U.S. patent application number 11/637196 was filed with the patent office on 2009-03-26 for heterologous g protein coupled receptors expressed in yeast, their fusion with g proteins and use thereof in bioassay.
This patent application is currently assigned to Wyeth. Invention is credited to Deborah Tardy Chaleff, John Richard Hadcock, Eileen Marie Kajkowski, Donald Richard Kirsch, Bradley Alton Ozenberger, Mark Henry Pausch, Laura Alicia Price.
Application Number | 20090081764 11/637196 |
Document ID | / |
Family ID | 22722547 |
Filed Date | 2009-03-26 |
United States Patent
Application |
20090081764 |
Kind Code |
A1 |
Pausch; Mark Henry ; et
al. |
March 26, 2009 |
Heterologous G protein coupled receptors expressed in yeast, their
fusion with G proteins and use thereof in bioassay
Abstract
The present invention is directed to expression vectors and
yeast cells transformed therewith containing a first heterologous
nucleotide sequence which codes for a G protein-coupled receptor,
for example, the somatostatin receptor, and a second nucleotide
sequence which codes for all or a portion of a G protein
.alpha..beta..gamma. complex. Said heterologous protein is
physically expressed in a host cell membrane in proper orientation
for both stereoselective binding of ligands, as well as functional
interaction with G proteins on the cytoplasmic side of the cell
membrane. In some embodiments, a nucleotide sequence encoding a
heterologous or chimeric G.alpha. protein is expressed in
conjunction with nucleotide sequences from the yeast G protein
.beta..gamma. subunits. A second aspect of the present invention
provides expression vectors and yeast cells transformed therewith
encoding chimeric yeast/heterologous G protein coupled receptors. A
third aspect of the present invention is directed to methods of
assaying compounds using such expression constructs and yeast cell
expression systems to determine the effects of ligand binding to
the heterologous receptors expressed in the systems.
Inventors: |
Pausch; Mark Henry;
(Robbinsville, NJ) ; Ozenberger; Bradley Alton;
(Yardley, PA) ; Hadcock; John Richard; (Mount
Holly, NJ) ; Price; Laura Alicia; (Langhorne, PA)
; Kajkowski; Eileen Marie; (Ringoes, NJ) ; Kirsch;
Donald Richard; (Princeton, NJ) ; Chaleff; Deborah
Tardy; (Pennington, NJ) |
Correspondence
Address: |
WYETH/FINNEGAN HENDERSON, LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
Wyeth
American Cyanamid Company
|
Family ID: |
22722547 |
Appl. No.: |
11/637196 |
Filed: |
December 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10302524 |
Nov 25, 2002 |
7148053 |
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11637196 |
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09057473 |
Apr 9, 1998 |
6607906 |
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10302524 |
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08696924 |
Oct 15, 1996 |
6406871 |
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PCT/US95/02075 |
Feb 14, 1995 |
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09057473 |
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08195729 |
Feb 14, 1994 |
5691188 |
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08696924 |
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Current U.S.
Class: |
435/254.2 |
Current CPC
Class: |
C07K 14/723 20130101;
C07K 2319/00 20130101 |
Class at
Publication: |
435/254.2 |
International
Class: |
C12N 1/19 20060101
C12N001/19 |
Claims
1. A transformed yeast cell comprising a first nucleotide sequence
which codes for a heterologous G protein coupled receptor and a
second nucleotide sequence which codes for all or a portion of a G
protein complex.
2-29. (canceled)
Description
FIELD OF INVENTION
[0001] This invention relates to heterologous G protein-coupled
receptor expression constructs, yeast cells expressing such
receptors, vectors useful for making such cells, and methods of
making and using same.
BACKGROUND OF THE INVENTION
[0002] The actions of many extracellular signals, for example:
neurotransmitters, hormones, odorants and light, are mediated by
receptors with seven transmembrane domains (G protein-coupled
receptors) and heterotrimeric guanine nucleotide-binding regulatory
proteins (G proteins). G proteins are comprised of three subunits:
a guanyl-nucleotide binding .alpha. subunit; a .beta. subunit; and
a .gamma. subunit [for review, see Conklin, B. R and Bourne, H. R.
(1993 Cell 73, 631-641]. G proteins cycle between two forms,
depending on whether GDP or GTP is bound to the .alpha. subunit.
When GDP is bound, the G protein exists as a heterotrimer, the
G.alpha..beta..gamma. complex. When GTP is bound, the .alpha.
subunit disassociates, leaving a G.beta..gamma. complex.
Importantly, when a G.alpha..beta..gamma. complex operatively
associates with an activated G protein coupled receptor in a cell
membrane, the rate of exchange of GTP for bound GDP is increased
and, hence, the rate of disassociation of the bound G.alpha.
subunit from the G.beta..gamma. complex increases. The free
G.alpha. subunit and G.beta..gamma. complex are capable of
transmitting a signal to downstream elements of a variety of signal
transduction pathways. This fundamental scheme of events forms the
basis for a multiplicity of different cell signaling phenomena. For
a review, see H. G. Dohlman, J. Thorner, M. Caron, and R. J.
Lefkowitz, Ann. Rev. Biochem, 60, 653-688 (1991). G
protein-mediated signaling systems are present in organisms as
divergent as yeast and man. The yeast Saccharomyces cerevisiae is
utilized as a model eukaryotic organism. Due to the ease with which
one can manipulate the genetic constitution of the yeast
Saccharomyces cerevisiae, researchers have developed a detailed
understanding of many complex biological pathways. It has been
demonstrated in numerous systems that the evolutionary conservation
of protein structure is such that many heterologous proteins can
substitute for their yeast equivalents. For example, mammalian
G.alpha. proteins can form heterotrimeric complexes with yeast
G.beta..gamma. proteins [Kang, Y.-S., Kane, J., Kurjan, J., Stadel,
J. M., and Tipper, D. J. (1990) Mol. Cell. Biol. 10, 2582-2590].
The G protein-coupled receptors represent important targets for new
therapeutic drugs. Discovery of such drugs will necessarily require
screening assays of high specificity and throughput. For example,
therapeutic intervention in the somatostatin-growth hormone axis
requires new chemical agents that act in a somatostatin receptor
subtype-selective manner. The somatostatin receptor (SSTR) is a
prototype of the seven transmembrane-domain class of receptors in
mammalian cells. The cyclic tetradecapeptide somatostatin, first
isolated from hypothalamus and shown to be a potent inhibitor of
growth hormone release from the anterior pituitary, has been shown
to have broad modulatory effects in CNS and peripheral tissues. In
response to binding of somatostatin, SSTR activates a
heterotrimeric G protein, which in turn modifies the activity of a
variety of effector proteins including but not limited to adenylate
cyclases, ion channels, and phospholipases. The effects of
somatostatin are transduced through the action of gene products
encoded in five distinct receptor subtypes that have recently been
cloned [Strnad, J., Eppler, C. M., Corbett, M., and Hadcock, J. R.
(1993) BBRC 191, 968-976; Yamada, Y., Post, S. R., Wang, K., Tager,
H. S., Bell, G. I., and Seino, S. (1992) Proc. Natl. Acad. Sci. USA
89, 251-255; Meyerhof, W., Paust, H.-J., Schonrock, C., and
Richter, D. (1991); Kiuxen, F.-W., Bruns, C., and Lubbert, H.
(1992) Proc. Natl. Acad. Sci. USA 89, 4618-4622; Li, X.-J., Forte,
M., North, R. A., Rose, C. A., and Snyder, S. (1992) J. Biol. Chem.
267, 21307-21312; Bruno, J. F., Xu, Y., Song, J., and Berelowitz,
M. (1992) Proc. Natl. Acad. Sci. USA 89, 11151-11154; O'Carrol,
A.-M., Lolait, S. J., Konig, M., and Mahan, L. (1992) Mol.
Pharmocol. 42, 939-946). Screening assays utilizing yeast strains
genetically modified to accommodate functional expression of the G
protein-coupled receptors offer significant advantages in research
involving ligand binding to the somatostatin receptor, as well as a
host of other receptors implicated in various disease states.
SUMMARY OF THE INVENTION
[0003] A first aspect of the present invention is directed to
expression vectors and yeast cells transformed therewith,
containing a first heterologous nucleotide sequence which encodes
for a G protein-coupled receptor, for example, the somatostatin
receptor, and a second nucleotide sequence which encodes for all or
a portion of a G protein .alpha..beta..gamma. complex. In certain
embodiments, all or a portion of a nucleotide sequence encoding for
a heterologous G protein .alpha. subunit is fused to a nucleotide
sequence from the yeast G protein .alpha. subunit. In certain
preferred embodiments, the expression vectors and transformed cells
contain a third heterologous nucleotide sequence comprising a
pheromone-responsive promoter and an indicator gene positioned
downstream from the pheromone-responsive promoter and operatively
associated therewith. The vectors and cells may further contain
several mutations. These include 1) a mutation of the yeast
SCG1/GPA1 gene, which inactivates the yeast G.alpha. protein,
facilitating interaction of the heterologous receptor with the G
protein; 2) a mutation of a yeast gene to inactivate its function
and enable the yeast cell to continue growing in spite of
activation of the pheromone response signal transduction pathway,
preferred embodiments being mutations of the FAR1 and/or FUS3
genes; and, 3) a mutation of a yeast gene, the effect of the which
is to greatly increase the sensitivity of the response of the cell
to receptor-dependent activation of the pheromone response signal
transduction pathway, preferred genes in this regard being the
SST2, STE50, SGV1, STE2, STE3, PIK2, AFR1, MSG5, and SIG1
genes.
[0004] A second aspect of the present invention is a chimeric
expression construct and yeast cells transformed therewith
comprising a first nucleotide sequence encoding for a yeast G
protein coupled receptor in operative association with a
heterologous nucleotide sequence which encodes for a heterologous G
protein coupled receptor. The constructs and cells may contain a
second heterologous nucleotide sequence comprising a
pheromone-responsive promoter and an indicator gene positioned
downstream from the pheromone-responsive promoter and operatively
associated therewith. The constructs and cells may further contain
several mutations. These include 1) a mutation of a yeast gene to
inactivate its function and enable the yeast cell to continue
growing in spite of activation of the pheromone response signal
transduction pathway, preferred embodiments being mutations of the
FAR1 and/or FUS3 genes; and, 2) a mutation of a yeast gene, the
effect of the which is to greatly increase the sensitivity of the
response of the cell to receptor-dependent activation of the
pheromone response signal transduction pathway, preferred genes in
this regard being the SST2, STE50, SGV1, STE2, STE3, PIK1, AFR1,
MSG5, and SIG1 genes. A productive signal is detected in a bioassay
through coupling of the heterologous receptor to a yeast
protein.
