbers below
the line refer to amino acid sequence, numbers to the
right of the line refer to nucleotide number. Putative
transmembrane domains have been overlined, and putative
N-linked glycosylation sites are indicated by closed
circles.
Figure 6 illustrates a representative dose
response curve for varying concentrations of L-glutamic
acid. Error bars, where larger than the symbols,
represent SEM.
Figure 7 illustrates the DNA sequence and
deduced amino acid sequence of a subtype lb glutamate
receptor clone. Numbers below the line refer to amino
acid sequence. Numbers to the right of the line refer to
nucleotide sequence.
Figure 8 illustrates the DNA sequence and
deduced amino acid sequence of a subtype 2a glutamate
receptor clone. Numbers below the line refer to amino
acid sequence. Numbers to the right of the line refer to
nucleotide sequence.
Figure 9 illustrates the DNA sequence of a
partial subtype 2b glutamate receptor clone. Numbers
refer to the nucleotide sequence.
Description of the Specific Embodiments
Gh%R is a family of G protein—coupled membrane
receptors for the neurotransmitter glutamate. As
glutamate has been described as having a major role in
the control of neurons, particularly neuroendocrine
neurons, Gh%R may play a critical role in effectuating
such control. Consequently, the development of agonists
and antagonists of the GluGR-ligand interaction and
Gh%R-mediated metabolism is of great interest.
The present invention presents the means to
identify agonists and antagonists of the Gh%R-ligand
interaction by providing isolated Glugl. The term
"Gh%R" refers to any protein either derived from a
naturally occurring GluGR, or which shares significant
structural and functional characteristics peculiar to a
naturally occurring GluGR. Such a receptor may result
when regions of a naturally occurring receptor are
deleted or replaced in such a manner as to yield a
protein having a similar function. Homologous sequences,
allelic variations, and natural mutants; induced point,
deletion, and insertion mutants; alternatively expressed
variants; proteins encoded by DNA which hybridize under
high or low stringency conditions to nucleic acids which
encode naturally occurring GluGR—encoding nucleic acids:
proteins retrieved from naturally occurring materials;
and closely related proteins retrieved by antisera
directed against GlufiR proteins are also included.
By GlufiR "ligand" is meant a molecule capable
of being bound by the ligand—binding domain of Gh%R, a
Gh%R analog, or chimeric Glu@R as generally described in
U.S. Pat. No. 4,859,609, incorporated by reference
herein. The molecule may be chemically synthesized or
may occur in nature. Ligands may be grouped into
agonists and antagonists. Agonists are those molecules
whose binding to a receptor induces the response pathway
within a cell. Antagonists are those molecules whose
binding to a receptor blocks the response pathway within
a cell.
By "isolated" Gluglis meant to refer to a
Gh%R which is in other than its native environment such
as a neuron, including, for example, substantially pure
Gh%R as defined hereinbelow. More generally, isolated
is meant to include a GluGR as a heterologous component
of a cell or other system. For example, a GluGR may be
expressed by a cell transfected with a DNA construct
which encodes the Glugk, separated from the cell and
added to micelles which contain other selected receptors.
In another example described below, a Glugi is expressed
by a cell which has been co—transfected with a gene
encoding muscarinic receptor. Thus, in this context, the
environment of the isolated Gh%R is not as it occurs in
its native state, particularly when it is present in a
system as an exogenous component.
The invention provides cloned GlufiR coding
sequences which are capable of expressing GlufiR proteins.
Complementary DNA encoding Gh%R may be obtained by
constructing a CDNA library from mRNA from, for example,
brain tissue. The library may be screened by
transcribing the library and injecting the resulting mRNA
into oocytes and detecting, by functional assays, those
oocytes which express the Gh%R. Alternatively, the
clones may be screened with a complementary labeled
oligonucleotide probe.
The present invention relates to successfully
isolating a CDNA encoding a GluGR. Functional cloning of
GluGR was accomplished by substantial modifications and
improvements to a number of CDNA cloning and molecular
biology techniques. Initially, an enriched source of
GluGR mRNA prepared by sucrose gradient centrifugation of
>4kb length rat cerebellum poly(A)+ mRNA was used as
template for cDNA synthesis. Further, a CDNA cloning
vector that was employed included a poly(A) tail, thereby
increasing by 40-fold the translational efficiency of the
transcription product of the CDNA insert and a polylinker
site to allow the directional cloning of the CDNA into
the vector between the promoter and the poly(A) tail.
Vector construction for directional cloning is described
in US—A—5 075 227, incorporated herein by reference.
The CDNA cloning vector was also used with two
transcriptional terminators, in tandem, following the
po1y(A) sequences, efficiently generating a unit length
transcript product without non—coding plasmid or viral
sequences, and without requiring a restriction
endonuclease to linearize the DNA template (a standard
practice that will often prevent functional cloning
strategies from working due to the presence of the
endonuclease site within the coding region of the CDNA).
The CDNA synthesis strategy maximized insert size and
recreation of the 5' ends of the cDNA's, without
introduction of homopolymer tails. CDNA inserts were
size-selected to be greater than 4 kb in length before
insertion into the vector. A library of 106 cDNA inserts
in pools of 100,000 was replica plated to reduce the
number of amplification steps in the fractionation of
sequentially smaller pools. Moreover, m1 muscarinic CDNA
(another G protein-coupled receptor coupled to
phosphoinositol metabolism) template was included in
transcription reactions of the subfractionated pools so
that before injection the in vitro transcripts from each
pool could be assayed by Northern analysis to assess
relative quantity and quality of the mRNA, and by
voltage-clamp of oocytes as an internal positive control
for each oocyte not responding to quisqualate or
glutamate. The inclusion of a dilution of SEAP-VEGT‘ (a
secreted form of alkaline phosphatase) template in
transcriptions was also employed so that oocytes selected
for voltage-clamp analysis were those synthesizing higher
levels of the co-injected GluGR mRNA. And further, low
noise electrical recording techniques were used to
monitor the small signals initially generated from rare
transcripts.
The above-described methods were used to
isolate a CDNA clone encoding a GlufiR designated "subtype
la." Oligonucleotide probes based on the sequence of the
subtype la clone were used to probe additional brain and
cerebellum cDNA libraries. These libraries yielded
clones encoding additional subtypes, which were
designated lb, 2a and 2b.
with the GlufiR and cDNA clones thereof provided
herein, nucleotide and amino acid sequences may be
determined by conventional means, such as by dideoxy
sequencing. See generally, Sambrook et al., Molecular
Cloning‘ A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989,
incorporated by reference herein. Genomic or CDNA
sequences encoding GlunR and homologous receptors of this
family may be obtained from libraries prepared from other
mammalian species according to well known procedures.
For instance, using oligonucleotide probes from rodent
Gh%R, such as whole length CDNA or shorter probes of at
least about fourteen nucleotides to twenty-five or more
nucleotides in length; often as many as 40 to 50
nucleotides, DNA sequences encoding GlunR of other
mammalian species, such as lagomorph, avian, bovine,
porcine, murine, etc. may be obtained. If partial clones
are obtained, it is necessary to join them in proper
reading frame to produce a full length clone, using such
techniques as endonuclease cleavage, ligation and loopout
mutagenesis.
A DNA sequence encoding GlunR is inserted into
a suitable expression vector, which in turn is used to
transfect eukaryotic cells. Expression vectors for use
in carrying out the present invention will comprise a
promoter capable of directing the transcription of a
cloned DNA and a transcriptional terminator.
To direct proteins of the present invention for
transport to the plasma membrane, at least one signal
sequence is operably linked to the DNA sequence of
interest. The signal sequence may be derived from the
Gh%R coding sequence, from other signal sequences
described in the art, or synthesized Qg 3939.
confers chloramphenicol resistance on yeast cells.
Additional vectors, promoters and terminators
for use in expressing the receptor of the invention in
yeast are well known in the art and are reviewed by, for
example, Emr, Meth. Enzygol. 1852231-279, (1990),
incorporated herein by reference. The receptors of the
invention may be expressed in Aspergillus spp. (McKnight
and Upshall, described in U.S. Patent 4,935,349, which is
incorporated herein by reference). Useful promoters
include those derived from Aspergillus nidulans
glycolytic genes, such as the AQQ; promoter (McKnight et
al., EMBO J. 4:2093-2099, 1985) and the tp;A promoter.
An example of a suitable terminator is the Agfig
terminator (McKnight et al., ibid.). Techniques for
transforming fungi are well known in the literature, and
have been described, for instance by Beggs (ibid.),
Hinnen et al. (Proc. Natl. Acad. Sci. USA 75:1929-1933,
1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-
1747, 1984), and Russell (Nature 301:167-169, 1983) each
of which are incorporated herein by reference.
Mammalian expression vectors for use in
carrying out the present invention will include a
promoter capable of directing the transcription of a
cloned gene or CDNA. Preferred promoters include viral
promoters and cellular promoters. Viral promoters
include the immediate early cytomegalovirus promoter
(Boshart et al., gel; 41: 521-530, 1985) and the SV40
promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864,
1981). Cellular promoters include the mouse
metallothionein-1 promoter (Palmiter et al., U.S. Patent
No. 4,579,821), a mouse V‘ promoter (Bergman et al.,
Proc. Natl. Acad. Sci. USA 81: 7041-7045, 1983; Grant et
al., Nuc. Acids Res. 15: 5496, 1987) and a mouse VH
promoter (Loh et al., gel; 33: 85-93, 1983). A
particularly preferred promoter is the major late
promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell.
glel. 2: 1304-13199, 1982). Such expression vectors may
also contain a set of RNA splice sites located downstream
from the promoter and upstream from the DNA sequence
encoding the peptide or protein of interest. Preferred
RNA splice sites may be obtained from adenovirus and/or
immunoglobulin genes.
Also contained in the expression vectors is a
polyadenylation signal located downstream of the coding
sequence of interest. Polyadenylation signals include
the early or late polyadenylation signals from SV40
(Kaufman and Sharp, ibid.), the polyadenylation signal
from the Adenovirus 5 B1B region and the human growth
hormone gene terminator (DeNoto et al., Nuc. Acids Res.
9: 3719-3730, 1981). The expression vectors may include
a noncoding viral leader sequence, such as the Adenovirus
2 tripartite leader, located between the promoter and the
RNA splice sites. Preferred vectors may also include
enhancer sequences, such as the SV4O enhancer and the
mouse u enhancer (Gillies, gel; 33: 717-728, 1983).
Expression vectors may also include sequences encoding
the adenovirus VA RNAs.
Cloned DNA sequences may be introduced into
cultured mammalian cells by, for example, calcium
phosphate-mediated transfection (wigler et al., gel; 14:
, 1978: Corsaro and Pearson, Somatic Cell Genetics 7:
, 1981; Graham and Van der Eb, Virology 52: 456,
1973.) other techniques for introducing cloned DNA
sequences into mammalian cells, such as electroporation
(Neumann et al., EMBO J. 1: 841-845, l982), may also be
used. In order to identify cells that have integrated
the cloned DNA, a selectable marker is generally
introduced into the cells along with the gene or CDNA of
interest. Preferred selectable markers for use in
cultured mammalian cells include genes that confer
resistance to drugs, such as neomycin, hygromycin, and
methotrexate. The selectable marker may be an
amplifiable selectable marker. Preferred amplifiable
selectable markers are the DHFR gene and the neomycin
resistance gene. Selectable markers are reviewed by
Thilly (Mammalian Cell Technology, Butterworth
Publishers, Stoneham, MA, which is incorporated herein by
reference). The choice of selectable markers is well
within the level of ordinary skill in the art.
Selectable markers may be introduced into the
cell on a separate plasmid at the same time as the gene
of interest, or they may be introduced on the same
plasmid. If on the same plasmid, the selectable marker
and the gene of interest may be under the control of
different promoters or the same promoter, the latter
arrangement producing a dicistronic message. Constructs
of this type are known in the art (for example, Levinson
and Simonsen, U.S. Patent No. 4,713,339). It may also be
advantageous to add additional DNA, known as "carrier
DNA" to the mixture which is introduced into the cells.
Transfected mammalian cells are allowed to grow
for a period of time, typically 1-2 days, to begin
expressing the DNA sequence(s) of interest. Drug
selection is then applied to select for growth of cells
that are expressing the selectable marker in a stable
fashion. Transfected cells may also be selected in the
presence of antagonist to inhibit the activity of the
receptor. Suitable antagonists in this context include
D, L, 2-aminophosphonopropionate. For cells that have
been transfected with an amplifiable selectable marker
the drug concentration may be increased in a stepwise
manner to select for increased copy number of the cloned
sequences, thereby increasing expression levels.
Promoters, terminators and methods suitable for
introducing expression vectors encoding recombinant Glugl
into plant, avian and insect cells are known in the art.