[0005] A third aspect of the present invention is a method of
assaying compounds to determine effects of ligand binding to the
heterologous receptors by measuring effects on cell growth. In
certain preferred embodiments, yeast cells of the kind described
above are cultured in appropriate growth medium to cause expression
of heterologous proteins, embedded in agar growth medium, and
exposed to compounds applied to the surface of the agar plates.
Effects on the growth of embedded cells are expected around
compounds that activate the heterologous receptor. Increased growth
may be observed with compounds that act as agonists, while
decreased growth may be observed with those that act as
antagonists.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1. Strains containing the indicated G.alpha. expression
plasmids are treated with mating pheromone (.alpha. factor). A
measure of resulting signal transduction is provided by a reporter
plasmid carrying FUS1-lacZ. Data are represented as a percent of
.beta.-galactosidase activity measured in a strain expressing
solely the wild type G.alpha. protein.
[0007] FIG. 2. Strains containing the indicated CUP1p-G.alpha.
expression plasmid and grown in medium containing the indicated
concentration of copper are treated with mating pheromone (.alpha.
factor). A measure of resulting signal transduction is provided by
a reporter plasmid carrying FUS1-lacZ. Data are represented as a
percent of .beta.-galactosidase activity measured in a strain
expressing no exogenous G.alpha. protein.
[0008] FIG. 3. Saturation binding of .sup.3H-spiperone to yeast
membrane fractions prepared from a strain (CY382) expressing the
5HT1a serotonin receptor. Bmax=3.2 pmol/mg, protein; Kd-115 nM.
[0009] FIG. 4. Amino-terminal chimeric Ste2/5HT1a receptors. The
CHI11 receptor contains the first 14 amino acids of the yeast Ste2
protein. The CHI17 receptor has a replacement of the amino-terminus
of the 5HT1a receptor through the first two transmembrane domains
with the corresponding region of the Ste2 receptor. The CHI18
receptor has the same Ste2 sequences fused directly to the
amino-terminus of the 5HT1a receptor to create a receptor predicted
to span the cellular membrane nine times. Bmax values were
determined by measuring maximal binding of the radiolabeled ligand
3H-spiperone. Values are given as pmol radioligand bound per mg
total protein.
[0010] FIG. 5. Competition binding analysis of the agonists
isoproterenol or epinephrine against .sup.125I-cyanopindolol with
crude membrane extracts prepared from a wild-type yeast strain
expressing the .beta..sub.2-adrenergic receptor. Data are presented
as percent maximal radioligand binding. IC.sub.50 values=10 nM,
isoproterenol; 200 nM, epinephrine.
[0011] FIG. 6. Competition binding analysis of the agonists
isoproterenol or epinephrine against .sup.125I-cyanopindolol with
extracts prepared from a yeast strain coexpressing the
.beta..sub.2-adrenergic receptor and mammalian G.sub.as. Data are
presented as percent maximal radioligand binding. IC.sub.50
values=10 nM, isoproterenol; 60 nM, epinephrine.
[0012] FIG. 7. Saturation binding of [.sup.125I]tyr.sup.11S-14 to
membranes of yeast cells coexpressing the SSTR2 subtype and
Scgl/G.sub.i.alpha.2. Membranes from yeast cells expressing the
SST2 subtype were prepared as described in Experimental Procedures.
Saturation binding was performed with 20-1600 pM
[.sup.125I]tyr.sup.11S-14. Non-specific binding for each point as
cpm bound in the presence of 1 .mu.M cold S-14 ranged from 10 to
40%. Displayed is a representative experiment performed in
duplicate.
[0013] FIG. 8. Immunoblot showing somatostatin receptor expression.
Membrane fractions are isolated from the indicated yeast strains.
Aliquots of 5 to 30 .mu.g of protein are examined by polyacrylamide
gel electrophoresis/Western blot analysis. Molecular weight markers
are indicated in kilodaltons. Arrows mark somatostatin receptor
protein bands. Several species of this receptor are observed;
doublet and triplet bands in addition to the 43 kd single receptor.
Lanes, 1+2, CY602 (see Table 1); 3+4. CY603; 5+6, CY624; 7,
congenic strain expressing no receptor.
[0014] FIG. 9. Immunoblot showing muscarinic acetylcholine receptor
(mAchR) expression. Membrane fractions are isolated from the
indicated yeast strains. Aliquots of 30 .mu.g of protein are
examined by polyacrylamide gel electrophoresis/Western blot
analysis as described in the text. Molecular weight markers are
indicated in kilodaltons. Arrows mark mAchR protein bands.
[0015] FIG. 10. Immunoblot showing .alpha.2-AR expression. Membrane
fractions are isolated form the indicated yeast strains. Aliquots
of 30 .mu.g of protein are examined by polyacrylamide gel
electrophoresis/western blot analysis. Molecular weight markers are
indicated in kilodaltons. Arrows mark .alpha.2-AR protein
bands.
[0016] FIG. 11. Somatostatin receptor expression plasmid, pJH1.
[0017] FIG. 12. G protein expression plasmid, pLP82.
[0018] FIGS. 13 (A&B). Dose dependent growth response of yeast
cells to somatostatin. Cultures of yeast strain LY268 are embedded
in agar (top plate) or spread evenly on the surface of agar plates
(bottom plate) and exposed to the indicated amounts of designated
compounds spotted on paper disks placed on top of the agar. Plates
are incubated at. 30.degree. C.
[0019] FIGS. 14 (A,B,C,&D). Growth response of yeast strains
exposed to somatostatin, is dependent on amount of chimeric G
protein expressed. Cultures of yeast strains described in the text
are embedded in agar and exposed to the indicated amounts of
designated compounds spotted on paper disks placed on top of the
agar. Plates are incubated at 30.degree. C.
[0020] FIGS. 15 (A,B,C,&D) Growth response of yeast strains
exposed to somatostatin is dependent on amount of yeast G protein
expressed. Cultures of yeast strains described in the text are
embedded in agar and exposed to the indicated amounts of designated
compounds spotted on paper disks placed on top of the agar. Plates
are incubated at 30.degree. C.
[0021] FIGS. 16 (A&B). Yeast cells bearing a mutation in the
sst2 gene exhibit elevated resistance to AT when exposed to mating
pheromone. Cultures of yeast strains are embedded in agar and
exposed to the indicated amounts of a mating factor spotted on
paper disks placed on top of the agar. Plates are incubated at
30.degree. C.
[0022] FIGS. 17 (A,B,C,&D). A mutation in the sst2 gene
enhances the growth of the yeast cells exposed to somatostatin.
Cultures of yeast strains described in the text are embedded in
agar and exposed to the indicated amounts of designated compounds
spotted on paper disks placed on top of the agar. Plates are
incubated at 30.degree. C.
[0023] FIG. 18. Ligand-binding to the rat CCK.sub.B receptor
expressed in yeast. Crude membrane-fractions from overnight liquid
cultures of LY631 cells were prepared, and agonist saturation
binding assays conducted as described in Methods and Materials.
Saturation binding was performed with 4-60 nM [3H] CCK-8 (25 .mu.g
protein/tube). Non-specific binding for each point as cpm bound in
the presence of 1 .mu.M cold CCK-8 ranged from 20 to 60%. Displayed
is a representative experiment performed in duplicate.
[0024] FIGS. 19 (A&B). Growth of yeast in response to CCK.sub.B
receptor agonists. Yeast strains that functionally express the rat
CCK.sub.B receptor (LY628, LLY631) were cultured as described in
Materials and Methods were plated in SC Galactose (2%)-ura, trp,
his agar medium (2.times.10.sup.4 cells/ml). Sterile filter disks
were placed on the surface of the solidified agar and saturated
with 10 .mu.l of DMSO containing 10 .mu.l amounts of the indicated
compounds. The plates were then incubated at 30.degree. C. for 3
days. (A) CCK-8, (B) CCK-4.
[0025] FIG. 20. A.sub.2a-adenosine receptor saturation binding
assay. Radioligand binding assays were performed in 96-well
microliter plates using binding buffers (50 nM HEPES, pH 7.4, 10 mM
MgCl.sub.2, 0.25% BSA) containing protease inhibitors (5 .mu.g/ml
leupeptin, 5 .mu.g/ml aprotinin, 100 .mu.g/ml bacitracin, and 100
.mu.g/ml benzamidine). All components were diluted in binding
buffer containing protease inhibitors and added to the microliter
plate wells in the following order: binding buffer, cold competitor
(NECA, 1 .mu.M final concentration), [.sup.3H]NECA (1-50 nM).
Binding reactions were initiated by adding 82 .mu.g of membrane
protein in a 170-11 volume. Final reaction volume was 200
.mu.l/well. All incubations were carried out at room temperature
for 2 hours. Free radioligand was separated from bound ligand by
rapid filtration through a glass fiber filter using an Inotech cell
harvester. The filter disks were then washed several times with
cold (4.degree. C.) binding buffer lacking BSA prior to
counting.
[0026] FIG. 21. Growth of yeast in response to A adenosine receptor
agonists. LY595 cells cultured as described in Materials and
Methods were plated in SC Galactose (2%)-ura, trp, his agar medium
(2.times.10.sup.4 cells/ml). Sterile filter disks were placed on
the surface of the solidified agar and saturated with 10 .mu.l of
DMSO containing the 10 .mu.g of the indicated compounds. The plates
were then incubated at 30.degree. C. for 3 days. (A, B) CGS-21680,
(B) NECA (C) DPMA.
[0027] FIG. 22. Growth of yeast cells containing SSTR5 in response
to somatostatin receptor agonists. LY620 cells cultured as
described in Materials and Methods were plated in SC Galactose
(2%)-ura, trp, his agar medium (2.times.10.sup.4 cells/ml). Sterile
filter disks were placed on the surface of the solidified agar and
saturated with 10 .mu.l of sterile, water containing the indicated
amounts of the indicated compounds. The plates were then incubated
at 30.degree. C. for 3 days. (A) 60 nmol S-14, (B) 30 nmol
S-28.
[0028] FIG. 23. Growth of yeast cells containing porcine SSTR2 in
response to somatostatin receptor agonists. LY474 (two independent
isolates: 21,22) were cultured as described in Materials and
Methods were plated in SC Galactose (2%)-ura, trp, his agar medium
(2.times.10.sup.4 cells/ml). Sterile filter disks were placed on
the surface of the solidified agar and saturated with 10 .mu.l of
sterile water containing the indicated amounts of the indicated
compounds. The plates were then incubated at 30.degree. C. for 3
days. (1) 600 pmol, (2) 60 pmol.
[0029] FIG. 24. Deletion of MSG5 increases the sensitivity of the
yeast bioassay. Cultures of yeast strains were induced to express
the SSTR2 as described in Materials and Methods and were plated in
SC Galactose (2%)-ura; trp, his agar medium (2.times.10.sup.4
cells/ml). Sterile filter disks were placed on the surface of the
solidified agar and saturated with 10 .mu.l of sterile water
containing the indicated amounts of S-14. The plates were then
incubated at 30.degree. C. for 3 days. (A). MPY459 sst2.DELTA.ADE2
msg5.DELTA.LEU2, (B) MPY458 SST2 msg5.DELTA.LEU2, (C) LY288 SST2
MSG5, (D) LY268 sst2.DELTA.ADE2 MSG5.
[0030] FIGS. 25 (A&B). Growth of yeast in response to GRF
receptor agonists. CY990 cells cultured as described in Materials
and Methods were plated in SC Galactose (2%)-ura, trp, his agar
medium (2.times.10.sup.4 cells/ml). Sterile filter disks were
placed on the surface of the solidified agar and saturated with 10
.mu.l of sterile water containing 20 nmol of the indicated
compounds. The plates were then incubated at 30.degree. C. for 3
days. (A) hGRF(1-29)-NH.sub.2, (B) hGRF (1-29),
(D-arg.sup.2)-hGRF(1-29).
[0031] FIGS. 26 (A&B). Effect of STE50 overexpression on SSTR2
bioassay. Assay medium and yeast strains were prepared as described
in Materials and Methods. Plate A contains the STE50 overexpression
strain CY560; plate B contains the control strain CY562. Filter
discs saturated with solutions of the following peptides were
applied to each plate: 1 mM yeast a pheromone (lefthand center), 1
.mu.g/ml somatostatin-14 (righthand top), 100 .mu.g/ml
somatostatin-14 (righthand bottom). Plates were incubated at
30.degree. C. for 3 days.
[0032] FIGS. 27 (A&B) Bioassay of compounds with somatostatin
receptor and/or antagonist properties. LY36.4 cells were plated in
SC Galactose (2%)-ura, trp, his agar medium (2.times.10.sup.4
cells/ml). For assay of antagonists, somatostatin (20 nM S-14) was
added to the molten agar prior to pouring. Sterile filter disks
were placed on the surface of the solidified agar and saturated
with 10 .mu.l of sterile water containing test compounds. The
plates were then incubated at 30.degree. C. for 3 days (Left) Assay
for somatostatin agonists. Somatostatin (S-14) was applied to
positions on the bottom row, left side (6 nmol, 600 pmol, 60 pmol,
600 pmol), (Right) Assay for somatostatin antagonists. Somatostatin
(S-14) was applied to positions on the bottom row, left side (6
nmol, 600 pmol, 60 pmol, 600 pmol).
[0033] FIGS. 28 (A&B) Fusion of STE2 sequences to the amino
terminal of SSTR2 reduces-signaling efficiency in response to
somatostatin. LY268 and LY322 cells were plated in SC Galactose
(2%)-ura, trp, his agar medium (2.times.10.sup.4 cell/ml). Sterile
filter disks were placed on the surface of the solidified agar and
saturated with 10 .mu.l of somatostatin (S-14). The plates were
then incubated at 30.degree. C. for 3 days. (A) LY268. S-14 was
applied to filter disks clockwise from the top: carrier, 60=mol, 6
nmol, 600 pmol, 60 pmol, 6 pmol. (B) LY322. S-14 was applied to
filter disks clockwise from the top: 0.6 pmol, carrier, 60 mmol, 6
nmol, 600 pmol, 60 pmol, 6 pmol.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Nucleotide bases are abbreviated herein as follows: [0035]
A-Adenine G-Guanine [0036] C-Cytosine T-Thymine [0037] U-Uracil
(sometimes herein abbreviated as "ura")
[0038] Amino acid residues are abbreviated herein to either three
letters or a single letter as follows: [0039] Ala; A-Alanine Leu;
L-Leucine [0040] Arg; R-Arginine Lys; K-Lysine [0041] Asn;
N-Asparagine Met; M-Methionine [0042] Asp; D-Aspartic acid Phe;
F-Phenylalanine [0043] Cys; C-Cysteine Pro; P-Proline [0044] Gln;
Q-Glutamine Ser; S-Serine [0045] Glu; E-Glutamic acid Thr;
T-Threonine [0046] Gly; G-Glycine Trp; W-Tryptophan [0047] His;
H-Histidine Tyr; Y-Tyrosine [0048] Ile; I-Isoleucine Val;
V-Valine
[0049] The terms "DNA" and "nucleotide sequence" are used
interchangably and are meant to include all forms of linear
polymers comprising nucleotide bases, without limitation, including
RNA when appropriate.
[0050] The term "mammalian" as used herein refers to any mammalian
species (e.g. human, mouse, rat, and monkey).
[0051] The term "heterologous" is used herein with respect to
yeast, and hence refers to DNA sequences, proteins, and other
materials originating from organisms other than yeast (e.g.,
mammalian, avian, amphibian, insect, plant), or combinations
thereof not naturally found in yeast.
[0052] The term "upstream" and "downstream" are used herein to
refer to the direction of transcription and translation, with a
sequence being transcribed or translated prior to another sequence
being referred to as "upstream" of the latter.
[0053] Any G protein-coupled receptor, or portions thereof, as well
as the nucleotide sequences encoding same, may be employed in
practicing the present invention.
[0054] Examples of such receptors include, but are not limited to,
adenosine receptors, somatostatin receptors, dopamine receptors,
cholecystokinin receptors, muscarinic cholinergic receptors,
.alpha.-adrenergic receptors, .beta.-adrenergic receptors, opiate
receptors, cannabinoid receptors, growth hormone releasing factor,
glucagon, and serotonin receptors. The term receptor as used herein
is intended to encompass subtypes of the named receptors, and
mutants and homologs hereof, along with the nucleotide sequences
encoding same. One skilled in the art will also understand that in
some instances, it may not be necessary that the entire receptor be
expressed to achieve the purposes desired. Accordingly, the term
receptor is meant to include truncated and other variant forms of a
given receptor, without limitation.
[0055] Any DNA sequence which codes for a G.alpha. subunit
(G.alpha.) may be used to practice the present invention. Examples
of G.alpha. subunits include, but are not limited to Gs subunits,
Gi subunits, Go subunits, Gz subunits, Gq, G11, G16 and transducing
subunits. G proteins and subunits useful for practicing the present
invention include subtypes, and mutants and homologs thereof, along
with the DNA sequences encoding same.
[0056] One skilled in the art will understand from the teachings as
presented herein that the G proteins useful in the constructs and
yeast cells of the present invention may comprise heterologous
G.alpha. subunits, yeast G.alpha. subunits, or chimeric
yeast/heterologous versions. One can easily determine which
configuration is best suited for adequate coupling to a particular
heterologous receptor by simply constructing vectors as taught
herein and measuring the signaling of ligand binding in response to
a given assay. In certain preferred embodiments, G.alpha..sub.i2 is
the G.alpha. subunit of choice, particularly when the heterologous
G coupled protein is all or a portion of a somatostatin receptor.
It is particularly preferred in this instance that the
G.alpha..sub.i2 subunit be coupled to a yeast G.beta..gamma.
complex. Certain chimeric constructs may also provide enhanced
signal transduction with regard to particular heterologous
receptors. Particularly preferred is a chimeric construct formed
from fusion of the amino terminal domain of yeast GPA1/SCG1 with
the carboxy terminal domain of a heterologous G.alpha..sub.i,
G.alpha..sub.s, and especially G.alpha..sub.i2.
[0057] Any DNA sequence which codes for a G.beta..gamma. subunit
(G.beta..gamma.) may be used to practice the present invention. G
proteins and subunits useful for practicing the present invention
include subtypes, and mutants and homologs thereof, along with the
DNA sequences encoding same. The host cells may express endogenous
G.beta..gamma., or may optionally be engineered to express
heterologous G.beta..gamma. (e.g., mammalian) in the same manner as
they would be engineered to express heterologous G.alpha..
[0058] Heterologous DNA sequences are expressed in a host by means
of an expression "construct" or "vector". An expression vector is a
replicable DNA construct in which a DNA sequence encoding the
heterologous DNA sequence is operably linked to suitable control
sequences capable of affecting the expression of a protein or
protein subunit coded for by the heterologous DNA sequence in the
intended host. Generally, eukaryotic control sequences include a
transcriptional promoter, however, it may be appropriate that a
sequence encoding suitable mRNA ribosomal binding sites be
provided, and (optionally) sequences which control the termination
of transcription. Vectors useful for practicing the present
invention include plasmids, viruses (including bacteriophage) and
integratable DNA fragments (i.e., fragments integratable into the
host genome by genetic recombination). The vector may replicate and
function independently of the host genome, as in the case of a
plasmid, or may integrate into the genome itself, as in the case of
an integratable DNA fragment. Suitable vectors will contain
replicon and control sequences which are derived from species
compatible with the intended expression host. For example, a
promoter operable in a host cell is one which binds the RNA
polymerase of that cell, and a ribosomal binding site operable in a
host cell is one which binds the endogenous ribosomes of that
cell.