The use of baculoviruses, for example, as vectors for
expressing heterologous DNA sequences in insect cells has
been reviewed by Atkinson et al. (Pestic. Sci. 28:
215-224,l990). The use of Agrobacterium rhizogenes as
vectors for expressing genes in plant cells has been
reviewed by Sinkar et al. (J. Biosci. (Banqlaorel 11:
47-58, 1937).
Host cells containing DNA constructs of the
present invention are then cultured to produce
recombinant Glugh The cells are cultured according to
accepted methods in a culture medium containing nutrients
required for growth of mammalian or other host cells. A
variety of suitable media are known in the art and
generally include a carbon source, a nitrogen source,
essential amino acids, vitamins, minerals and growth
factors. The growth medium will generally select for
cells containing the DNA construct by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker on the DNA
construct or co-transfected with the DNA construct.
Transfected cells expressing a cloned Gh%R can
be detected by several methods. By transfecting cells
with an expression vector containing expression units for
both the GluGR and a reporter gene (e.g. luciferase), the
activity of the reporter gene provides an indicator of
expression of the cotransfected GluGR clone. By
including one or more cyclic AMP response elements (CRE)
in the reporter gene expression unit, clones encoding
receptors coupled to either the stimulation or inhibition
of the second messenger adenylate cyclase can be
identified by a change in reporter gene expression in
response to added ligand. DNA constructs comprising a
linked CRE and reporter gene are known in the art. See,
for example, Mellon et al., Proc. Natl. Acad. Sci. USA
86: 4887-4891 (1989), incorporated herein by reference.
Cell lines expressing functional receptors can also be
detected by electrophysiological measurements of agonist-
induced channel activity. Receptor activity can also be
assayed by measuring cytosolic free calcium
concentrations in transfected cells. See, for example,
Thastrup et al., Proc. Natl. Acad. Sci. USA 87: 2466-
2470 (1990) and Picard et al., science 247: 327-329
(1990), which are incorporated herein by reference. A
preferred method for measuring cytosolic free calcium is
by scanning cells with a fluorescent microscope coupled
to a video camera. The cells are injected with a
fluorescent Cab indicator (e.g. Fluo-3 or Fura-2,
Molecular Probes, Inc., Eugene, OR) and exposed to
ligand.
The Gh%R produced according to the present
invention may be purified from the recombinant expression
systems or other sources using purification protocols
that employ techniques generally available to those
skilled in the art. The most convenient sources for
obtaining large quantities of GluGR are cells which
express the recombinant receptor. However, other
sources, such as tissues, particularly brain tissues of
the cerebellum which contain Glugh may also be employed.
Purification may be achieved by conventional
chemical purification means, such as liquid
chromatography, lectin affinity chromatography, gradient
centrifugation, and gel electrophoresis, among others.
Methods of protein purification are known in the art (see
generally, Scopes, R., Protein Purification,
Springer—Verlag, NY (1982), which is incorporated herein
by reference) and may be applied to the purification of
the GluGR and particularly the recombinantly produced
Gh%R described herein. In a preferred embodiment
immunoaffinity chromatography is employed using
antibodies directed against Gh%R as herein described.
In another method of purification, a recombinant gene
encoding GlJ%R or portions thereof can be modified at the
amino terminus, just behind a signal peptide, with a
sequence coding for a small hydrophilic peptide, such as
described in U.S. Patent Nos. 4,703,004 and 4,782,137,
incorporated herein by reference. Specific antibodies
for the peptide facilitate rapid purification of Glugn
and the short peptide can then be removed with
enterokinase.
Thus, as discussed above, the present invention
provides Glx%R isolated from its natural cellular
environment, substantially free of other G protein
coupled glutamate receptors. Purified Glugz is also
provided. Substantially pure GluGR of at least about 50%
is preferred, at least about 70-80% more preferred, and
95-99% or more homogeneity most preferred, particularly
for pharmaceutical uses. Once purified, partially or to
homogeneity, as desired, the recombinant GluGR or native
Gh%R may then be used to generate antibodies, in assay
procedures, etc.
In another aspect, the invention concerns
polypeptides and fragments of Glugl. Polypeptides and
fragments of GlufiR may be isolated from recombinant
expression systems or may be synthesized by the solid
phase method of Merrifield, Fed. Proc. 21:412 (1962),
Merrifield, J. Am. Chem. Soc. 85:2l49 (1963), or Barany
and Merrifield, in The Pe tides, vol. 2, pp. 1-284 (1979)
Academic Press, NY, each of which are incorporated herein
by reference, or by use of an automated peptide
synthesizer. By "polypeptides" is meant a sequence of at
least about 3 amino acids, typically 6 or more, up to
100-200 amino acids or more, including entire proteins.
For example, the portion(s) of Glu¢R proteins which bind
ligand may be identified by a variety of methods, such as
by treating purified receptor with a protease or a
chemical agent to fragment it and determine which
fragment is able to bind to labeled glutamate in a ligand
blot. Polypeptides may then be synthesized and used as
antigen, to inhibit ligand—G1unR interaction, etc. It
should be understood that as used herein, reference to
Gh%R is meant to include the proteins, polypeptides, and
fragments thereof unless the context indicates otherwise.
In another aspect, the invention provides means
for regulating the GluGR-ligand interaction, and thus
treating, therapeutically and/or prophylactically, a
disorder which can be linked directly or indirectly to a
Gh%R or to its ligands, such as glutamate and other
endogenous excitatory amino acids. By virtue of having
the receptors of the invention, agonists or antagonists
may be identified which stimulate or inhibit the
interaction of ligand with a Glugh with either agonists
or antagonists the metabolism and reactivity of cells
which express the receptor are controlled, thereby
providing a means to abate or in some instances prevent
the disease of interest.
In one functional screening assay, the
initiation of fertilization activation events are
monitored in eggs which have been injected with, e.g.,
mRNA which codes for Glugi and subsequently exposed to
selected compounds which are being screened, in
conjunction with or apart from an appropriate ligand.
See generally, Kline et al., Science 241:464—467 (1988),
incorporated herein by reference. Oocytes injected with
mRNA coding for Glu¢R can also be assayed by measurement
of free cytosolic cap as described above.
Another screening assay is based on the use of
mammalian cell lines which express GluGR functionally
coupled to a mammalian G protein. In this assay,
compounds are screened for their relative affinity as
receptor agonists or antagonists by comparing the
relative receptor occupancy to the extent of ligand
induced stimulation or inhibition of second messenger
metabolism. For example, activation of phospholipase C
leads to increased inositol monophosphate metabolism.
Means for measuring inositol monophosphate metabolism are
generally described in Subers and Nathanson, J. Mol.
Cell, Cardiol. 20:13l-140 (1988), incorporated herein by
reference. As noted previously, receptor subtypes that
are coupled to the stimulation or inhibition of the
second messenger adenylate cyclase can be used in assay
systems wherein reporter gene (e.g. luciferase) activity
is linked to receptor—ligand interactions.
The screening procedure can be used to identify
reagents such as antibodies which specifically bind to
the receptors and substantially affect their interaction
with ligand, for example. The antibodies may be
monoclonal or polyclonal, in the form of antiserum or
monospecific antibodies, such as purified antiserum or
monoclonal antibodies or mixtures thereof. For
administration to humans, e.g., as a component of a
composition for in yiyg diagnosis or imaging, the
antibodies are preferably substantially human to minimize
immunogenicity and are in substantially pure form. By
substantially human is meant generally containing at
least about 70% human antibody sequence, preferably at
least about 80% human, and most preferably at least about
-95% or more of a human antibody sequence to minimize
immunogenicity in humans.
Antibodies which bind GluGR may be produced by
a variety of means. The production of non-human antisera
or monoclonal antibodies, e.g., murine, lagomorpha,
equine, etc. is well known and may be accomplished by,
for example, immunizing the animal with the receptor
molecule or a preparation containing a desired portion of
the receptor molecule, such as that domain or domains
which contributes to ligand binding. Receptor subtype-
specific antibodies can be generated by immunizing with
specific peptides. Small peptides (e.g., about 14-20
amino acids) can be coupled to keyhole limpet hemocyanin,
for example, to enhance immunogenicity. For the
production of monoclonal antibodies, antibody producing
cells obtained from immunized animals are immortalized
and screened, or screened first for the production of
antibody which binds to the receptor protein and then
immortalized. As the generation of human monoclonal
antibodies to human GluGR antigen may be difficult with
conventional techniques, it may be desirable to transfer
antigen binding regions of the non-human antibodies, e.g.
the F(ab')2 or hypervariable regions, to human constant
regions (Fc) or framework regions by recombinant DNA
techniques to produce substantially human molecules.
Such methods are generally known in the art and are
described in, for example, U.S. Patent No. 4,816,397 and
EP publications 173,494 and 239,400, which are
incorporated herein by reference. Alternatively, one may
isolate DNA sequences which code for a human monoclonal
antibody or portions thereof that specifically bind to
the human receptor protein by screening a DNA library
from human B cells according to the general protocol
outlined by Huse et al., Science 246:1275-1281 (1989),
incorporated herein by reference, and then cloning and
amplifying the sequences which encode the antibody (or
binding fragment) of the desired specificity.
In other embodiments, the invention provides
screening assays conducted in vitro with cells which
express the receptor. For example, the DNA which encodes
In yet another aspect, the screening assays
provided by the invention relate to transgenic mammals
whose germ cells and somatic cells contain a nucleotide
sequence encoding GluGR protein or a selected portion of
the receptor which, e.g., binds ligand, GTP binding
protein, or the like. There are several means by which a
sequence encoding, for example, the human GlunR may be
introduced into a non-human mammalian embryo, some of
which are described in, e.g., U.S. Patent No. 4,736,866,
Jaenisch, Science 240—l468—1474 (1988) and Westphal et
al., Annu. Rev. Cell Biol. 5:181-196 (1989), which are
incorporated herein by reference. The animal's cells
then express the receptor and thus may be used as a
convenient model for testing or screening selected
agonists or antagonists.
In another aspect the invention concerns
diagnostic methods and compositions. By means of having
the Gh%R molecule and antibodies thereto, a variety of
diagnostic assays are provided. For example, with
antibodies, including monoclonal antibodies, to Glugh
the presence and/or concentration of receptor in selected
cells or tissues in an individual or culture of interest
may be determined. These assays can be used in the
diagnosis and/or treatment of diseases such as, for
example, cerebral ischemia, Parkinsons, senile dementia
and other cognitive disorders, Huntington's chorea,
amyotrophic lateral sclerosis, emesis, migraine, and
others.
Numerous types of immunoassays are available
and are known to those skilled in the art, e.g.,
competitive assays, sandwich assays, and the like, as
generally described in, e.g., U.S. Pat. Nos. 4,642,285;
4,376,110: 4,016,043; 3,879,262; 3,852,157; 3,850,752;
3,839,153; 3,791,932; and Harlow and Lane, Antibodies, A
Laboratory Manual, Cold Spring Harbor Publications, N.Y.
(1988), each incorporated by reference herein. In one
assay format Gh%R is identified and/or quantified by
using labeled antibodies, preferably monoclonal
antibodies which are reacted with brain tissues, e.g.,
cortex, striatum, hippocampus, cerebellum, and
determining the specific binding thereto, the assay
typically being performed under conditions conducive to
immune complex formation. Unlabeled primary antibody can
be used in combination with labels that are reactive with
primary antibody to detect the receptor. For example,
the primary antibody may be detected indirectly by a
labeled secondary antibody made to specifically detect
the primary antibody. Alternatively, the anti-Glugl
antibody can be directly labeled. A wide variety of
labels may be employed, such as radionuclides, particles
(e.g., gold, ferritin, magnetic particles, red blood
cells), fluorophores, chemiluminescers, enzymes, enzyme
substrates, enzyme cofactors, enzyme inhibitors, ligands
(particularly haptens), etc.
The Gh%R DNA may be directly detected in cells
with a labeled Gh%R DNA or synthetic oligonucleotide
probe in a hybridization procedure similar to the
Southern or dot blot. Also, the polymerase chain
reaction (Saiki et al., Science 239:487 (1988), and U.S.
Pat. No. 4,683,195) may be used to amplify DNA sequences,
which are subsequently detected by their characteristic
size on agarose gels, Southern blots of these gels using
GluGR DNA or a oligonucleotide probe, or a dot blot using
similar probes. The probes may comprise from about 14
nucleotides to about 25 or more nucleotides, preferably,
40 to 60 nucleotides, and in some instances a substantial
portion or even the entire CDNA of Glu¢R may be used.
The probes are labeled with a detectable signal, such as
an enzyme, biotin, a radionuclide, fluorophore,
chemiluminescer, paramagnetic particle, etc.