[0059] DNA regions are operably associated when they are
functionally related to each other. For example: a promoter is
operably linked to a coding sequence if it controls the
transcription of the sequence; a ribosome binding site is operably
linked to a coding sequence if it is positioned so as to permit
translation. Generally, operably linked means contiguous and, in
the case of leader sequences, contiguous and in reading phase.
[0060] Transformed host cells of the present invention are cells
which have been transformed or transfected with the vectors
constructed using recombinant DNA techniques and express the
protein or protein subunit coded for by the heterologous DNA
sequences. A variety of yeast cultures, and suitable expression
vectors for transforming yeast cells, are known. See e.g., U.S.
Pat. No. 4,745,057; U.S. Pat. No. 4,797,359; U.S. Pat. No.
4,615,974; U.S. Pat. No. 4,880,734; U.S. Pat. No. 4,711,844; and
U.S. Pat. No. 4,865,989. Saccharomyces cerevisiae is the most
commonly used among the yeasts, although a number of other yeast
species are commonly available. See. e.g., U.S. Pat. No. 4,806,472
(Kluveromyces lactis and expression vectors therefore); 4,855,231
(Pichia pastoris and expression vectors therefore). Yeast vectors
may contain an origin of replication from the endogenous 2 micron
yeast plasmid or an autonomously replicating sequence (ARS) which
confers on the plasmid the ability to replicate at high copy number
in the yeast cell, centromeric (CEN) sequences which limit the
ability of the plasmid to replicate at only low copy number in the
yeast cell, a promoter, DNA encoding the heterologous DNA
sequences, sequences for polyadenylation and transcription
termination, and a selectable marker gene. Exemplary plasmids and
detailed of materials and methods for making and using same are
provided in the Examples section.
[0061] Any promoter capable of functioning in yeast systems may be
selected for use in the constructs and cells of the present
invention. Suitable promoting sequences in yeast vectors include
the promoters for metallothionein, 3-phosphoglycerate kinase (PGK)
[Hitzeman et al., (1980) J. Biol. Chem. 255, 2073] or other
glycolytic enzymes [(Hess et al., (1968) J. Adv. Enzyme Reg. 7,
149]; and Holland et al., (1978) Biochemistry 17, 4900], such as
enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. Suitable vectors and promoters for use in yeast
expression are further described in R. Hitzeman et al., EPO Publn.
No. 73,657. Other promoters, which have the additional advantage of
transcription controlled by growth conditions, are the promoter
regions for alcohol dehydrogenase, 1,2,-isocytochrome C, acid
phosphates, degradative enzymes associated with nitrogen
metabolism, and the aforementioned metallothionein and
glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes
responsible for maltose and galactose utilization, such as the
galactose inducible promoter, GAL1. Particularly preferred for use
herein are the PGK, GAL1, and alcohol dehydrogenase (ADH)
promoters. Finally, in constructing suitable expression plasmids,
the termination sequences associated with these genes may also be
ligated into the expression vector 3' of the heterologous coding
sequences to provide polyadenylation and termination of the mRMA.
In preparing the preferred expression vectors of the present
invention, translational initiation sites are chosen to confer the
most efficient expression of a given nucleic acid sequence in the
yeast cell [see Cigan, M. and T. F. Donahue 1987, GENE, Volume 59,
pp. 1-18, for a description of suitable translational initiation
sites).
[0062] A particularly preferred nucleotide expression vector useful
for carrying out the present invention comprises such an
aforementioned promoter sequence, positioned upstream to the
translational initiation site of the heterologous nucleotide
sequence encoding for the heterologous G protein coupled receptor
it is desired to express, and in correct reading frame therewith.
Particularly preferred promoters in this regard are the GAL1, PGK,
and ADH promoters. Positioning of the aforementioned promoter
upstream to the chosen translational initiation site may enhance
expression of a heterologous protein. In these preferred
embodiments, no yeast G protein coupled receptor segment is fused
to the heterologous G protein coupled receptor segment. The present
inventors have discovered that such hybrid receptors are not
critical to achieve receptor expression in yeast. This is contrary
to the art accepted teaching in this regard [see King, et al. cited
infra].
[0063] In certain other embodiments however, at least a fragment of
the 5'-untranslated region of a yeast gene is positioned upstream
from the heterologous G protein coupled segment and operatively
associated therewith. To that end, the present invention also
provides constructs having suitable promoters and translational
initiation sites as described above, but these constructs include a
yeast segment comprising at least .alpha.-fragment of the extreme
amino-terminal coding nucleotide sequence of a yeast G
protein-coupled receptor and a second segment downstream from said
first segment and in correct reading frame therewith, the second
segment comprising a nucleotide sequence encoding a heterologous G
protein-coupled receptor. The yeast segment in this regard may be
provided to actually act as a reporter sequence, rather than to
serve to enhance effective expression of the heterologous G protein
in the yeast system. Thus, certain embodiments comprise a gene
sequence encoding a yeast segment of a yeast G protein-coupled
receptor, that acts as a reporter segment, in that it encodes a
peptide that may be detected through conventional means, such as
antibody binding, and the like. Preferred in this regard is all or
a portion of a yeast pheromone receptor fused to a heterologous G
protein coupled receptor, which may be used primarily as an
"epitope tag" for the highly specific detection of expression of
the desired heterologous receptor using antibodies directed
specifically to the epitope sequence expressed. In constructing
such a vector, the yeast segment may be positioned upstream to the
heterologous protein, or alternatively, a fragment of the extreme
amino-terminal coding sequence of the heterologous G
protein-coupled receptor may be deleted, and the yeast segment
fused directly thereto. In some cases, one or more of the amino
terminal transmembrane domains or intracellular domains of the
heterologous protein are deleted. Alternatively, the yeast segment
may be added directly to the amino terminus of the heterologous
receptor, thereby elongating the overall chimeric receptor
construct.
[0064] The first and second segments are operatively associated
with a promoter, such as the GAL1 promoter, which is operative in a
yeast cell. Coding sequences for yeast G protein-coupled receptors
which may be used in constructing such vectors are exemplified by
the gene sequences encoding yeast pheromone receptors (e.g., the
STE2 gene, which encodes the .alpha.-factor receptor, and the STE3
gene, which encodes the .alpha.-factor receptor).
[0065] Certain preferred chimeric receptors provided herein
comprise a yeast Ste2 protein segment fused directly to all or a
portion of a heterologous G protein receptor, and preferably, the
5HT1a receptor, muscarinic receptor, .alpha.-adrenergic receptor,
or a somatostatin receptor.
[0066] Any of a variety of means for detecting the effects of
ligand binding can be utilized. For example, measurement of the
disassociation of G.alpha. from G.beta..gamma. can be made through
conventional biochemical techniques. However, it should be noted
that the binding of ligand to a receptor may either trigger or
block a detectable biological response, which may also lend itself
to measurement. One such biological response is the ability of
yeast cells to mate. Use of the pheromone induced mating signal
transduction pathway is a preferred method of detecting the effects
of ligand binding in the assay systems herein presented, the basic
premise of which is discussed in more detail, as follows.
[0067] G protein-coupled pheromone receptors in yeast control a
developmental program that culminates in mating (fusion) of a and
.alpha. haploid cell types to form the a/.alpha. diploid (for a
review, see G. F. Sprague, Jr. and J. W. Thorner, in the Molecular
Biology and Cellular Biology of the Yeast Saccharomyces: volume II,
Gene Expression). The process of mating is initiated by
extracellular peptides, the mating pheromones. Cells of the a
mating type secrete .alpha.-factor, which elicits a response in
.alpha.-cells; cells of the .alpha.-mating type secrete
.alpha.-factor which acts only on a cells. Haploid cells respond to
the presence of the peptide mating pheromones through the action of
endogenous G protein-coupled pheromone receptors (STE2: the
.alpha.-factor receptor, expressed, only in .alpha. cells and STE3:
the .alpha.-factor receptor, expressed only in .alpha.-cells). Both
receptors interact with the same heterotrimeric G proteins and a
signal transduction cascade that is, common to both haploid cell
types. Upon pheromone-binding to receptor, the receptor presumably
undergoes a conformational change leading to activation of the G
protein. The .alpha.-subunit, SCG1/GPA1, exerts a negative effect
on the pheromone response pathway, which is relieved by
receptor-dependent activation. The complex of .beta..gamma.
subunits (STE4, STE18) is thought to transmit the positive signal
to an effector, possibly STE20, a putative protein-kinase [Leberer,
E., Dignard, D., Harcus, D., Thomas, D. Y., Whiteway, M. (1992)
EMBO J. 11, 4815-4824]. The effector in turn activates downstream
elements of the signal transduction pathway which include STE5, and
a presumptive protein kinase cascade composed of the products of
the STE11, STE7, FUS3 and KSS1 genes, eventually resulting in cell
cycle arrest and transcription induction. The primary interface
between elements of the pheromone response pathway and cell cycle
regulatory machinery is the FAR1 gene product. Certain recessive
alleles of FAR1 and FUS3 fail to undergo-cell cycle arrest in
response to pheromone, while permitting pheromone dependent
transcription to occur. Pheromone-dependent transcription is
mediated through the action of the sequence-specific DNA-binding
protein STE12. Activation of STE12 results in transcription of
genes possessing a cis-acting DNA sequence, the pheromone response
element. These pheromone responsive genes encode products that are
required for pheromone synthesis (MFa1, MEa2, MFA1, MFA2, STE6,
STE13) and the response to pheromone (STE2, STE3, SCG1/GPA1, FUS3),
facilitate or participate in cell association and fusion (FUS1),
cell cycle arrest (FAR1), and the morphological events required for
mating. In the event that the mating process is not consummated,
yeast cells become adapted to the presence of pheromone and resume
mitotic growth. Thus, in certain preferred embodiments, the FUS3 or
FAR1 gene is mutated or deleted altogether, thereby disconnecting
the cell cycle arrest pathway from the signal transduction pathway,
and allowing continued growth of the cells in response to mating
pheromone binding to the heterologous receptor. Since FAR1 is a
primary factor in the cell cycle regulatory pathway, its deletion
or mutation is preferred in the expression constructs of the
present invention. Yeast cells transformed with such constructs
yield superior yeast strains for ligand-binding assays.
[0068] The mating signal transduction pathway is known to become
desensitized by several mechanisms including pheromone degradation
and modification of the function of the receptor, G proteins and/or
downstream elements of the pheromone signal transduction by the
products of the SST2, STE50, AFR1 [Konopka, J. B. (1993) Mol. Cell.
Biol. 13, 6876-6888] and SGV1, MSG5, and SIG1 genes. Selected
mutations in these genes can lead to hypersensitivity to pheromone
and an inability to adapt to the presence of pheromone. For
example, introduction of mutations that interfere with function
into strains expressing heterologous G protein-coupled receptors
constitutes a significant improvement on wild type strains and
enables the development of extremely sensitive bioassays for
compounds that interact with the receptors. Other mutations e.g.
STE50, sgvl, ste2, ste3; pik1, msg5, sig1, and afr1, have the
similar effect of increasing the sensitivity of the bioassay. One
skilled in the art will understand that increased sensitivity of
the assay systems is attained through deletion of one or more of
these aforementioned genes, introduction of mutations that
down-regulate their expression, or in certain instances, effecting
their overexpression. For example, in the STE50 construct,
overexpression of the gene is desired, not deletion of the
gene.
[0069] Introduction of a constellation of mutations in the mating
signal transduction pathway results in a yeast cell well suited to
expression of heterologous G protein-coupled receptors, which are
able to functionally respond to their cognate ligands, while
providing a biological response that signals the binding of the
receptor to the ligand.
[0070] In conjunction with one or more of the above-referenced
mutations, a particularly convenient method for detecting
ligand-binding to heterologous receptor expressed in yeast cells is
to utilize a conventional genetic indicator system. Thus, in
certain preferred embodiments, the cells are provided with an
additional heterologous nucleotide sequence, comprising a
pheromone-responsive promoter and an indicator gene positioned
downstream from the pheromone-responsive promoter and operatively
associated therewith. With such a sequence in place, the detecting
step can be carried out by monitoring the expression of the
indicator gene in the cell. Any of a variety of pheromone
responsive promoters could be used, examples being promoters
driving any of the aforementioned pheromone responsive genes (e.g.
mFa1, mFa2, MFA1, MFA2, STE6, STE13), the BAR1 gene promoter, and
the FUS1 gene promoter. Likewise, any of a broad variety of
indicator genes could be used, with examples including the HIS3,
G418r, URA3, LYS2, CAN1, CYH2, and LacZ genes. A particularly
preferred reporter gene construct is utilized by fusing
transcription control elements of a FUS1 gene to HIS3 protein
coding sequences, and replacing the original FUS1 gene with this
reporter construct. Expression of the HIS3 gene product is thereby
placed under the control of the pheromone signal transduction
pathway. Yeast strains (his3) bearing this construct are able to
grow poorly on supplemented minimal medium lacking histidine, and
are sensitive to an inhibitor of the HIS3 gene product. In other
preferred embodiments, plasmids carry a FUS1-lacZ gene fusion.
Expression of the FUS1 gene is stimulated in response to receptor
activation by binding of pheromone. Therefore, signal transduction
can be quantitated by measuring .beta.-galactosidase activity
generated from the FUS1-lacZ reporter gene.
[0071] Other useful reporter gene constructs, still under the
control of elements of the pheromone signal transduction pathway,
but alternative to the above-discussed reporter systems, may
involve signals transduced through other heterologous effector
proteins that are coexpressed. For example, 1) ligand-dependent
stimulation of a heterologous adenylylcyclase may permit a yeast
strain lacking its own adenylylcyclase due to mutation in the cdc35
gene to survive, 2) ligand-dependent stimulation of a heterologous
G protein-coupled potassium channel may permit a yeast strain
unable to grow in medium containing low potassium concentration
[(trk1, trk2), for example, see-Anderson, J. A. et al (1992) (Proc.
Natl. Acad. Sci. USA 89, 3736-3740] to survive, or 3)
ligand-dependent stimulation of a heterologous phospholipase C
(especially PLC-.beta.) may permit a yeast strain lacking its own
PLC [(plc), for example, see Payne, W. E. and Fitzgerald-Hayes, M.
(1993) Mol. Cell. Biol. 13, 4351-4363] to survive.
[0072] Any DNA sequence which codes for an adenylylcyclase may be
used to practice the present invention. Examples of adenylylcyclase
include the product of the D. melanogaster Rutabaga gene and the
mammalian subunit types I-VIII [for review see, Tang, W.-J. and
Gilman, A. G. (1992) Cell 70, 869-872], and mutants and homologs
thereof, along with the DNA sequences encoding same, which are
useful for practicing the present invention.
[0073] Any DNA sequence which codes for a G protein-gated potassium
channel may be used to practice the present invention. Examples of
G protein-coupled potassium channel include GIRK1 [Kubo, Y.
Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1992)
Nature 365, 802-806], subunits useful for practicing the present
invention, and mutants and homologs thereof, along with the DNA
sequences encoding same.
[0074] Any DNA sequence which codes for a phospholipase protein may
be used to practice the present invention. Examples of
phospholipase (PLC) proteins include the D. melanogaster norpA gene
product and the PLC-.beta. proteins (for review, see Rhee, S. G.,
and Choi, K. D. (1992) J. Biol. Chem. 267, 12392-123961, subunits
useful for practicing the present invention, and mutants and
homologs thereof, along with the DNA sequences encoding same.
[0075] A particularly preferred yeast expression system is
described herein, having yeast cells bearing SSTR and chimeric
G-protein, and dependent upon the presence of somatostatin for
continued growth. As noted above, transformed host cells of the
present invention express the proteins or protein subunits coded
for by the heterologous DNA sequences. When expressed, the G
protein-coupled receptor is located in the host cell membrane
(i.e., physically positioned therein in proper orientation for both
the stereoselective binding of ligands and for functional
interaction with G proteins on the cytoplasmic side of the cell
membrane). Implementation of the sensitive and specific yeast
expression system described herein will facilitate description of
structural and functional aspects of receptor-ligand and receptor-G
protein interactions. Powerful genetic selection schemes, made
possible by modification of elements of the mating signal
transduction pathway, may be employed to identify aspects of the
receptor that have effects on agonist selectivity, ligand stereo
selectivity, and determinants of agonist/antagonist binding. The
role of proteins that modify the response of receptors and G
proteins to ligand may be worked out in detail with the assistance
of this powerful genetic system. Importantly, the system provides a
generalized approach to the study of the functioning and components
of the G protein-coupled signal transduction system, as well as a
generalized approach to screening assays utilizing the G protein
coupled signal transduction system. The present invention provides
expression constructs and assay systems adapted to receive any of a
variety of heterologous G protein coupled receptors, in the form of
"expression cassettes". The heterologous G protein-coupled receptor
it is desired to study is simply inserted into the vectors herein
provided, and expressed in yeast cells. Ligands that may bind to
the expressed receptor are allowed to come into contact with the
cells in any conventional assay manner, and the effects of the
interaction are easily monitored. The systems presented herein thus
provide tremendous utility in the identification of ligands for
orphan G protein-coupled receptors and for discovering novel
therapeutically useful ligands for receptors of medical,
veterinary, and agricultural importance.
[0076] The following Examples are provided to further illustrate
various aspects of the present invention. They are not to be
construed as limiting the invention.
EXAMPLE 1
Functional Expression of Mammalian G.alpha. Proteins in
Saccharomyces cerevisiae
[0077] A sensitive bioassay is utilized to measure interference of
yeast G.alpha. and G.beta..gamma. interactions by expression of
heterologous G.alpha. proteins. Mammalian G.alpha. genes are
expressed from 2.mu. or centromere-bearing plasmids under the
control of the constitutive PGK or the inducible CUP1 promoter. The
data demonstrates that the rat G.alpha..sub.s, G.alpha..sub.i2, and
chimeric yeast/mammalian G.alpha. can effectively interact with
yeast G.beta..gamma..
[0078] Media and Strains. Growth of bacterial strains and plasmid
manipulations are performed by standard methods (Maniatis T.,
Molecular Cloning, (Cold Spring Harbor Laboratory Press, 1982).
Growth and transformation of yeast strains are performed as
described in Rose et al. (Rose M. D., Methods in yeast genetics,
Cold Spring Harbor Laboratory Press, 1990). The yeast strains used
in these studies (CY414, MATa ura3-52 trpl leu2 his3 pep4::HIS3)
originate from strains described by E. Jones (Jones, E. W., Ann,
Rev. Genet. 18:233, 1984). CY414 is sequentially transformed with
the FUS1-lacZ fusion plasmid pSB234 (Trueheart J., et al Mol. Cell.
Biol. 7(7): 2316-2328, 1987) and G.alpha. expression plasmids.
[0079] Construction of G.alpha. expression plasmids. Rat cDNA
clones for G.alpha..sub.s and G.alpha..sub.i2 and for fusions with
the yeast SCG1 gene are described elsewhere [Kang, Y.-S., Kane, J.,
Kurjan, J., Stadel, J. M., and Tipper, D. J. (1990) Mol. Cell.
Biol. 10, 2582-2590]. To express these genes from low-copy-number
plasmids, XhoI-SalI fragments containing each expression cassette
(including the PGK promoter and terminator sequences) are isolated
and cloned into the CEN plasmid pRS414 digested with XhoI. For
inducible expression, the DNA segment containing PGK promoter
sequences are replaced with upstream activating sequences form the
CUP1 gene.
[0080] .beta.-galactosidase assays. Cultures are . . . diluted to
5.times.10.sup.7 cells/ml and aliquotted into separate tubes.
Pheromone is added to a final concentration of 10-IM to one sample.
Cultures are then incubated for 4 hrs at 30.degree. C. Subsequent
measurement of .beta.-galactosidase activity is conducted as
described elsewhere (Rose M. D., Cold Spring Harbor Laboratory
Press, 1990).