Kits can also be supplied for use with the
receptor of the subject invention in the detection of the
presence of the receptor or antibodies thereto, as might
be desired in the case of autoimmune disease. Thus,
antibodies to Glugz, preferably monospecific antibodies
such as monoclonal antibodies, or compositions of the
receptor may be provided, usually in lyophilized form in
a container, either segregated or in conjunction with
additional reagents, such as anti-antibodies, labels,
gene probes, polymerase chain reaction primers and
polymerase, and the like.
The following examples are offered by way of
illustration, not by limitation.
EXAMPLE I
Preparation of GlgcR enriched mRNA
Total RNA was prepared from the cerebellum of
rats using guanidine isothiocyanate (Chirgwin et al.
Biochemistry 18:52-94 (1979)) and CsCl centrifugation.
Poly(A)+ RNA was isolated using oligo d(T) cellulose
chromatography. After 2 rounds of chromatography on
oligo d(T) cellulose the RNA (800 pg) was divided into
two aliquots and layered over 10-40% linear sucrose
gradients in tubes for an SW 28 rotor. The gradients
were centrifuged for 28 hours at 25,000 rpm to pellet RNA
greater than 4 kb in size. The enriched RNA was injected
into frog oocytes and assayed for the presence of the
GluGR.
Injection of oocytes and voltage-clamp assay of Gh%R
activity
oocytes were prepared from ovarian lobes that
were surgically removed from anesthetized Xenopus
females. The ovarian lobes were washed, pulled apart
into small clumps and dissociated by treatment with
collagenase for 2-3 hours at 20°C with constant, gentle
agitation. The dissociation and defolicularization of
the oocytes is completed manually after removal of the
collagenase. oocytes that were judged healthy and
greater than 1 mm in diameter were transferred to a 50 mm
sterile tissue culture dish and incubated in sterile,
antibiotic-supplemented Barth's medium (88 mM NaCl, lmM
KCl, 0.82 mM MgSO,, 0.33 mM Ca(N03)2, 0.41 mM CaClZ, 2.4
mM NaHCO3, 10 mM HEPES, pH 7.4, 0.1 mg/ml gentamicin,
0.01 mg/ml penicillin, 0.01 mg/ml streptomycin, 0.5 mM
theophylline, and 2.5 mM Na pyruvate) at 19’C.
Injection pipettes were pulled from hard glass
tubing (Drummond) on a modified 700C Kopf vertical
puller. The tip was broken and bevelled using a List
Medical microforge. Tip diameters of the pipettes ranged
from 20-30 mM. Injection pipettes were made RNase free
by heating to 285°C overnight.
Following overnight incubation, healthy oocytes
were selected for injection. RNA, which was stored at
-70°C in DEPC-treated water, was thawed and centrifuged
at 15,000 g for five minutes. Injection was performed
using a modified pipetting device (Drummond). After
injection, the oocytes were incubated in fresh, sterile
Barth's medium which was changed daily, and unhealthy
oocytes were removed.
I Voltage-clamp assays were carried out on
injected oocytes which were each placed in a small
chamber of approximately 500 ul in volume and which was
continuously perfused with standard frog Ringer's (115 mM
NaCl, 2.5 mM KC1, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2) at
1-6 ml/min. The oocyte was impaled with two glass
microelectrodes for recording which, when filled with 3 M
KC1, had a tip resistance of 0.5 to 7.0 megaohms. One of
the two electrodes was connected to a differential
amplifier via a silver/silver chloride half cell. The
bath potential was measured by connecting the other side
of the differential amplifier to the bath via a
silver/silver chloride pellet and a Ringer/Agar bridge.
A low noise, high compliance, voltage—clamp system (NPI)
was used to control the membrane potential and to measure
membrane current. The oocyte membrane potential was
maintained at -60 mV (inside cell negative). one
millimolar glutamate (Sigma), 100 pM quisqualate (Sigma),
1 mM carbamylcholine (Sigma), and the other drugs used in
this assay were applied by switching the perfusing medium
to a medium containing a drug for approximately three
minutes, and the membrane current was recorded on a chart
recorder (Linear Instruments).
After impaling the oocyte with the two
microelectrodes, and imposing the voltage-clamp, the
membrane current (the holding current) gradually declines
to a steady state over a period of several minutes. When
the holding current stabilizes, so that the chart record
is horizontal, the drug is applied for one to three
minutes. An oocyte is judged to have a positive response
if a rapid inward current spike (downward deflection on
the chart), followed by slow current oscillations of
decreasing magnitude, is observed. Our lower limit of
detection depended on the steadiness of the holding
current prior to drug application, but was in the range
of 5-10 nA.
Construction of pVEGT'
To permit transcription of cloned CDNA without
prior endonuclease digestion, bacteriophage T7
transcriptional terminators were added to a cloning
vector. Plasmid pVEGT' is described in WO—A—
92/04444, which is incorporated by reference
herein. The sequence of the putative T7 RNA
transcription terminator, which lies between gene 10 and
gene 11 of bacteriophage T7, is disclosed by Dunn and
Studier (J. Mol. Biol. 166: 477-536 (1983)). As shown in
Figure 5, four synthetic oligonucleotides were designed
from this sequence and ligated into the vector pGEM-l
(obtained from Promega Biotec, Madison, WI), a plasmid
containing a bacterial origin of replication, ampicillin
resistance gene, and the T7 promoter adjacent to a
multiple cloning site. Terminal phosphates were added to
the 5' ends of oligonucleotides ZC776 and ZC777 with T4
polynucleotide kinase and ATP, under standard conditions
(Maniatis et al. ibid). (The sequences of these and
other oligonucleotides referred to herein are shown in
Table 1.) After the incubation, the kinase was heat
killed at 65°C for 10 min. Twenty~five nanograms of
oligonucleotide ZC775 and 25 ng of oligonucleotide ZC776
were annealed by incubation at 65°C for 15 minutes, then
allowed to cool to room temperature in 500 ml of water.
oligonucleotides ZC777 and ZC778 were similarly annealed.
The annealed oligonucleotides were stored at -20'C until
use. The vector pGEM-l was digested with Pst I and Hind
III, and the linearized vector DNA was purified by
agarose gel electrophoresis. The synthetic T7 terminator
(annealed oligonucleotides ZC775, ZC776, ZC777 and ZC778;
was then cloned into pGEM-l.
Twenty-five nanograms of vector plus an equal molar
amount of each of the annealed oligonucleotides
ZC775/ZC776 and ZC777/ZC778 were combined in a 10 pl
reaction mix. After an overnight ligation at 14'C, the
DNA was transformed into competent E. ggli JM83 cells,
and the transformed cells were selected for ampicillin
resistance. Plasmid DNA was prepared from selected
transformants by the alkaline lysis procedure (Birnboim
and Doly, Nuc. Acids Res. 7:1513-1523 (1979)). A portion
of the DNA from these samples was cut with Pst I and Hind
III and analyzed on a 4% polyacrylamide gel to identify
clones that released an 80 bp Pst I—Hind III fragment.
Other diagnostic cuts, such as Eco RI and Not I, were
also made. One of the isolates, designated pGEMT, was
shown by restriction analysis to contain the T7
terminator fragment.
ZC775:
Table 1
Oligonucleotide Seggences (5' - 3')
GCT AGC ATA ACC CCT TGG GGC CTC TAA ACG GGT CT
ZC776:
CTC AAG
ZC777:
TGA GGG
ZC778:
AGC TTG
ZCl75l:
AAT TCT
ZC1752:
GAT CCT
ZC2063:
GAT CCA
ZC2064:
CTT TTA
ZC2938:
GAC_AGA
TTA GAG GCC CCA AGG GGT TAT GCT AGC TGC A
TGC TGA AAG GAG GAA CTA TGC GGC CGC A
CAT AGT TCC TCC TTT CAG CAA AAA ACC C
TGT CAA G
AGC ACA G
TAA AAG AGC T
ATT CAC TAG TGA GCT CTT TTT TTT TTT TTT T
(Table 1, continued)
ZC3015:
TTC CAT GGC ACC GTC AAG GCT
ZC30l6:
AGT GAT GGC ATG GAC TGT GGT
ZC3652:
ACA TGC ACC ATG CTC TGT GT
ZC3654:
AGT GAT GGC ATG GAC TGT GGT
The native T7 terminator from plasmid pAR2529
(Rosenberg et al., Qggg 56:125-135 (1987)) was added to
plasmid pGEMT. Plasmid pGEMT was digested with Bam HI and
plasmid pAR2529 was digested with Bam HI and Bgl II
(Figure 1). The Bam HI—Bg1 II terminator fragment from
pAR2529 was purified by agarose gel electrophoresis. The
terminator fragment was ligated to Bam HI digested pGEMT,
and the DNA was transformed into competent E. 99;; LM1035
cells. Colonies that were ampicillin resistant were
inoculated into 5 ml cultures for overnight growth.
Plasmid DNA prepared by the alkaline lysis procedure was
screened for proper terminator orientation by Bam HI-Sal
I digestion and electrophoresis on an 8% polyacrylamide
gel. A clone that contained the terminator in the
correct orientation, as evidenced by the presence of a
130 bp Bam HI—Sal I fragment, was chosen and named pGEMTT
(Figure 1).
To allow pGEMTT to be packaged as single-stranded
DNA in the presence of M13 phage proteins, the M13
intergenic region from pUC382 (similar to pUC118 and 119
as disclosed by Vieira and Messing, Methods Enzymol. 153:
3-11 (1987) was added to pGEMTT (Figure 1). Plasmid
pGEMTT was digested with Fsp I and Nar I, and the
fragment containing the T7 promoter and transcription
terminator was purified. Plasmid pUC382 was digested
with Fsp I and Nar I, and the fragment encoding the
ampicillin resistance gene and the M13 intergenic region
was gel purified. These fragments were then ligated
together in the presence of T4 DNA ligase. The ligated
DNA was transformed into competent E; ggli LM1035 cells.
Plasmid DNA from twelve ampicillin-resistant colonies was
prepared by the alkaline lysis method, and the DNA was
screened by digestion with Ava I. The appropriate
construction gave two bands, one of 2430 bp and another
of 709 bp. one such isolate was chosen and named pVEG.
Synthetic oligonucleotides encoding the prime sequence
were added to pVEG between the Bam HI and Eco RI sites
(Figure 1). Plasmid pVEG was digested with Bam HI and
Eco RI and the vector fragment was gel purified.
Ninety-six nanograms each of oligonucleotides ZCl751 and
ZC1752 were annealed in 4.5 ul of 10 mM Tris pH 7.5, 20
mM MgCl2 and 10 mM Nacl at 65°C for 20 minutes, then the
mixture was cooled to room temperature over a period of
minutes. The annealed oligonucleotides were ligated
to the pVEG vector fragment with T4 DNA ligase and then
transformed into competent E. ggli LM1035 cells. After
growing overnight to develop the colonies, a filter lift
was taken of the colonies on the agar plate. The filter
was probed with 32P-labeled oligonucleotide ZC1751. All
of the colonies were positive. Plasmid DNA was prepared
from cultures grown from 12 of the colonies. The plasmid
DNA was screened by digestion with Sst I to verify the
absence of the Sst I site between the Eco RI and Bam HI
sites of pVEG. All 12 of the plasmid DNAs were negative
for Sst I digestion. one of these 12 isolates was chosen
and named pVEG‘.
A polyadenylate sequence derived from an
Aspergillus alcohol dehydrogenase CDNA was added to pVEG.
As shown in Figure 1, plasmid pM098 (disclosed in
published European patent application EP 272,277 and
deposited with American Type Culture Collection under
accession number 53428) was digested with Dra I and Bam
HI, and the approximately 150 bp poly(A) fragment was
purified by agarose gel electrophoresis. This fragment
contained mostly po1y(A) sequence with very little
flanking CDNA. To clone the poly(A) cDNA fragment into
pVEG, pVEG was digested with Bam HI and Sma I, and the
3.4 kb vector fragment was gel purified. The vector and
poly(A) fragments were ligated together with T4 DNA
ligase to produce vector pVEGT (Figure 1).
Synthetic oligonucleotides encoding the prime
sequence were added to pVEGT. To accomplish this, pVEGT
was digested with Not I and Sst I, and the 370 bp
fragment containing the poly(A) sequence and the two T7
transcriptional terminators was purified by agarose gel
electrophoresis. Plasmid pVEG' was digested with Not I
and Bam HI, and the 3.2 kb vector fragment was
gel-purified. Two oligonucleotides (ZC2063 and ZC2064)
that formed, when annealed, a Bam HI-Sst I adapter were
synthesized. The two oligonucleotides were individually
kinased and annealed, and ligated with the linearized
vector and the poly(A)—terminator fragment. The
resultant vector, designated pVEGT' (Figure 1), contained
a T7 RNA transcription promoter, an Eco RI cloning site
flanked by the prime sequence, a poly(A) tract, and two
T7 RNA polymerase terminators.