[0081] High and low-copy-number plasmids carrying the yeast SCG1 or
mammalian G.alpha..sub.s or G.alpha..sub.i or chimeric
yeast/mammalian G.alpha. genes expressed from the yeast PGK
promoter are transformed into a wild-type yeast strain also
containing a plasmid carrying a FUS1-lacZ gene fusion. Expression
of the FUS1 gene is stimulated in response to receptor activation
by binding of pheromone. Therefore, signal transduction can be
quantitated by measuring .beta.-galactosidase activity generated
from the FUS1-lacZ reporter gene. Interference of normal signal
transduction by expression of a heterologous G.alpha. protein is
observed as a decrease in .beta.-galactosidase activity.
[0082] Strains expressing introduced G.alpha. genes are assayed for
pheromone-induced gene activation. Data are represented as percent
of wild-type response in FIG. 1. Expression from all G.alpha.
plasmids reduced FUS1-lacZ expression levels demonstrating that the
G.alpha. proteins functionally coupled to yeast G.beta..gamma.. A
dose dependence was observed for Scgl, G.alpha..sub.s and
G.alpha..sub.i2. Expression from high-copy-number plasmids greatly
reduces signaling suggesting that a large excess of heterologous
G.alpha. protein is present. The Scg-G.alpha..sub.i2 chimeric
protein reduces signaling to near the unstimulated background
levels even from the low-copy-number plasmid. Other CEN expression
plasmids reduce signaling to 53 to 84% of wild-type levels.
[0083] To achieve more precise control of G.alpha. expression and
reduce expression to a level sufficiently low that minimal effects
on pheromone induced signaling will occur, G.alpha. genes (except
G.alpha..sub.s) are placed under the control of the inducible CUP1
promoter and transformed into yeast on low-copy-number plasmids.
The level of signaling repression mediated by these plasmids is
dependent on the concentration of Cu++4 added to the medium (FIG.
2). However, basal expression (no Cu++4 added) was equivalent to
levels observed from the PGK promoter (FIG. 1). As in the previous
experiment, the SCG-G.alpha.i2 chimeric protein reduces signaling
to almost background levels.
[0084] The data presented in FIGS. 1 and 2 indicates that all
G.alpha. expression plasmids examined produce functional G.alpha.
proteins in that all inhibit the signal transduction pathway. Using
a constitutive promoter (PGK), most G.alpha. genes exhibit a
dose-dependent effect with high-copy-number 2 micron plasmids
drastically reducing signaling (FIG. 1). Lower expression from CEN
plasmids reduce signaling levels as little as 16% (See G.sub.i,
FIG. 1). Expression of the G.alpha. genes from the CUP1 promoter
shows expected dose/response effects with reduced signaling
correlated to increased Cu++ concentrations (FIG. 2).
EXAMPLE 2
Pharmacological Evaluation of Heterologous G Protein-Coupled
Receptors Expressed in Saccharomyces cerevisiae
[0085] Yeast strains. Growth and transformation of yeast strains
are performed as described (Rose, M. D., Methods in Yeast Genetics,
Cold Spring Harbor Laboratory Press, 1990). The yeast strains used
in these studies (CY414; MATa ura3-52 trpl leu2 his3
pep4.DELTA.HIS3) originate from strains described by E. Jones
(Jones, E. W., Ann. Rev. Genet, 18:233, 1984).
[0086] Nucleic acid manipulation. Growth of bacterial strains and
plasmid manipulations are performed by standard methods [Sambrook,
J., Fritsch, E. F., and Maniatis, T., Molecular Cloning, 2nd ed.
(Cold Spring Harbor Laboratory Press, 1989)]. DNA sequencing is
performed by high temperature cycle sequencing (Applied
Biosystems).
[0087] Protein analysis. Receptor expression strains are grown in
synthetic complete medium lacking specific nutrients to select for
plasmid retention and containing 3% galactose to induce receptor
gene expression. Cells are pelleted and washed in lysis buffer (10
mM sodium bicarbonate, pH 7.2, 1 mM EGTA, 1 mM EDTA) then
resuspended in lysis buffer plus protease inhibitors (5 .mu.g/ml
leupeptin, 10 .mu.g/ml benzamidine, 10 .mu.g/ml Bacitracin, 5
.mu.g/ml pepstatin, 5 .mu.g/ml aprotinin) and lysed by physical
disruption with glass beads. Debris is removed by centrifugation at
1000.times.g for 10 min. The membrane fraction is isolated by
centrifugation at 100,000.times.g for 10 min. This pellet is washed
once in lysis buffer plus inhibitors. Polyacrylamide gel
electrophoresis of yeast extracts is performed by standard methods
except without boiling of samples. Proteins are transferred to
Immobilon-P millipore filters by the semi-dry technique. Receptor
protein is visualized using ECL reagents with rabbit anti-Ste2
antibodies.
[0088] Radioligand binding assays. Reactions are performed in a
volume of 0.2 .mu.l with 5 to 50 .mu.g of protein. Binding assays
for 5HT1a receptor or .beta.2-adrenergic receptor ligands use
buffer of 50 mM Tris, pH 7.4, 10 mM MgCl.sub.2. Somatostatin
binding is performed in a buffer of 50 mM HEPES, pH 7.4, 5 mM
MgCl.sub.2. After allowing ligand binding to reach equilibrium at
room temperature, membrane fractions are isolated on GFC glass
fiber filters. The following final concentrations of ligands are
used: radioligands-3H spiperone, 80 nM; .sup.125I-cyanapindolol,
250 .mu.M; [.sup.125I-tyr.sup.11]-somatostatin 14, 250 pM;
competitors-serotonin, 10 .mu.M; propranolol, 20 .mu.M somatostatin
14, 1 .mu.M. The guanosine triphosphate analog Gpp(NH)p is used at
100 .mu.M.
[0089] Expression of the human 5HT1a serotonergic receptor. The
gene encoding the human 5HT1a receptor is modified to add the first
14 amino acids of the yeast Ste2 protein, cloned into the
expression plasmid pMP3 and, designated pCHI11. This strain,
designated CY382, is grown in medium containing galactose to induce
receptor expression, fractioned and tested for receptor activity by
binding of the radiolabelled antagonist .sup.3H-spiperone.
Saturation binding demonstrates that the receptor is expressed at
high levels (Bmax=3.2 pmol/mg protein) and that it binds spiperone
with an affinity (Kd=115 nM; FIG. 3) similar to that observed in
mammalian tissues (Kd-20 to 100 nM).
[0090] Two chimeric receptor genes are engineered; in pCH117,
sequences encoding the N-terminus including the first two
transmembrane domains of the 5HT1a receptor are replaced with the
corresponding sequences of the Ste2 receptor, and in pCH118, these
Ste2 sequences are added directly to the N-terminus of the 5HT1a
receptor to create a novel nine-transmembrane-domain receptor (FIG.
4). Strains expressing these receptors are examined for binding of
radiolabelled ligand. Both receptors demonstrate specific binding
of the 5HT receptor antagonist .sup.3H-spiperone (FIG. 4).
Replacement of the first two transmembrane domains with those of an
unrelated receptor does not apparently affect binding of this
ligand. Addition of transmembrane domains do not effect binding,
suggesting that this unusual receptor can attain a functional
conformation in the cell membrane. Strains carrying pCHI11, pCHI17,
or pCHI18 produce Bmax values of 3.1, 1.6, or 0.7 pmol/mg,
respectively. Although these chimeric receptors produce interesting
results regarding receptor structure, they do not enhance overall
levels of functional receptors in the cell.
[0091] All intracellular sequences of the 5HT1a receptor are
replaced with corresponding sequences of the yeast Ste2 protein to
directly couple the receptor to the yeast G protein. The resultant
chimeric receptor, CHI16, is expressed in a wild-type yeast strain
and examined for high affinity binding of 5HT1a receptor agonists.
Agonist binding is not detected. However, the level of
radiolabelled spiperone binding is equal to CHI11, indicating that
this receptor is expressed at high levels and in a functional
conformation.
[0092] Expression of the human .beta.2-adrenergic receptor. The
human adrenergic receptor is expressed in yeast with the intention
of using it as a model to optimize expression and G protein
coupling. A yeast strain expressing the receptor is examined by
Scatchard analysis for binding of the ligand
.sup.125I-cyanopindolol. Binding is saturable and demonstrated a Kd
(23 pM)i similar to that reported in mammalian tissues. Strains
with or without coexpressed G.alpha..sub.s are then examined in
competition assays in which binding of this radioligand is competed
with the agonists isoproterenol or epinephrine. High affinity
binding, which is only expected to occur if the receptor is
actively coupled to G protein, was observed in both strains (FIGS.
5 and 6). The extrapolated Ki values for these ligands
(isoproterenol=10 mM; epinephrine=60 nM) are consistent with
affinities observed in mammalian tissues and exhibit the expected
order of potency. However, other data suggest that the high
affinity binding of agonists to the .beta.2-adrenergic receptor in
yeast is anomalous and not a result of coupling to G protein. In
particular, the third intracellular loop (containing the primary.
G.alpha. contact points) of the .beta.2-adrenergic receptor is
replaced with the corresponding domain of the yeast Ste2 receptor.
This receptor exhibits the same affinities for adrenergic agonists
suggesting that the .beta.2-adrenergic receptor takes on an
inappropriate conformation in yeast.