Construction of CDNA library from rat cerebellum polv
A + RNA
Because there was evidence suggesting that the
Gh%R was encoded a very large mRNA of 7 kb (Pong,
Davidson, and Lester, Synapse 2:657 (1988)) and because
full length cDNA encompassing the coding sequence is
required for functional cloning of CDNA, measures were
taken to optimize for synthesis of large cDNA. A novel
method of CDNA synthesis was developed which yielded
large full length cDNA. This was evident by
demonstration that full length 7.5 kb CDNA could be
synthesized from a model 7.5 kb mRNA and that large full
length CDNA were present in a library constructed from
poly(A)+ RNA as demonstrated by Southern blot analysis.
In addition, all enzymes which were important in this
method were pretested and selected from a large number of
lots of enzymes available from commercial suppliers.
Once a satisfactory lot was identified, a large amount of
the enzyme was purchased and the enzyme was stored at
—70°C until used. Once used, the enzyme was stored at
-20°C for a few months and then discarded. Different
"lots" of enzymes from commercial suppliers, including
lots of Superscript reverse transcriptase (BRL), E. 99;;
DNA polymerase I (Amersham) and Mung bean nuclease (NEB),
which were used in the cDNA synthesis, were screened for
quality in test synthesis assays. Superscript reverse
transcriptase lots were assayed for the ability to
synthesize unit length (7.5 kb) first strand cDNA from
7.5 kb RNA (BRL) control. Conditions for first strand
synthesis with Superscript reverse transcriptase lots
were prepared as described below. Radiolabeled first
strand CDNA was analyzed by alkaline agarose gel
electrophoresis. Superscript lots capable of producing
unit length, 7.5 kb cDNA were selected for use.
. 99;; DNA polymerase I lots were assayed for the
ability to produce, by hairpin DNA formation, full—length
second strand CDNA from the 7.5 kb unit—length first
strand CDNA. The second strand CDNA syntheses were
carried out as described below. The quality of the
second strand syntheses were assessed by alkaline agarose
electrophoresis of the radiolabeled product. DNA
polymerase I lots capable of producing 15 kb second
strand DNA from the 7.5 kb unit length first strand cDNA
were selected for use.
Mung bean nuclease lots were tested for the
ability to clip the hairpin DNA formed during second
strand synthesis without degrading the CDNA. In
addition, varying concentrations of enzyme were added to
determine the optimum enzyme concentration for the
conditions set forth below. The reactions were assessed
by alkaline agarose electrophoresis. Lots and
concentrations resulting in the production of 7.5 kb unit
length CDNA were selected for use.
First strand CDNA was synthesized from one time
poly d(T)-selected cerebellum poly(A)+ RNA in two
separate reactions. One reaction, containing
radiolabeled dATP, was used to assess the quality of
first strand synthesis. The second reaction was carried
out in the absence of radiolabeled dATP and was used, in
part, to assess the quality of second strand synthesis.
Superscript reverse transcriptase (BRL) was used
specifically as described below. A 2.5x reaction mix was
prepared at room temperature by mixing, in order, 10 pl
of 5x reverse transcriptase buffer (BRL; 250 mM Tris-Hcl
pH 8.3, 375 mM KCl, and 15 mM MgCl2), 2.5 pl 200 mM
dithiothreitol (made fresh or stored in aliquots at
-70°C) and 2.5 pl of a deoxynucleotide triphosphate
solution containing 10 mM each of dATP, dGTP, dTTP and
—methyl dCTP (Pharmacia). The reaction mix was
aliquoted into two tubes of 7.5 pl each. To the first
tube, 1.3 pl of 10 pCi/pl anP-dATP (Amersham) was added
and 1.3 pl of water was added to the second reaction
tube. Seven microliters from each tube was transferred
to reaction tubes. Fourteen microliters of a solution
containing 10 pg of cerebellum poly(A)+ RNA diluted in 14
pl of 5 mM Tris-Hcl pH 7.4, 50 pM EDTA was mixed with 2
pl of 1 pg/pl first strand primer, ZC2938 (Table 1), and
the primer was annealed to the RNA by heating the mixture
to 65°C for 4 minutes, followed by chilling in ice water.
Eight microliters of the RNA-primer mixture was added to
each of the two reaction tubes followed by 5 pl of 200
U/pl Superscript reverse transcriptase (BRL). The
reactions were mixed gently, and the tubes were incubated
at 45°C for 30 minutes. After incubation, 80 pl of 10 mM
Tris-HCl pH 7.4, 1 mM EDTA was added to each tube, the
samples were vortexed and centrifuged briefly. Three
microliters of each reaction was removed to determine
total counts and TCA precipitable counts (incorporated
counts). Two microliters of each sample was analyzed by
alkaline gel electrophoresis to assess the quality of
first strand synthesis. The remainder of each sample was
ethanol precipitated. The nucleic acids were pelleted by
centrifugation, washed with 80% ethanol and air dried for
ten minutes. The first strand synthesis yielded 1.4 pg
of cerebellum CDNA or a 28% conversion of RNA into DNA.
Second strand cDNA synthesis was performed on the
RNA-DNA hybrid from the first strand reactions under
conditions which encouraged first strand priming of
second strand synthesis resulting in DNA hairpin
formation. The nucleic acid pellets containing the first
strand cDNA were resuspended in 71 pl of water. To
assess the quality of second strand synthesis, anP-dATP
was added to the unlabeled first strand CDNA. To
encourage formation of the hairpin structure, all
reagents except the enzymes were brought to room
temperature, and the reaction mixtures were set up at
room temperature. (Alternatively, the reagents can be on
ice and the reaction mixture set up at room temperature
and allowed to equilibrate at room temperature for a
short time prior to incubation at 16°C.) Two reaction
tubes were set up for each synthesis. One reaction tube
contained the unlabeled first strand CDNA and the other
reaction tube contained the radiolabeled first strand
CDNA. To each reaction tube, 20 pl of 5x second strand
buffer (100 mM Tris, pH 7.4, 450 mM KCl, 23 mM MgCl2, 50
mM (NHg2SO,), 3 pl of beta-NAD and 1 pl of a
deoxynucleotide triphosphate solution containing 10 mM
each of dATP, dGTP, dTTP and dCTP (Pharmacia), 1 pl
After incubation, 3 pl was taken from each reaction tube
to determine total and TCA precipitable counts. Two
microliters of each sample was analyzed by alkaline gel
electrophoresis to assess the quality of second strand
synthesis by the presence of a band of approximately
twice unit length. To the remainder of each sample, 2 pl
of 2.5 pg/pl oyster glycogen, 5 pl of 0.5 M EDTA and 200
pl of 10 mM Tris-HC1 pH 7.4, 1 mM EDTA were added, the
samples were phenol-chloroform extracted, and isopropanol
precipitated. The nucleic acids were pelleted by
centrifugation, washed with 80% ethanol and air dried.
The yield of double stranded CDNA in each of the
reactions was approximately 2 pg.
The single-stranded DNA in the hairpin structure
was clipped using mung bean nuclease. Each second strand
DNA sample was resuspended in 12 pl of water. Two
microliters of 10x mung bean buffer (0.3 M NaOAC, pH 4.6,
3 M Nacl, 10 mM znsog, 2 pl of 10 mM dithiothreitol, 2
pl of 50% glycerol, and 2 pl of 10 U/pl mung bean
nuclease (NEB, lot 7) were added to each tube, and the
reactions were incubated at 30°C for 30 minutes. After
incubation, 80 pl of 10 mM Tris-HC1 pH 7.4, 1 mM EDTA was
added to each tube, and 2 pl of each sample was subjected
to alkaline gel electrophoresis to assess the cleavage of
the second strand product into unit length CDNA. one
hundred microliters of 1 M Tris-HC1 pH 7.4 was added to
each sample, and the samples were twice extracted with
phenol-chloroform. Following the final phenol-chloroform
extraction, the DNA was isopropanol precipitated. The
DNA was pelleted by centrifugation, washed with 80%
ethanol and air dried. Approximately 2 pg of DNA was
obtained from each reaction.
The CDNA was blunt—ended with T4 DNA polymerase
after the cDNA pellets were resuspended in 12 pl of
water. Two microliters of 10x T4 DNA polymerase buffer
(330 mM Tris-acetate, pH 7.9, 670 mM KAC, 100 mM MgAc, 1
mg/ml gelatin), 2 pl of 1 mM dNTP, 2 pl 50 mM
dithiothreitol, and 2 pl of 1 U/pl T4 DNA polymerase
(Boehringer-Mannheim) were added to each tube. After an
incubation at 15°C for 1 hour, 180 pl of 10 mM Tris-Hcl
pH 7.4, 1 mM EDTA was added to each sample, and the
samples were phenol-chloroform extracted followed by
isopropanol precipitation. The CDNA was pelleted by
centrifugation, washed with 80% ethanol and air dried.
Eco RI adapters (Invitrogen, Cat. # N409—20) were ligated
to the blunted cDNA after the DNA from each reaction was
resuspended in 6.5 pl water.
The first strand primer encoded an 8st I cloning
site to allow the CDNA to be directionally cloned into an
expression vector. The CDNA was digested with Sst I
followed by phenol-chloroform extraction and isopropanol
precipitation. After digestion, the CDNA was
electrophoresed in a 0.8% low melt agarose gel, and the
cDNA over 4.2 kb was electroeluted using an Elutrap
(Schleicher and Schuell, Keene, NH). The electroeluted
cDNA in 500 pl of buffer was isopropanol precipitated and
the CDNA was pelleted by centrifugation. The cDNA pellet
was washed with 80% ethanol.
A cerebellum CDNA library was established by
ligating the cDNA to the Eco RI-Sst I digested, agarose
gel purified pVEGT'.
Ten sublibraries of one million clones each were
constructed representing a library of ten million
independent clones. To prepare each sublibrary, 80 ng of
linearized vector were ligated to 40 ng of cDNA. After
incubation at room temperature for 11 hours, 2.5 pg of
oyster glycogen and 80 pl of 10 mM Tris-HCl, 1 mM EDTA
was added and the sample was phenol-chloroform extracted
followed by ethanol precipitation. The DNA was pelleted
by centrifugation, and the DNA pellet washed with 80%
ethanol. After air drying, the DNA was resuspended in 3
pl of water. Thirty-seven microliters of
electroporation-competent DHl0B cells (BRL) was added to
the DNA and electroporation was completed using a BioRad
electroporation unit. After electroporation, 4 ml of SOC
(Maniatis et al.) was added to the cells, and 400 pl was
spread on each of 10-150 mm LB ampicillin plates. Each
plate represented a sublibrary of 100,000 clones. After
an overnight incubation, the cells were harvested by
adding 10 ml of LB ampicillin media to each plate and
scraping the cells into the media. Glycerol stocks and
plasmid DNA were prepared from each plate. The library
background (vector without insert) was established at
about 15%.
The Xenopus oocyte efficiently translates
exogenously added mRNA. Preliminary experiments were
done using the mouse ml muscarinic receptor CDNA (a G
protein coupled receptor that can be detected by voltage-
clamp) cloned into pVEGT'. Injection of RNA transcribed
ig vitro from increasing dilutions of the ml template DNA
indicated that ml agonist induced activity could be
detected for one clone in a pool size of 100,000. A
cerebellum sublibrary was plated into ten pools of
100,000 unique clones.
The pools could also be replica plated onto a
nitrocellulose filter and the original and replica
allowed to grow for a few hours. The original plate is
scraped to harvest all the colonies. Plasmid DNA is
prepared and purified by cesium chloride gradient
ultracentrifugation. The DNA from each pool is
transcribed in vitro with T7 RNA polymerase in the
presence of 7—methy1-G, the capped nucleotide, to
increase translation efficiency. Template DNA
transcription reactions are spiked with a dilution of two
control genes cloned into pVEGT': the mouse ml gene and a
secreted version of the human placental alkaline
phosphatase gene (SEAP; Tate et al., Fed. Am. soc. Exp.
Eigly 8: 227-231 (1990), incorporated by reference
herein). Transcription from the control genes would
allow selection of oocytes that more efficiently
translate the injected RNA, and a determination whether
oocytes that are negative for the GlunR are true
negatives, that is, still having a detectable ml agonist—
induced response.
Plasmid DNA prepared from each of the 10 pools of
100,000 clones, which in total represented one sublibrary
of one million clones of the cerebellum cDNA library, was
purified by cesium chloride gradient ultracentrifugation.