[0093] Expression of rat somatostatin receptor. High affinity
binding of somatostatin to SSTR2 is dependent on formation of a
receptor/G protein complex (Strnad et al., 1993). When SSTR2 and G
protein are uncoupled from each other, high affinity binding of
[.sup.125I]tyr.sup.11S-14 is attenuated. As shown in FIG. 8,
binding of [.sup.125I]tyr.sup.11S-14 to SSTR2 expressed in yeast
coexpressing Scgl/G.sub..alpha.i2 is saturable and of high
affinity. The calculated K.sub.d of [.sup.125I]tyr.sup.11S-14
binding SSTR2 expressed in yeast is 600 pM. Similar binding
affinities are observed when G.sub..alpha.i2 is coexpressed with
SSTR2 rather than Scgl/G.sub..alpha.i2. The K.sub.d observed in
yeast is in close agreement to the calculated K.sub.d of
[.sup.125I]tyr.sup.11S-14 binding of SSTR2 expressed in mammalian
cells (Strnad et al., 1993). It is demonstrated that addition of
the yeast Ste2 receptor to the N-terminus of this receptor has no
affect on its ability to bind S-14. The Ste2 sequences act as a tag
for immunochemical examination of receptor expression. The
immunoblot shown in FIG. 7 illustrates the high level of expression
of SSTR2 in three different strains. Yeast strains expressing the
SSTR2 somatostatin receptor and different G.alpha. proteins are
derived from strain YPH500. These strains share the genotype MATa
scgl.DELTA.hisG lys2-801 ura3-52 leu2.DELTA.1 trpl.DELTA.63
his3.DELTA.200 ade2 SSTR2. Strains designated CY624 (G.alpha.i2),
CY602 (G.alpha.s, and CY603 Scgl) are examined for specific binding
of radiolabelled somatostatin-14 (S-14). All three exhibit some
degree of somatostatin binding (Table 1). High affinity binding of
this ligand requires coupling of the receptor to G protein,
normally G.alpha.i (Luthin D. R. 1993; Strnad J. 1993), so the high
level of binding in the absence of G.alpha.i is unexpected. As
confirmation that the receptor is coupled to G proteins, binding is
examined in the presence of the nonhydrolyzable guanosine
triphosphate analog Gpp (NH)p. The addition of this compound
eliminates ligand binding (Table 1), demonstrating that the SSTR2
receptor is coupled to G protein. These data demonstrate that a
mammalian G protein-coupled receptor can functionally interact with
a G protein composed of its favored G.alpha. protein plus yeast
G.beta. and G.gamma. subunits and that a heterologous receptor can
functionally couple to a G protein entirely composed of yeast
subunits.
TABLE-US-00001 TABLE 1 Binding of .sup.125I-somatostatin
STRAIN.sup.a G.alpha. Bmax.sup.b +Gpp (NH) p.sup.c CY624 G.alpha.i
94.5 22% CY602 G.alpha.s 13.7 0% CY603 Scgl 24.7 4% .sup.aCrude
membrane extracts were prepared from yeast strains expressing the
rat SSTR2 somatostatin receptor subtype and the indicated G.alpha.
protein. The maximal binding of radiolabeled somatostatin 14 was
measured as described in the text. .sup.bBmax values are given as
fmol/mg total protein. .sup.cThe non-hydrolyzable GTP analog
Gpp(NH)p was added to samples to uncouple receptor and G protein.
Data are presented as percent radioligand bound compared to
untreated samples.
[0094] Expression of Drosophila msscarinic acetylcholine receptor.
DNA sequences encoding a Drosophila muscarinic acetylcholine
receptor (Dm mAChR) are modified by addition of a SalI site in the
5' coding sequences through the use of PCR. DNA sequences encoding
the first 23 amino acids of the STE2 gene product are added to the
5' end of Dm mAChR as a BamHI/SalI fragment. The modified Dm mAChR
is inserted into the BamHI site in plasmid pMP3, placing expression
of the receptor under the control of the GAL1 promoter, forming
plasmid pMP3-Dm mAChR. Strain CY414 is transformed with this
plasmid and cultured for receptor expression by standard methods.
Crude membrane preparations are prepared from these cells and
tested for the presence of specific binding sites for the
muscarinic antagonist 3H-quinuclidinyl benzilate (10 nM) competed
with atropine (50 .mu.M) Specific binding sites (Bmax 10 and 30
fmol/mg) are observed.
[0095] Drosophila mAchR expression is also detectable by
immunoblotting methods. An abundant 75 kDa polypeptide, consistent
with the predicted molecular weight from the primary sequence of
mAchR, is detected in samples of protein (30 .mu.g/lane) from crude
membrane preparations from cells expressing mAchR from pMP3 using
an antibody directed against the associated Ste2 epitope (FIG. 9).
Substantially less protein is detected when mAchR is expressed from
pMP2, a derivative of pMP3 lacking GAL4 sequences, which is not
expected to confirm high level expression of mAchR.
[0096] Expression of an .alpha.2-adrenergic receptor (2.alpha.-AR).
An EcoRI-NarI fragment from plasmid pMP3, including the GAL1,10
promoter EcoRI-BamHI fragment, DNA sequences encoding the first 23
amino acids of the STE2 gene product present on a BamHI-SalI
fragment, SalI-SphI polylinker fragment from YEp352, and STE7
terminator sequences, is transferred to pRS424, forming pLP15. A
PstI-PvuII fragment encoding a porcine .alpha.2A-AR [Guyer, C. A.,
Horstman, D. A., Wilson, A. L., Clark, J. D., Cragoe, E. J., and
Limbird, L. E. (1990) J. Biol. Chem. 265, 17307-17317] is inserted
into the PstI-SmaI sites of pLP15, forming pLP50. An EcoRI fragment
of GAL4 is inserted into the EcoRI site of pLP50, forming pLP60.
Strain LY124 [a derivative of YPH500 (Stratagene) containing the
scgl.DELTA.hisG allele and bearing plasmid pLP10 [pLP10: pUN75
(Elledge, S. J. and Davis, R. W. Genetics 87, 189-194) containing
the PGK-Scgl-G.alpha.i2 XhoI-SalI fragment from
pPGKH-Scgl-G.alpha.i2 inserted into the SalI site] is transformed
with pLP60 and cultured for receptor expression by standard
methods. Crude membrane preparations are prepared from these cells
and tested for the presence of specific binding sites for the
.alpha.2-AR antagonist 3H-rauwolscine (200 nM) competed with
phentolamine (10 .mu.M). Specific binding sites with a Bmax of
between 10 and 84 fmol/mg were observed.
[0097] Porcine .alpha.2AR expression is also detectable by
immunoblotting methods (FIG. 10). Several abundant polypeptides are
detected in samples of protein (30 .mu.g/lane) from crude membrane
preparations from cells expressing .alpha. 2AR from pMP3 using an
antibody directed against the associated Ste2 epitope.
EXAMPLE 3
Agonist Dependent Growth of Yeast in Response to Somatostatin
Receptor Agonists
[0098] Yeast strains that respond to somatostatin are created by
introducing several modifications into typical laboratory yeast
strains. First, a cDNA encoding the somatostatin receptor subtype 2
(SSTR2) is placed under the control of the galactose-inducible GAL1
promoter in a multicopy yeast plasmid (FIG. 11). High level
expression of receptor is accomplished by inducible
co-overexpression of the transcriptional activating protein GAL4
from the same plasmid. Second, the endogenous G.alpha. protein
gene, GPA1/SCG1, is replaced with a chimeric gene composed of an
amino terminal domain from GPA1/SCG1, and C-terminal sequences from
rat G.alpha.i2 (FIG. 12). Expression of chimeric G proteins in
yeast have previously been shown to suppress the growth defect of
scgl/gpal mutant cells [Kang, Y.-S., Kane, J., Kurjan, J., Stadel,
J. M., and Tipper, D. J. (1990) Mol. Cell. Biol. 10, 2582-2590].
Third, the FAR1 gene is deleted, permitting continued growth in the
presence of an activated mating signal transduction pathway. The
FAR1 protein is thought to serve as the primary interface between
the mating signal transduction pathway and cell cycle machinery
[Chang, F. and Herskowitz, I. (1990) Cell 63, 999-1012; Peter, M.,
Gartner, A., Horecka, J., Ammerer, G., and Herskowitz, I. (1993)
Cell. Biol. 13, 5659-5669]. Fourth, the FUS1 gene is replaced with
a reporter gene construct made by fusing transcription control
elements of the FUS1 gene to HIS3 protein coding sequences, thereby
placing expression of the HIS3 gene product under the control of
the pheromone signal transduction pathway. Yeast strains (his3)
bearing this construct are able to grow poorly on supplemented
minimal medium lacking histidine and are sensitive to
3-amino-1,2,4-triazole (AT), an inhibitor of the HIS3 gene product.
Receptor activation by agonist-binding leads to increased HIS3
protein expression, a corresponding increase in resistance to AT,
and, therefore, the ability to grow on medium lacking histidine
and/or in the presence of the inhibitor. Adjusting the pH of the
growth medium to >pH 5.5 enhances the ability of such cells to
grow in presence of agonist, presumably due to an increased ability
of somatostatin to bind to SSTR2.
[0099] The utility of the yeast expression system lies in its
adaptability to rapid mass screening. To facilitate screening for
novel therapeutics directed at the SSTRs, a convenient agar plate
bioassay is developed in which functional coupling of somatostatin
binding to receptor and subsequent activation of the mating signal
transduction pathway is detected as a zone of growth (halo) around
applied compounds (FIG. 13). Overnight liquid cultures of LY268 (a
derivative of YPH500 (Stratagene) MATa ura3-52 lys2-801 ade2
trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1 farl.DELTA.LYS2
scgl.DELTA.hisG fusl.DELTA.FUS1-HIS3 sst2.DELTA.ADE2 bearing the
SSTR2 expression plasmid and the SCG1-G.alpha.i2 expression plasmid
pLP82 in SC dextrose (2%) lacking ura, trp were transferred to SC
Lactate medium (2%) lacking ura and trp and subsequently SC
galactose (2%) medium lacking ura and trp. Cells (2.times.10.sup.5)
are then plated in 30 ml of SC galactose (2%) lacking ura, trp, and
his agar (FIG. 13, top panel), or spread evenly on the surface
(FIG. 13, bottom panel), the indicated amounts of selected
compounds applied to paper disks situated on the surface of the
agar plate, and incubated at 30.degree. C. for 3-5 days. Halos of
growth are observed around disks saturated with varying
concentrations of somatostatin (S-14) and MK678 [a hexapeptide
analog of somatostatin that exhibits high affinity binding to SSTR2
[Verber, D., Saperstein, R., Nutt, R., Friedinger, R., Brady, P.,
Curley, P., Perlovi, D., Paleveda, W., Zacchei, A., Cordes, E.,
Anderson, P., and Hirschmann, R. (1981) Life Sci, 35, 1371-1378].
Halo size increases in proportion to the amount of agonist applied
and a significant response is observed even at the lowest amount
applied (6 nmol S-14), demonstrating the exquisite sensitivity of
the assay. No detectable response is observed with carrier alone,
or to met-enkephalin, an opiate receptor agonist, demonstrating the
high specificity of the assay.