The DNA was transcribed in vitro with T7 RNA polymerase
(Pharmacia) in the presence of capped nucleotide (GpppG,
Pharamcia). The presence of a poly(A) sequence and two
T7 RNA polymerase terminators in pVEGT' resulted in RNA
with a capped 5' end, the sequence of the CDNA insert,
and 3' poly(A) tails. Capped RNA is believed necessary
for efficient translation in oocytes (Noma et al. Nature
319:640 (1986)) and the poly(A) sequence has been shown
to increase the synthesis of a protein in oocytes by more
then 40 fold. The transcription reaction tubes were set
up by adding 12 pl of 5x transcription buffer (Stratagene
Cloning Systems, La Jolla, CA), 3 pl each of 10 mM ATP,
CTP, GTP, and UTP, 6 pl of 10 mM GpppG (Pharmacia), 6 pl
of 1 mg/ml BSA, 3 pl of 200 mM DTT, 1.5 pl of 40 U/pl
RNasin (ProMega Biotech, Madison, WI), 8.5 pl of water,
pl of cDNA containing 5 to 10 pg DNA, and 1 pl of 70
U/pl T7 RNA polymerase. After mixing, 10 pl of the
reaction was transferred to a tube containing 0.5 pci of
anP-UTP to determine the total counts and counts
incorporated into RNA. The samples were incubated at
37°C for one hour. The CDNA in the unlabeled samples was
degraded with the addition of 1 pl of 200 mM DTT, 2 pl of
U/pl DNase I, and 0.5 pl of 40 U/pl RNasin and the
incubation was continued at 37°C for 15 minutes. Forty
microliters of water was added to the radiolabeled
reactions, and 1 pl was removed from each sample and
counted to determine total counts. The remainder of the
labeled samples were ethanol precipitated. The samples
were centrifuged to collect the RNA and the RNA pellets
were counted to determine the counts incorporated into
RNA. After the DNA degradation reaction in the unlabeled
samples, 70 pl of 10 mM Tris-Hcl, 1 mM EDTA was added to
each sample, and the samples were twice-extracted with
phenol—chloroform followed by one chloroform extraction.
The RNA was ethanol precipitated. After centrifugation
to collect the RNA, the pellets were washed with 80%
ethanol, followed by air drying for 10 minutes. A
typical yield of the unlabeled RNA was 20 to 30 pg. The
unlabeled RNA was resuspended at 2 pg/pl in
diethylpyrocarbonate (DEPC, Sigma) treated water and
stored at -70°C.
Prior to microinjection into oocytes, the RNA
samples were thawed and centrifuged in a microfuge for 5
minutes to remove any particles that might clog a
microinjection pipet. After centrifugation, 80% of each
sample was removed and split into two tubes.
The RNA from each of the 10 sublibraries were
injected into oocytes as described above and translation
was allowed for four days. Expression of Glugl activity
was assessed by voltage-clamp assay as described above.
One of the 10 sublibraries, Z93-1.9, produced a signal
with administration of quisqualate to the oocyte.
Subdivision of the cDNA library pool to obtain pure Glugi
elfl
The DNA pool (Z93—1.9) was subdivided by plating
clones from the glycerol stock onto LB ampicillin plates.
To determine the number of clones that should be plated
for the subdivision of the 100,000 clone pool to identify
a positive clone, the probability equation N = ln (1- P)
/ ln (1 - f) (Maniatis et al., ibid.) was used, where P
is the desired probability of including the clone of
interest, f is the fraction of positive clones in the
pool, and N is the number of clones to be plated to
provide the given probability. For a probability of
99.8% for a pool size of 100,000 to contain one positive
clone, 621,461 clones should be plated.
Forty-eight 150 mm LB ampicillin plates were
plated with the glycerol stock representing the 100,000
positive pool, Z93-1.9, at a density of approximately
14,000 clones per plate to give a total of 670,000
clones. After an overnight incubation 37°C, the bacteria
on each plate were harvested into 10 ml of Solution I (as
described by Birnboim and Doly, Nuc. Acids Res. 7:l513
(1979)), incorporated by reference herein). A glycerol
stock was prepared from a portion of the cells, and
plasmid DNA was prepared from the remainder of the cells.
Six pools of DNA representing eight of the LB ampicillin
plates each were prepared by combining one tenth of the
plasmid DNA from groups of eight plates into each pool.
The plasmid DNA from these six pools was purified by
cesium chloride gradient centrifugation. The DNA was
transcribed into RNA as outlined above. Transcription of
the parent pool Z95-1.9 was included as the positive
control. Oocytes were injected with the RNA and
voltage-clamp assays on the oocytes identified pool
Z9932 as positive for Gh%R. Pool Z99—25—32
contained DNA prepared from plates 25 through 32.
Plasmid DNA from plates 25 to 32 were cesium
chloride banded and transcribed into RNA as described
above along with the positive parent pool Z99—25-32.
Oocytes were injected with the RNA and voltage clamp
assays, carried out as described above, identified pools
Z104-25 and Z111—32 as being weakly positive, Z106—27 and
Z109-30 as intermediately positive, and Z108-29 and
Z110-31 as the most positive. The pool resulting in
Z110-31 was chosen for further subdivision.
Identification of positive pools from the
subdivision of the positive pool of 14,000 (Z110-31) from
the glycerol stock was unsuccessful. Therefore, plasmid
DNA prepared from the pool resulting in Z110-31 was
electroporated into bacteria and plated on 60 plates at a
density of 1,000 clones/plate. Plasmid DNA was prepared
from the bacteria harvested from each plate. Aliquots of
the plasmid DNA from each plate were mixed to make six
pools representing ten plates each. The plasmid DNA was
cesium chloride banded, and the RNA was transcribed as
described above. RNA was transcribed from pools Z108-29,
Z110-31, and a muscarinic receptor cDNA, ml, for use as
positive controls. The RNA was injected into oocytes and
voltage-clamp assays were carried out as described above.
The assays identified pool Z133-21 to 30 as positive.
Plasmid DNA from plates 21 to 30 were cesium
chloride banded and transcribed as described above. The
transcribed RNA and the RNA from the parent pool Z133-21
to 30 were injected into oocytes and assayed as described
above. The voltage—clamp assay identified pool Z142-22
as positive.
Identification of positive pools by the
subdivision of the positive pool Z142-22 from a glycerol
stock proved unsuccessful. Restriction analysis of
plasmid DNA prepared from randomly selected clones from
pools Z110-31 (the pool of 14,000) and Z142-22 (the pool
of 1,000) indicated that 50% of pool Z110 - 31 and 68% of
pool Z142 - 22 were clones without inserts.
To assess physical methods for enriching for the
Gh%R clone and to establish how many clones from pool
Z142-22 needed to be assayed to include a Glugz clone,
undigested plasmid DNA from pool Z142—22 was
electrophoresed on an agarose gel. The super-coil band
representing vector without insert was cut out and the
remainder of the DNA was eluted from the gel. The DNA
was then electroporated into bacteria cells, and plated
at densities of 3,400, 6,900, and 13,800 clones per
plate. The plates were replica plated and grown
overnight. Plasmid DNA was prepared from the cells
harvested from the replica of each plate. The plasmid
DNA was transcribed, and the RNA was assayed in oocytes
as described above. As a control, each pool contained
the equivalent of one colony of ml as an internal
positive control. In addition, ml was used as an
external positive control. The voltage—clamp assays
identified the DNA from the 6,900 clone pool (Z167~7) as
positive.
The clones represented on the 6,900 clone plate
that resulted in the positive pool Z167-7 were subdivided
by replica plating the master plate onto a Biodyne-A
nylon membrane on an LB ampicillin plate. The replica
plate was incubated four hours at 37°C. After
incubation, sub-pools were prepared by removing the
membrane from the plate, taping the membrane to a sterile
glass plate on a light box, and overlaying the membrane
with a grid which divided the membrane into 100 sections.
The sections of the grid and underlying membrane were
then cut out with a razor blade that had been dipped in
alcohol and flamed between each cut. Alcohol-treated,
flamed forceps were used to transfer each membrane
section to a test tube containing 12.5 ml of LB
ampicillin media. The cultures containing the membrane
sections were incubated overnight at 37’C. After
incubation, 0.5 ml of each culture was mixed with 0.5 ml
of 50% glycerol and stored at -70°C to establish glycerol
stocks of each sub-pool. Aliquots of the 100 cultures
were pooled in a 10 X 10 matrix with samples (1) through
(10) on the abscissa and samples (a) through (j) on the
ordinate. For example, 1 ml of cultures (1) through (10)
were added to tube 1 and 1 ml of cultures (1), (11),
(21), (31), (41), (51), (61), (71), (81), and (91) were
added to tube (a) and so on until 10 rows of 10 and 10
columns containing pools of 10 cultures each were
completed. Ten microliters of an overnight culture
containing ml-transformed bacteria was added to each pool
as an internal control. Plasmid DNA was prepared from
the 20 sub-pools, and the DNA was purified by cesium
chloride gradient centrifugation. RNA was transcribed
from the plasmid DNA and was assayed in oocytes as
described above. Positive controls were the parent pool
Z167-7 and pure m1 RNA. The voltage—clamp assays
indicated that only pools Z175—1 and Z191-g were
positive. Consulting the matrix, this indicated that the
membrane section number (7) contained the Glu@R clone.
To subdivide the clones contained in section (7),
a piece of Biodyne A membrane was applied to the master
plate containing section (7), the membrane extending
beyond section (7) on each side by half the width of
section (7). The membrane was removed from the plate,
applied to a fresh LB ampicillin plate colony side up,
and incubated overnight at 37°C. The membrane was
subdivided as described above with the central region of
the membrane, the actual section (7) area, divided into 9
small, equivalent—sized squares and the membrane on each
side of section (7) was taken as four additional areas.
Each membrane section was used to inoculate a 10 ml
liquid culture. Bacteria transformed with the ml clone
were used as an internal control in each culture as
described above. After overnight incubation at 37°C,
plasmid DNA was prepared, and the DNA was purified by
cesium chloride gradient centrifugation. RNA was
transcribed and assayed in oocytes as described above
using RNA from m1 and the parent pool number (7) as
positive controls. Gh%R activity was found in only pool
Z203-7 corresponding to membrane section number (7).
Pool Z203—7 was subdivided by electroporating the
plasmid DNA prepared from the membrane section number (7)
into DH10B electroporation-competent cells. The
transformants were plated at a density enabling
individual colonies to be picked. Individuals clones
were picked to a master plate and into 2 ml of LB
ampicillin media. The cultures were incubated overnight,
and plasmid DNA was prepared by the method essentially
described by Holms and Quigley (Anal. Bioc. 114: 193,
(1981)). Restriction analysis suggested that the clones
were grouped into 7 different classes of clones. Plasmid
DNA, prepared from each class, representing fifty total
clones were prepared, transcribed, and assayed in oocytes
as described above. However, none of the clones were
positive.
To screen for positive clones, electroporation-
competent E. ggl; DH10B cells were electroporated with
the DNA prepared from membrane section number (7)
(Z203-7) and were plated at 180, 360, 900, and 1800
colonies per plate. The plates were incubated overnight,
and replica plates were prepared as described above.
Plasmid DNA prepared from each replica plate was combined
with 1 to 1000 parts of ml as an internal control. The
DNA pools, the ml clone and the parent pool Z203-7 were
transcribed, and the RNA was assayed by oocyte injection.
The first transcription and injection showed no
positives, however, upon retranscription and reanalysis
the 1800 clone pool (2264-1800) was positive for Glugk
activity.
To subdivide the positive pool of 1800
(Z264-1800), all of the colonies from the plate of 1800,
1528 in total, were each picked to two 100 mm LB
ampicillin agar plates on a 100 colony grid. After
overnight growth, one set of the duplicate plates was
designated as a master set and was placed at 4°C. The
other set was replica plated to a third set of plates.
After overnight incubation of these plates, the cells on
the replica plates were harvested into media and plasmid
DNA was prepared from the pooled cells. As described
above, an internal ml control was included in each DNA
preparation. ml DNA and the parent Z264-1800 DNA were
used as external positive controls. Plasmid DNA prepared
from the 16 plates was transcribed, and the RNA was
assayed in oocytes as described above. one of the pools
ofl00 clones, Z256-I produced Gh%R activity.
row and column of the 10 x 10 matrix. As described
previously, plasmid DNA encoding ml was used as an
internal positive control. Plasmid DNA prepared from
each tube, ml DNA and DNA from the parent pool 2264-1800
were transcribed and assayed in oocytes as described
above. G1l%R activity was identified only in row (5) and
column (e). Thus, the positive clone number 45 was
identified as containing the GlunR activity.
To confirm the result, plasmid DNA from clone #45
was prepared, transcribed and assayed in oocytes as
described above. The results of the assay indicated that
clone #45 was capable of producing Gluglactivity.
Figure 2 illustrates the data taken from voltage-clamp
recordings at several stages in the subfractionation of
the cerebellum library. Panel (a) is a recorded response
to quisqualate of an oocyte previously injected with in
yitgg transcribed RNA from a rat cerebellum sublibrary of
100,000 independent colonies; panel (b) shows the
response to quisqualate in a cell previously injected
with RNA transcribed from a subfractionated pool of
14,000 colonies. The peak current was truncated by the
chart recorder, but the actual peak current (estimated
from a digital panel meter) was approximately 1300 nA.
Panel (c) shows the response to quisqualate in a cell
injected with pure Glugz RNA from clone 45—A. The
amount of RNA injected per oocyte was approximately 100
ng, except in panel (c) where the amount of RNA was 50
P9-
The following describes an alternative means for
subdividing and screening a positive pool. Working with
CDNA inserts in a plasmid based rather than a lambda-
based vector influences the subfractionation protocol.
Once a positive pool is identified, the replica filter is
overlayed with another sterile nitrocellulose filter.
The filter is cut into 88 pieces by using evenly spaced
cuts of 10 rows and 10 columns to form a grid. Each of
the 88 pieces is transferred to 10 ml of sterile LB +Amp
and grown for several hours. Twenty pools are formed: C
1-10 (corresponding to column number) and R 1-
47
(corresponding to row number). An aliquot of each of the
88 subfractions is pipetted into 2 tubes, corresponding
to its position in a row and a column. DNA is isolated
from the 20 pools, purified on CsCl gradients and
transcribed in an in yiggg reaction that includes the
control ml and SEAP plasmids. After injection into
oocytes and voltage—clamp recording there are 2 positive
pools, pinpointing the location of 1 of the 88 original
subfractions.
Because the positive clone is still part of a pool
it must be further subdivided. The probability equation
described above is used to determine the number of clones
to be plated for the next subdivision of the pool. The
glycerol stock from the positive pool is plated out at,
e.g., 3000, 6000 and 18,000 clones per plate. After
replica plating the DNA is harvested, transcribed,
injected and assayed. The pool which is positive is
subdivided into a grid of 88 as described above. The
assay is repeated, and a single square of the grid is
positive. At the next step of subdivision of the pool,
100 individual colonies to a plate are picked, replica
plated, and 20 pools are made for transcription and
assay. Positive clones are streaked out, several
colonies picked and restriction mapped and template and
transcript prepared for injection and assay.
Characterization of Glugz
To establish that the GlunR encoded by clone 45—A
groups were incubated for 24 hours at 19°C as described
by sugiyama et al., Nature 325:53l (1987) and Moriarity
et al., J. Biol. Chem. 264:l3521 (1989), both of which
are incorporated by reference herein. The oocytes from
both the control and toxin-treated groups were subjected
to voltage-clamp assays as described previously. In one
example, oocytes perfused as described previously with
100 uM L—glutamic acid showed a mean L—glutamic
acid—induced current of 264.2 nA +/- 73 nA in control
oocytes (SEM, n=6) and 57.7 nA +/- 19 nA (n=9) in
toxin-treated oocytes. The mean membrane current in the
toxin-treated group was significantly smaller (p < 0.01)
than in the control group suggesting that oocytes
injected with 45—A RNA coupled to a pertussis
toxin-sensitive G protein.
L-glutamic acid and some of its structural
derivatives that are known to activate GlucR currents in
a dose-dependent manner were applied to oocytes that had
been injected with RNA transcribed from the 45-A clone.
RNA was transcribed and oocytes were prepared and
injected as previously described. Dose dependent
responses were measured using voltage clamp assays were
carried out in the presence of increasing concentrations
of L—glutamic acid (Sigma), quisqualic acid (Sigma),
ibotenic acid (Sigma), or trans 1-amino-cyclopentyl-1,3
dicarboxylic acid (tACPD; Tocris Neuramin, Essex,
England). Four or five separate oocytes were perfused
with increasing concentrations of a particular drug with
minutes between consecutive applications of the drug
to minimize any interference from desensitization. The
responses were normalized to a subsequent response to 100
pM L—glutamic acid. The data were analyzed using the
following equation:
(Fractional current) = (Dose")/(Dose”) + (ECm)",
where:
Dose = a dose of drug normalized to that evoked by
a subsequent application of 100 uM L—glutamic acid:
Fractional current = the peak current evoked by a
dose, as defined above;
Ecw = effective concentration that evokes a 50%
response (a measure of the potency of an agonist): and
n = the Hill coefficient, a measure of the
cooperativity of the reaction.
Using this equation, the effective concentration
at 50% stimulation relative to 100 uM L-glutamic acid was
determined for each dose response experiment. Figure 6
shows a representative dose response curve for varying
concentrations of L-glutamic acid. The potency series of
glutamate analogs and their associated ECm's are listed
in Table 2.
Table 2
Glutamate Analoq Potencies (Ecml
Quisqualic acid 0.681 uM
L-glutamic acid 12.32 uM
Ibotenic acid 32.37 uM
tACPD 3 7 6 um
In addition, oocytes were exposed to the following
L-glutamic acid analogs: aspartic acid (Tocris Neuramin),
kainic acid, N-methyl-D-aspartic acid (NMDA; Sigma),
2-amino—4-phosphonobytyric acid (APB; Sigma),
a-aminohydroxymethyl-isoxazolepropionic acid
(AMPA; Research Biochemicals Inc., Wayland, MA) at
saturating concentrations and the responses were each
normalized to a subsequent response to 100 pM
L-glutamate. The L-glutamic acid analogs that were found
to be ineffective were 1 mM aspartic acid, 1 mM kainic
acid, 100 pM NMDA + 10 pM glycine, 100 uM APB and 100 uM
AMPA.
Voltage clamp assays were also carried out on
injected oocytes to measure the inhibition by the
putative glutamate G protein coupled receptor antagonist,
2—aminophosphonopropionic acid (AP3). Voltage clamp
assays showed that at 1 mM, DL-AP3 (Sigma) reduced the
current evoked by 10 uM glutamic acid to 59.3 +/- 7.3% of
the control.
Clone 45 cells were streaked out on LB Amp plates
and several colonies were picked, grown up and the DNA
isolated. Pure 45-A DNA was prepared and restriction
mapped by standard procedures. Clone 45-A has been
deposited with the American Type Culture Collection,
12301 Parklawn Drive, Rockville, MD, 20852, under ATCC
Accession No. 68497. DNA was digested with single or
multiple enzymes. The fragments were separated on both
1% agarose and 4% Nusieve gels by electrophoresis. After
electrophoresis the DNA was transferred to nitrocellulose
filters using standard protocols for Southern transfer.
Restriction sites were mapped based on size and based on
hybridization to Pst I subclones of 45-A DNA.
Additionally, the entire 45-A cDNA insert can be isolated
by digestion with Not I restriction endonuclease. The
Not I insert was kinased with 1-RP ATP, and after
digestion of half of the sample with Bam HI to remove the
3' label, both samples were subjected to digestion with a
number of enzymes known to be present once in the insert.
In this way the unique sites could be localized. A
restriction map of GluGR clone 45-A is shown in Figure 3.
The entire 4S—A clone was sequenced in both
directions using the dideoxynucleotide chain termination
method (Sanger and Coulson, J. Mol. Biol. 94:441 (1975),
incorporated herein by reference). Figure 5 shows the
DNA sequence and deduced amino acid sequence of clone
45-A. Figure 5 also shows the location of putative N-
linked glycosylation sites, which have been predicted to
occur at the amino acid sequence Asn-X-Thr.
As shown in Figure 5, seven putative transmembrane
domains have been predicted from the deduced amino acid
sequence of clone 45-A using the method described by
Eisenberg et al. J. Mol. Biol. 179:125—142, (1984),
incorporated herein by reference. Only those predicted
to be transmembrane multimeric domains were included. An
additional transmembrane domain (the third) was predicted
using the method of Hopp and Woods, Proc. Natl. Acad.
Sci. USA 78:3824-3838 (1981). Based on these
predictions, the protein encoded by clone 45-A appears to
have two unusually large domains on the amino- and
carboxy-termini that are not found in any of the other
reported G protein coupled receptors which have the
common structural feature of seven predicted membrane
spanning regions. Analysis of the deduced amino acid
sequence of clone 45-A predicts three other hydrophobic
stretches including one at the amino-terminus of the
sequence. This amino-terminal hydrophobic stretch may be
a signal sequence, although no signal cleavage site is
predicted downstream of the sequence.
Poly(A)+ RNA was isolated from total rat brain and
rat cerebellum using oligo d(T) cellulose chromatography
as described by Aviv and Leder (ibid.). Poly(A)+ RNA
from rat retina, rat heart, rat lung, rat liver, rat
kidney, rat spleen, rat testis, rat ovary and rat
pancreas were purchased from Clonetech. The poly(A)+ RNA
samples were analyzed by northern analysis (Thomas, Proc.
Natl. Acad. Sci. USA 77:5201-5205 (1980), which is
incorporated by reference herein). The RNA was denatured
in glyoxal, electrophoresed in agarose and transferred to
a nitrocellulose membrane essentially as described by
Thomas (ibid.). The northern blot was hybridized with a
radiolabeled 3473 bp Eco RI-Xba I fragment from the 45-A
clone. Autoradiography of the blot showed hybridization
to a major band of approximately 7 kb and a smaller band
of approximately 3.8 kb in the total rat brain and rat
cerebellum RNA.
Single-stranded cDNA was synthesized using 1 pg of
the poly(A)+ RNA using Superscript reverse transcriptase
(BRL) under conditions described by the manufacturer.
one fourth of the CDNA was used as a template for PCR
amplification using 40 pmoles each of the GluGR-specific
primers ZC3652 (Table 1) and ZC3654 (Table 1) and 2.5 U
Taq I polymerase (Perkin Elmer Cetus, Norwalk, VA) and
conditions specified by the manufacturer. As an internal
control, the PCR reaction also contained 2 pmoles each of
the glucose—6—phosphate dehydrogenase-specific primers
ZC3015 (Table 1) and ZC3016 (Table 1). After thirty
cycles (one minute at 94°C, one minute at 60°C, ninety
seconds at 72°C), the samples were phenol-chloroform
extracted and 20% of each reaction was electrophoresed in
agarose. The DNA was bidirectionally transferred to
nitrocellulose membranes, and the filters were hybridized
with either radiolabeled ZC3652, ZC3654, ZC3015 and
ZC3016 or with the radiolabeled Eco RI—Xba I fragment of
clone 45-A described above. Autoradiography of the
hybridized blot showed that GlufiR transcript was mainly
confined to total rat brain and rat cerebellum; however,
longer exposures showed a GludR-specific transcript in
both retina and testis.
Total RNA was prepared, as described above, from
specific rat brain regions including frontal cortex,
cerebellum, hippocampus, cortex, striatum, pons medulla,
and the remainder of the brain. Single-stranded cDNA was
synthesized as described previously using 20 pg of total
RNA in 50 pl using Superscript reverse transcriptase
(BRL) under conditions described by the manufacturer.
After a one hour incubation at 42°C, the samples were
treated with RNAse (Boehringer Mannheim Biochemicals,
Indianapolis, IN), phenol-chloroform extracted, and
ethanol precipitated. The samples were resuspended in
water and half of each sample was subjected to PCR
amplification. Each PCR amplification contained 40
pmoles of each of the GlucR-specific primers zC3652 and
ZC3654 described above, 2 pmoles of each of the
glucose—6—phosphate dehydrogenase-specific primers ZC3015
and ZC3016 and 2.5 U Taq I polymerase (Perkin Elmer
Cetus) and conditions described by the manufacturer.
After 35 cycles (one minute at 94°C, one minute at 60°C,
ninety seconds at 72°C), the samples were
phenol-chloroform extracted, and 20% of each reaction was
electrophoresed in agarose. The DNA was transferred to a
nitrocellulose membrane, and the filter was hybridized
with the radiolabeled Eco RI-Xba I fragment of clone 45-A
described above. Autoradiography of the hybridized blots
showed a broad distribution of the Gh%R transcript
throughout the brain, although the frontal cortex and
cerebellum appear to be somewhat enriched.
Southern analysis of rat and human genomic DNA was
carried out using the method essentially described by
Blin et a1. (Nuc. Acids Res. 3:2303 (1976), which is
incorporated by reference herein). Briefly, rat and
human genomic DNA was prepared from the rat cell line UMR
106 (ATCC CRL 1661) and a human hepatoma cell line (ATCC
HTB 52), respectively. The genomic DNA was digested with
either Eco RI or Pst I, and electrophoresed through
agarose. The DNA was transferred to a nitrocellulose
membrane, and the membrane was hybridized with a
radiolabeled 1.6 kb Pst I fragment from clone 45-A.
Autoradiography of the hybridized blot suggest that the
human gene has a similar sequence to the rat Glugk
sequence, the GludR gene contains at least one intron,
and that there are a small number of closely related
genes.
Expression in Mammalian Cells
The entire GluGR cDNA insert was removed from the
pVEGT' cloning vector by digestion with Not I and Xba I.
The ends were blunted with DNA polymerase I (Klenow
fragment) and dNTPs, and were then ligated with Eco RI
(Smart) linkers. After linker ligation, the insert with
Eco RI ends in kinased and ligated to Eco RI—cut and
capped Zem228 expression vector. Bacteria were
transformed with the ligation reaction and clones were
characterized by restriction analysis and partial
sequencing (see Fig. 4).
Cultured mammalian cells, such as BHK 570 and BHK
ts13 served as host cells for expression. Twenty five pg
of CsCl—purified DNA was precipitated with calcium
phosphate and added to tissue culture cells in a 150 mm
plate. After 4 hours the cells were subjected to a
glycerol shock and were then put into non-selective
medium. In some cases it may be necessary to include an
Transfected cell lines expressing GlufiR were
identified by the binding of 3H—glutamate to membrane
preparations from transfected cells. Cell lines
expressing low to moderate levels of GlufiR are used to
set up functional screening assays.
Clones of BHK 570 and BHK TKts13 cells expressing
the rat G protein coupled glutamate receptor CDNA were
plated in two or three 150 mm maxi-plates culture dishes
and were grown to confluency. The cells from each plate
were scraped in 5 ml of PBS (phosphate buffered saline,
Sigma Chemical Co., St. Louis, MO), which was was
pre-chilled to 4°C. The cells were removed to a chilled
centrifuged tube, and the plates were each rinsed with 5
ml of chilled PBS and pooled with the cells. The chilled
tubes were spun at 1,000 rpm for two minutes, and the
supernatant was discarded. The cells were frozen at
either -70°C or on dry ice. In some cases, the cells
were left overnight at -70°C. The cells were thawed on
ice and were resuspended in 10 ml of a buffer containing
mM Tris, pH 7.0, 2.5 mM Caclz, 1 mM PMSF, which was
pre-chilled to 4°C, by homogenizing the cells for about
seconds. The suspension was poured into chilled
centrifuge tubes. The homogenizer was rinsed with 10 ml
of the same chilled solution, and the rinse was combined
with the suspension. The centrifuge tubes were spun for
fifteen minutes at 40,000 x g at 4°C, and the supernatant
was discarded. The pellet was homogenized with a buffer
containing 30 mM Tris, pH 7.0, 2.5 mM Caclz, which was
pre-chilled to 4°C. The homogenizer was rinsed with the
chilled buffer, and the rinse was combined with the
homogenate. The homogenate was spun as described above.
The second homogenization was repeated on the resulting
pellet. The final pellet was resuspended in between two
and five milliliters of 30 mM Tris, pH 7.0, 2.5 mM Cacly
which was pre-chilled to 4°C. Triplicate samples were
prepared for each plus and minus quisqualate assay point
such that 250 ul aliquots of each homogenate sample were
added to the wells of a 96-well microtiter plate. To a
buffer containing 30 mM Tris, pH 7.0, 2.5 mM Caclz, which
was pre-chilled to 4°C, a final concentration of 10 nM
tritiated glutamic acid was added, and the solution was
split in half. To one half, quisqualate was added to a
final concentration of 1 mM. Two hundred and fifty
microliter aliquots of either 30 mM Tris, pH 7.0, 2.5 mM
Caclz, 5 nM tritiated glutamic acid and 500 mM
quisqualate, or 30 mM Tris, pH 7.0, 2.5 mM CaCl2, 5 nM
tritiated glutamic acid were added to the triplicate
samples. The samples were incubated for thirty minutes
at room temperature. The samples were harvested onto
glass filters and were immediately washed with ice—cold
mM Tris, pH 7.0, 2.5 mM CaCl2 under vacuum using an
LKB 1295-001 automated cell harvester (Pharmacia LKB,
Piscataway, NJ). The filters were dried in a microwave
oven and counted in a gamma counter.
Protein determinations were carried out using a
Coomassie Blue-based assay from Pierce Chemical Company
(Rockford, IL) under conditions set forth by the
manufacturer. One hundred microliters of undiluted cell
homogenate or BSA standard was added to 2 ml of reagent
and the optical density was measured at 595 nm. Protein
concentrations of the samples were taken from a standard
curve generated using the BSA standards diluted in 30 mM
Tris, pH 7.0, 2.5 mM Caclr
The results of these assays showed that
quisqualate was able to competitively bind the glutamate
receptor expressed by the transfected BHK cells.
Functional screening of aqonists and antagonists
BHK 570 cells expressing GluGR or mock-transfected BHK
570 cells are plated into 24-well tissue culture dishes
at about 100,000 cells per well. After 24 hours, the
cells are labeled with 0.2 pci of myo-(2-}H) inositol
(specific activity — 20 Ci/mmol; New England Nuclear,)
per well. At the end of a 24 to 48 hour incubation, the
cells are washed with prewarmed DMEM (Dulbecco's Modified
Eagles Medium: Product No. 51-432, JRH Biosciences,
Lenexa, KS) which has been buffered to pH 7.4 with Hepes
buffer (Sigma Chemical Co.) containing 10 mM Licl, and
are incubated for five minutes at 37. The selected drugs
are then added and the cells are incubated for an
additional thirty minutes at 37°C. The reaction is
stopped by placing the cells on ice, and the cells are
lysed by aspirating off the media and adding 0.5 ml of
cold DMEM and 0.5 ml of ice-cold 10% perchloric acid.
After ten minutes the cell lysate is transferred to a
tube on ice containing 250 pl 10 mM EDTA, pH 7.0. The
samples are neutralized with 325 pl of 1.5 M KOH in 60 mM
Hepes Buffer. After the precipitates settles, 1.0 ml of
the supernatant is applied to an Amprep minicolumn
(Amersham, Arlington Heights, IL, RPN1908). Inositol
phosphates are eluted off the column and samples are
counted in a scintillation counter. A positive response
is indicated by an increase in labeled inositol phosphate
levels.
EXAMPLE II
Screening for additional glutamate receptor subtypes
Additional glutamate receptor subtypes were
isolated using probes derived from clone 45-A. Glutamate
receptor subtypes were isolated from a total rat brain
CDNA library in Lambda Zap II, which was size selected
for inserts of 3 kb before ligation (prepared for Terry
Snutch, Ph.D., University of British Columbia, Vancouver,
British Columbia, Canada by Stratagene Cloning Systems,
La Jolla, CA) and a rat cerebellum CDNA library in Lambda
Zap II, which was size selected for inserts of 3 kb
before ligation (Stratagene Cloning Systems, La Jolla,
CA) .
The total rat brain library and the rat cerebellum
library were plated out with g. 99;; XL-1 cells onto NZY
agar plates (Table 3) to obtain approximately 2.1 x 106
plaques. Clone 45-A, encoding subtype 1a, was digested
with Pst I to isolate the 1.3 and 1.6 kb fragments. The
45-A Pst I fragments were labeled by random priming using
the Amersham random—priming kit (Amersham, Arlington Hts,
IL). Duplicate lifts were prepared from the plates, and
the filters were hybridized with the probes in 50%
formamide at 37°C. After an overnight hybridization, the
filters were washed in 2x SSC + 0.1% SDS at 50°C.
Positive plaques were isolated by several rounds of
dilution plating and repeated screening with the
random—primed probes.
Table 3
NZY Agar
To 950 ml of deionized water, add:
g NZ amine: Casein hydrolysate enzymatic (ICN
Biochemicals)
g Nacl
g bacto-yeast extract
1 g casamino acids
g MgSO,. ' 7H2O
sterilize by autoclaving for 20 minutes.
X SSC
Dissolve 175.3 g Nacl and 88.2 g sodium citrate in
800 mllgo. Adjust the pH to 7.0 with a few drops of 10
N NaOH. Adjust the volume to 1 liter with Hgx
sterilize by autoclaving.
Plasmid DNA was prepared from positive plaques
using the Bluescript system (Stratagene Cloning Systems).
The plasmid DNA was subjected to restriction analysis and
Southern blot analysis (Sambrook et al., ibid., which is
incorporated herein by reference). Two clones, SN23,
derived from the total rat brain library, and SR2,
derived from the rat cerebellum library, were identified
as being different than the 45-A clone and were
sequenced. Sequence analysis showed that they
represented two additional subtypes. SN23 encodes
subtype lb, which contains an additional 85 bp exon that
encodes a new stretch of 20 amino acids and a stop codon
in the intracellular domain, is 292 amino acids shorter
than the 45-A clone. The nucleotide sequence and deduced
amino acid sequence of clone SN23 are shown in Fig. 7.
SR2 was found to contain a partial CDNA sequence encoding
subtype 2a, which is a novel sequence that shares a 42%
homology to the transmembrane domains and extracellular
domain of the 45-A clone.
A complete subtype 2a clone was obtained by
rescreening both libraries as described above with the
radiolabeled 1.3 kb Pst I fragment from clone 45-A and a
radiolabeled 1.4 kb Eco RI—Pvu II fragment from SR2. Two
additional clones were obtained. SN30, derived from the
total rat brain library, contained the entire subtype 2a
coding sequence. The nucleotide sequence and deduced
amino acid sequence of clone SN30 are shown in Fig. 8.
SR13, derived from the rat cerebellum library, contained
an incomplete sequence of a new receptor subtype, 2b.
Sequence analysis of SR13 showed that the coding sequence
was incomplete at the 3' end and was virtually identical
to the SN30 sequence except that it contained a 610 base
pair deletion within the 3' terminus of SN30. The DNA
sequence of the CDNA insert in clone SR13 is shown in
Figure 9.
The complete 3' end of the subtype 2a clone was
generated using PCR amplification and an oligonucleotide
containing a sequence unique to SR13 (ZC4520, Table 4)
and an oligonucleotide corresponding to a sequence near
the 3' end of the 3' non-translated region of SN30
(ZC4519, Table 4). DNA was prepared from plate lysates
of the original plating of each library. Each plate
produced a pool of clones. For the PCR reactions, ten
nanograms from each library and 100 pmol of each
oligonucleotide were combined in a reaction volume of 50
pl containing 50 mM KCl, 10 mM Tris-HCl pH 9.0, 1.5 mM
MgCl2, 0.1% Triton X-100, 0.01% gelatin, 0.2 mM each
deoxynucleotide triphosphate and 2.5 units of Thermus
aggaticus (Taq) DNA polymerase (Promega Corporation,
Madison, WI). The reaction mixture was overlaid with
mineral oil. After five cycles (30 seconds at 94°C, 30
seconds at 45°C, 1 minute at 50°C) and twenty-five cycles
(30 seconds at 94°C, 30 seconds at 45°C, 1 minute at
°C) the amplified DNA was removed for analysis.
Table 4
Degenerate Oliqonucleotide Primer Sequences (5' - 3')
ZC4519
TTT ATT AGA AAT GTT CTC GGT
ZC452O
CCT CTT CCA TAT TTT TCC ATT
ZC4559
ATA AGA ATT CAT NKR YTT NGC YTC RTT RAA
ZC4560
ATA AGA ATT CTT YRA YGA RAA NGG NGA YGC
ZC4561
ATA AGA ATT CGC NGG NAT HTT YYT NKG NTA
ZC4562
ATA AGA ATT CTA NCM NAR RAA DAT NCC NGC
ZC4563
ATA AGA AAT CAN GTN GTR TAC ATN GTR AA
An aliquot from each reaction was electrophoresed
on agarose and transferred to nitrocellulose for Southern
analysis. Southern analysis of the PCR products showed
that a 460 bp fragment corresponding to the 3' end of the
2b sequence was present in several pools. one of the
pools that produced the correct size PCR product encoding
the 3' sequence of the 2b subtype was diluted and
screened with radiolabeled ZC4519 and ZC4520 (Table 4).
Phage that hybridize to both radiolabeled ZC45l9 and
ZC452O are picked, eluted, diluted, plated and rescreened
with the oligonucleotide probes. The screening is
repeated until a pure clone is obtained. The pure clone
is sequenced, and a full-length clone is constructed
using the most convenient restriction enzyme(s).
Based on an alignment of the deduced amino acid
sequences of subtypes la and 2a, strategies were designed
for cloning additional subtypes using PCR amplification.
Degenerate oligonucleotide families were prepared to
encode conserved amino acid sequences in the sixth
transmembrane domain, a region surrounding the conserved
amino acid sequence Phe-Asp-Glu-Lys, the third
cytoplasmic loop, and the second transmembrane domain
(Table 4).
Glutamate receptor CDNA sequences were amplified
with pairs of degenerate primers from Table 4 using the
PCR method on cDNA from the total rat brain library, the
cDNA from the rat cerebellum library, a rat cortex CDNA
library or a rat hippocampus CDNA library (both obtained
from Michael Brownstein, National Institutes of Health,
Bethesda, MD). The primers also each contained a 5' tail
of 10 nucleotides, which provided convenient restriction
enzyme sites. For each PCR reaction, ten nanograms from
the library and 100 pmol of the oligonucleotide pools
ZC4563 and ZC4560 (Table 4) were combined in a reaction
volume of 50 pl containing 50 mM KCl, 10 mM Tris-HCl pH
9.0, 1.5 mM MgCl2, 0.1% Triton X-100, 0.01% gelatin, 0.2
mM each deoxynucleotide triphosphate and 2.5 units of Taq
DNA polymerase. The reaction mixture was overlaid with
mineral oil. After five cycles (30 seconds at 94°C, 30
seconds at 45°C, 1 minute at 50°C) and twenty—five cycles
(30 seconds at 94°C, 30 seconds at 45°C, 1 minute at
72°C) the amplified DNA was removed for analysis.
An aliquot from each reaction was electrophoresed
on an agarose gel. Southern analysis of the gel was
performed using essentially the method described by
Sambrook et al. (ibid.) and random—primed fragments
covering the entire coding regions from both the subtype
la and 2a clones. The autoradiographs showed that the
PCR reaction generated fragments of novel size that were
different from either the 1a or 2a subtype. The
PCR-generated fragments were electrophoresed on an
agarose gel. Regions corresponding to the unique-sized
receptor-related products were excised and
electrophoresed onto NA45 paper (Schleicher and Schuell,
Keene, NH). The purified fragments were recovered using
essentially the method described by the manufacturer,
digested with Eco RI and ligated to plasmid pVEGT' that
had been linearized by digestion with Eco RI and treated
with phosphatase to prevent recircularization. The
ligation mixtures were transformed into E. ggli strain
DH10b cells. Transformants were picked and replica
plated onto nitrocellulose filters and screened using
random-primed probes from the la and the 2a clones.
Forty-eight colonies were picked for restriction analysis
and sequencing.
DNA sequences from the cDNA from the total rat
brain library and the cDNA from the rat cerebellum
library were each amplified and analyzed using the
methods described above and oligonucleotide ZC4559 in
combination with either ZC456l or ZC4559 (Table 4).
A rat cortex CDNA library and a rat hippocampus
CDNA library (both obtained from Michael Brownstein, NIH)
are subdivided into 30 pools of 10,000 colonies. Plasmid
DNA is prepared from each pool, and the DNA is subjected
to Southern analysis after restriction digestion of the
pools with Bam HI and Xho I or by PCR amplification of
each pool using the degenerate oligonucleotides of Table
4. The library pools containing DNA that hybridize to
the probes and appear to contain a full-length cDNA are
subdivided. The plasmid DNA is prepared and screened as
described above. Positive pools are again divided and
the procedure is continued until the pool is reduced to
pure clones. The clones are subjected to restriction
analysis and partial sequence analysis. Clones that
represent distinct glutamate receptor homologs are
completely sequenced. Full length clones are generated
by subjecting the original pools to PCR amplification
using an oligonucleotide primer specific to the SP6
promoter at the 5' end of the cDNA insert and an
antisense oligonucleotide primer corresponding to the 5'
end of the most complete cDNA to identify pools that
contain the longest glutamate receptor homolog CDNA. The
pool is then diluted and rehybridized with the probes as
described above to isolate a full length cDNA clone.
Expression of Glutamate Receptor Subtvpeg
Complementary DNA sequences encoding subtypes lb
and 2a were subcloned first into the mammalian expression
vector Zem228R to obtain convenient terminal restriction
sites. The cDNAs were then subcloned into pVEGT'. The
CDNA sequence encoding subtype lb was constructed by
replacing the 3' terminal portion of subtype la described
in Example I with the analogous portion of subtype lb
from SN23. Plasmid SN23 was digested with Kpn I and Xba
I to isolate the fragment containing the 3' terminus of
the lb subtype. The plasmid containing the subtype la
coding sequence (45-A) in Zem228R was digested with Kpn I
and Xba I to isolate the vector containing fragment. The
vector containing fragment is ligated to the Kpn I-Xba I
fragment from SN23. The resulting plasmid comprises the
MT-1 promoter, the subtype lb cDNA and the hGH
terminator. This plasmid was transfected into the BHK
570 cell line essentially as described in Example I to
obtain stably transfected cell lines expressing the
subtype lb receptor. The subtype lb CDNA fragment was
isolated as a Bam HI fragment, which was ligated with
pVEGT' that had been linearized with Bam HI. A plasmid
containing the cDNA sequence in the correct orientation
was used to synthesize RNA in an in vitro system. The
RNA was injected into oocytes as described above.
Plasmid SN30, which comprises the subtype 2a cDNA,
was digested with Eco RI to isolate the subtype 2a cDNA.
The Eco RI fragment was ligated with Eco RI-linearized
Zem228R. A plasmid containing the insert in the correct
orientation was digested with Bam HI to isolate the cDNA
sequence. The Bam HI fragment comprising the subtype 2a
CDNA was ligated with Eco RI-linearized pVEGT'. A
plasmid containing the cDNA in the correct orientation
was used to synthesize RNA in an in vitro translation.
The RNA was injected into frog oocytes as described
above.
EXAMPLE III
Generation of antibodies to glutamate receptor subtypes
Receptor subtype-specific polyclonal antisera
were generated in rabbits using standard immunization
techniques. Synthetic peptides (Table 5) were designed
from the cloned receptor sequences. The peptides were
conjugated to keyhole limpet hemocyanin, and each antigen
was used to immunize two animals. For each peptide, the
animals were injected with 100-200 pg of conjugated
peptide divided among three subcutaneous sites. The
animals were immunized at three—week intervals and bled
via an ear vein 10 days after the third and subsequent
immunizations.
Table 5
Subtype Peptide Segpence Apparent Location
la RDSLISIRDEKDGLNRC extracellular
DRLLRKLRERLPKARV extracellular
EEVWFDEKGDAPGRYD extracellular
EFVYEREGNTEEDEL cytoplasmic
PERKCCEIREQYGIQRV extracellular
IGPGSSSVAIQVQNLL extracellular
IAYSATSIDLSDKTL extracellular
lb KKPGAGNAKKRQPEFS cytoplasmic
PEFSPSSQCPSAHAQL cytoplasmic
a DKIIKRLLETSNARG extracellular
VNFSGIAGNPVTFNEN extracellular
GEAKSELCENLETPAL cytoplasmic
b PARLALPANDTEFSAWV cytoplasmic
Anti-peptide antibodies were purified by affinity
TM Kit (Multiple Peptide
Systems (San Diego, CA). Purified antibodies were stored
in column elution buffer and neutralizing buffer
purification using the Proton
(supplied by Multiple Peptide Systems). Bovine serum
albumin was added to a concentration of 1 mg/ml, and
sodium azide was added to a concentration of 0.05%. The
antibodies were stored at 4°C or in small aliquots at
-20°C.
Antibodies generated from the peptides listed in
Table 6 were used to detect G protein-coupled glutamate
receptors by Western blot analysis of membranes prepared
from transfected cell lines that were stably expressing
the subtype la or subtype lb receptors. Control cell
lines were transfected with vector alone.
Table 6
Analysis of Antibodies Raised to Peptides
Antibodies to Location Western
Peptide Seggence
RDSLISIRDEKDGLNRC extracellular +++ with bkgd
DRLLRKLRERLPKARV extracellular +
EEVWFDEKGDAPGRYD extracellular ++++ low bkgd
BFVYEREGNTEEDEL cytoplasmic ++++ low bkgd
KKPGAGNAKKRQPEFS cytoplasmic + for la
- for lb
PEFSPSSQCPSAHAQL cytoplasmic +++ for lb low bkgd
Transfectants that were stably expressing either
the la or lb subtype were each grown to confluency in
five to ten 150 mm plates. Each plate was first washed
twice with 15 ml of cold PBS and then 20 ml of ice cold
mM NaHC03 was added to each plate. The cells from
each plate were scraped off the plates with a rubber
spatula and transferred to a glass dounce homogenizer on
ice. The cells were disrupted with ten strokes of the B
pestle. The homogenates from each plate were combined
and centrifuged for thirty minutes at 3000 rpm at 4‘C.
The pellets were resuspended in 4-8 ml of 10 mM NaHCO3
using a 22 g needle and syringe, and 69% sucrose was
added (6-12 ml) to each sample until an index of
refraction of 1.410 was reached. The samples were
transferred to a high speed centrifugation tube, and each
sample was overlayed with 42% sucrose. The samples were
centrifuged for two hours at 25,000 rpm at 4°C. The
samples were collected by gently floating the membranes
off the 42% sucrose layer by adding 1 ml of 10 mM NaHC03
and resuspending the membranes by carefully stirring the
upper layer. The upper layer was transferred to a fresh
tube on ice. The purified membranes were centrifuged at
,000 rpm at 4°C and the pellets resuspended in 10 mM
NaHCO3. The purified membranes were then adjusted to a
final protein concentration of 1-2 pg/ml.
Ten to twenty micrograms of each purified membrane
preparations were diluted with 2x SDS-mercaptoethanol
buffer (100 mM Tris HCl (pH 6.8), 200 mM dithiothreitol,
4% SDS, 0.2% bromophenol blue, 20% glycerol). The
samples were incubated for 15 minutes at 37°C followed by
boiling for 5 minutes. The samples were subjected to
SDS—PAGE on 4-15% gradient gel. The samples were
electrotransferred to nitrocellulose using the method
essentially described by Towbin (Proc. Natl. Acad. Sci.
us; 76: 4350-4354, 1979; which is incorporated herein by
reference in its entirety). After transfer, the
nitrocellulose was cut into strips such that each strip
contained a control and receptor samples. The
nitrocellulose was preincubated in blocking buffer and
then incubated with a dilution of either the preimmune
serum or the serum collected after antigenic stimulation
(serum from later bleeds (i.e. those after four antigen
stimulations) were diluted 1:1500). After washing, a
horse radish peroxidase-conjugated goat anti-rabbit
antibody (Bio-Rad Laboratories, Richmond, CA) diluted
1:2,500 was added and after incubation and washing, the
horse radish peroxidase substrate (Bio-Rad Laboratories)
was added and the color reaction was initiated. ‘The
reaction was stopped by rinsing the filters in distilled
water. Table 6 shows the results of the Western blot
analysis.
Claims (1)
1. An isolated mammalian G protein-coupled glutamate receptor or a glutamate- binding fragment thereof, wherein the receptor has an amino acid sequence encoded by a DNA molecule that hybridizes to an oligonucleotide of 50 or more continguous nucleotides that is complementary to the protein encoding sequence of
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US6190864B1 (en) * | 1991-05-08 | 2001-02-20 | Chiron Corporation | HCV genomic sequences for diagnostics and therapeutics |
IL105587A0 (en) | 1992-05-08 | 1993-09-22 | Lilly Co Eli | Human metabotropic glutamate receptor and related dna compounds |
CA2101586A1 (en) | 1992-08-05 | 1994-02-06 | Rajender Kamboj | Ampa-binding human glur4 receptors |
CA2105804C (en) | 1992-09-17 | 2003-04-29 | Rajender Kamboj | Kainate-binding, human cns receptors of the eaa5 family |
US5521297A (en) * | 1993-06-04 | 1996-05-28 | Salk Institute Biotechnology/Industrial Associates | Nucleic acids encoding human metabotropic glutamate receptors |
US5728545A (en) * | 1993-06-18 | 1998-03-17 | The Salk Institute Of Biological Studies | Cloning and recombinant production of CRF receptor (S) |
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1991
- 1991-12-10 NZ NZ240921A patent/NZ240921A/en not_active IP Right Cessation
- 1991-12-11 PT PT99770A patent/PT99770B/en not_active IP Right Cessation
- 1991-12-11 IE IE431491A patent/IE914314A1/en not_active IP Right Cessation
- 1991-12-11 IL IL10034391A patent/IL100343A/en not_active IP Right Cessation
- 1991-12-12 JP JP4502875A patent/JPH06503961A/en active Pending
- 1991-12-12 AU AU91356/91A patent/AU9135691A/en not_active Abandoned
- 1991-12-12 DE DE69132671T patent/DE69132671T2/en not_active Revoked
- 1991-12-12 CA CA002098295A patent/CA2098295C/en not_active Expired - Fee Related
- 1991-12-12 WO PCT/US1991/009422 patent/WO1992010583A1/en not_active Application Discontinuation
- 1991-12-12 EP EP92902633A patent/EP0577605B1/en not_active Revoked
-
1992
- 1992-06-30 AT AT92902633T patent/ATE203566T1/en not_active IP Right Cessation
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1995
- 1995-05-31 US US08/455,602 patent/US5747267A/en not_active Expired - Fee Related
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