[0100] Yeast medium and culture conditions are formulated according
to standard procedures and DNA-mediated transformation of yeast is
by the LiAc method (Sherman, F., Fink, G. R., and Hicks, J. B.
(1986) Methods in Yeast Genetics (Cold Spring Harbor Laboratory
Press]. LY2.68 is constructed by sequential insertional deletion
using recombinant scgl:.DELTA.hisG, farl.DELTA.LYS2,
FUS1.DELTA.HIS3, and sst2.DELTA.ADE2 alleles. The scgl.DELTA.hisG
allele is assembled by inserting the hisG-URA3-hisG fragment from
pNKY51 [Alani, E., Cao, L., and Kleckner, N. (1987) Genetics 116,
541-5451 between the 5' EcoRI-HindIII and 3' SphI-SnaBI fragments
of SCG1/GPA1. After DNA-mediated transformation of appropriate
yeast strains and selections for replacement of the chromosomal
allele, the URA3 gene is removed by inducing recombination between
hisG repeats by growth on 5-fluoroorotic acid (FOA)-containing
medium [Boeke, J., Lacroute, F., and Fink, G. (1984) Mol. Gen.
Genet. 197, 345-3461. The farl.DELTA.LYS2 allele is constructed by
amplifying two fragments of the FAR1 gene [Chang, F. and
Herskowitz, I. (1990) Cell 63, 999-10121 from yeast genomic DNA
(strain YPH501, Stratagene) using synthetic oligonucleotides that
introduce an EcoRI site at 1201 in the 5' fragment and an HindIII
site at position 2017 and a SalI site at 2821 in the 3' fragment.
The fragments are cloned into the EcoRI/SalI fragment of pBSK
(Stratagene). The completed farl.DELTA.LYS2 construct is digested
with EcoRI and used to transform yeast. An EcoRI fragment encoding
the FUS1-HIS3 reporter gene is released from pSL1497 [Stevenson, B.
J., Rhodes, N., Errede, B., and Sprague, G. F. (1992) Genes Dev. 6,
1293] and used to transform appropriate yeast strains. The
sst2.DELTA.ADE2 allele [Dietzel, C. and Kurjan, J. (1987) Mol.
Cell. Biol. 7, 4169-41771 is built from a 2.5 kb fragment of the
ADE2 gene amplified by PCR using oligos that placed a C1a site at
position 1 and an NheI site at position 2518. This fragment is used
to replace the internal ClaI-NheI fragment in SST2. The
sst2.DELTA.ADE2 fragment is released by digestion with SalI and
used to transform appropriate yeast strains.
[0101] The multistep construction of the SSTR2 expression plasmid,
pJH2 (FIG. 11), is initiated by inserting a SphI/NarI fragment of
3' untranslated region from the STE7 gene [Teague, M. A., Chaleff,
D. T., and Errede, B. (1986) Proc. Natl. Acad. Sci. USA 83,
7371-7375], and an EcoRI/BamHI fragment of the GAL1/10 promoter
[Yocum, R. R., Hanley, S., West, R., and Ptashne, M. (1984) Mol.
Cell. Biol. 4 1985-19.98] into appropriate sites in YEp352 (Hill,
J. E., Myers 2, A. M., Koerner, T. J., and Tzagoloff, (1986) Yeast
2, 163-167.], creating pEK1. A PCR product, encoding the open
reading frame and transcriptional termination sequences of the GAL4
gene, is amplified with oligos containing 5' EcoRI and 3' AatII
sites and inserted into pEK1, creating pMP3. The cDNA encoding rat
SSTR2 (Strnad, J., Eppler, C. M., Corbett, M., and Hadcock, J. R.
(1993.) BBRC, 191, 968-976] is modified by PCR using
oligonucleotides that add a BglII site in the DNA sequences
encoding the amino terminus of SSTR2 and a BglII site directly
after the translational stop site. SSTR2 coding sequences are
inserted as a BglII PCR fragment into the BamHI site of pMP3.
[0102] Plasmid pLP82 (FIG. 12) is constructed by first replacing
the XhoI/EcoRI promoter fragment in pPGKH-SCG1-G.alpha.s [Kang,
Y.-S., Kane, J., Kurjan, J., Stadel, J. M., and Tipper, D. J.
(1990) Mol. Cell. Biol. 10, 2582-2590], with a modified SCG1
promoter fragment [Dietzel, C. and Kurjan, J. (1987) Cell 50,
1001-1010] amplified from yeast genomic DNA using oligonucleotides
that introduce 5' XhoI and 3' EcoRI sites at positions -200 and
-42, forming plasmid pLP61. The BamHI fragment encoding G.alpha.s
domain is replaced with a comparable fragment encoding G.alpha.i2,
forming plasmid pLP71. The XhoI/SalI fragment of pLP71 encoding an
SCG1-G.alpha.i2 chimeric G protein expressed under the control of
the SCG1 promoter is transferred to the SalI site in pRS414
(Stratagene), forming pLP82.
[0103] Plasmid pLP83 is constructed by replacing the EcoRI fragment
in pLP71 with the EcoRI fragment encoding I from pPGKH-SCG1 [Kang,
Y.-S., Kane, J., Kurjan, J., Stadel, J. M., and Tipper, D. J.
(1990) Mol. Cell. Biol. 10, 2582-2590), forming plasmid pLP75. The
XhoI/SalI fragment encoding SCG1 is transferred to the SalI site in
pRS414 (Stratagene), forming plasmid pLP83.
EXAMPLE 4
Effects of G Protein Expression on the Sensitivity of the
Bioassay
[0104] Yeast strains LY268 (pLP82: CEN pSCG1-Scgl-G.alpha.i2),
LY262 (pRS414-PGK-Scgl-G.alpha.i2: pRS414 containing the
PGK-Scgl-G.alpha.i2 Xho/SalI fragment from pPGKH-Scgl-G.alpha.i2 in
the SalI site), LY324 (pLP84: 2.mu. pSCG1-Scg-G.alpha.i2), and
LY284 (pRS424-PGK-Scg-G.alpha.i2: pRS424 containing the
PGK-Scgl-G.alpha.i2 Xho/SalI fragment from pPGKH-Scgl-G.alpha.i2 in
the SalI site) were constructed by placing the designated plasmids
in strain LY260 [a derivative of YPH500 (Stratagene) MATa ura 3-52
lys2-801 ade2 trp1D63 his3.DELTA.200 leu2 ml farl.DELTA.LYS2
scgl.DELTA.hisG fusl.DELTA.FUS1-HIS3 sst2.DELTA.ADE2 bearing the
SSTR2 expression plasmid]. Overnight liquid cultures in SC-Dextrose
(2%) lacking ura and trp were transferred to Sc-Lactate (2%) medium
lacking ura and trp and subsequently SC-Galactose (2%) medium
lacking ura and trp. Cells (2.times.10.sup.5) are then plated in 30
ml of SC-Galactose (2%) lacking ura, trp, and his agar, the
indicated amounts of selected compounds applied to paper disks
situated on the surface of the agar plate, and incubated at
30.degree. C. for 3-5 days (FIG. 14). The extent of growth around
S-14 is dependent upon the G protein expression plasmid contained
in each strain. The most luxuriant growth is observed in response
to S-14 by LY268 (pLP82: CEN pSCG1-Scgl-G.alpha.i2), less growth is
seen in strains LY262 (pRS414-PGK-Scgl-G.alpha.i2) and LY324
(pLP84: 2.mu. pSCG1-Scg-G.alpha.i2), while little detectable growth
is exhibited by LY284 (pRS424-PGK-Scgl-G.alpha.i2). These results
are consistent with the observed inhibition of pheromone stimulated
transcriptional induction by elevated amounts of expressed G.alpha.
protein. Thus, precise regulation of G protein expression levels in
strains expressing heterologous G protein-coupled receptors is
critical to the design and successful implementation of a bioassay
for compounds that interact with the receptor.
[0105] Plasmid pLP84 is constructed by first replacing the
XhoI/EcoRI promoter fragment in pPGKH-SCG1-G.alpha..sub.s [Kang,
Y.-S., Kane, J., Kurjan, J., Stadel, J. M., and Tipper, D. J.
(1990) Mol. Cell. Biol. 10, 2582-2590], with a modified SCG1
promoter fragment [Dietzel, C. and Kurjan, J. (1987) Cell 50,
--1001-1010] amplified from yeast genomic DNA using
oligonucleotides that introduce 5' XhoI and 3' EcoRI sites at
positions -200 and -42, forming plasmid pLP61. The BamHI fragment
encoding G.alpha., domain is replaced with a comparable fragment
encoding G.alpha.i21 forming plasmid pLP71. The XhoI/SalI fragment
of pLP71 encoding a SCG1-G.alpha..sub.i2 chimeric G protein
expressed under the control of the SCG1 promoter is transferred to
the SalI site in pRS424 (Stratagene), forming pLP84.
EXAMPLE 5
Somatostatin Receptor is Capable of Transmitting Signal Through the
Endogenous Yeast G.alpha.
[0106] SSTR2 is thought to couple to G.alpha..sub.i2 and
G.alpha..sub.i3 in mammalian cells [Luthin, D. R., Eppler, C. M.,
and Linden, J. (1993) J. Biol. Chem. 268, 5990-59963. However, when
expressed in appropriate yeast strains (described below), SSTR2 is
shown to be capable of transmitting a signal through the endogenous
yeast Gm protein. Implicit in this observation is the necessary
coupling of SSTR2 to the endogenous Gm protein. The ability of
heterologous G protein-coupled receptors to couple to the
endogenous Gm protein is a significant improvement in existing
technology, and is thought not to be possible in the prior art
(King K. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz,
R. J. (1990) Science 250, 121-123). Yeast strains LY266 (pLP83: CEN
pSCG1-Scgl), LY280 (pRS414-PGK-Scgl: pRS414 containing the PGK-Scgl
Xho-SalI fragment from pPGKH-ScgI in the SalI site), LY326 (pLP86:
2.mu. p SCG1-Scg), and LY282 (pRS424-pPGK-Scg pRS424 containing the
PGK-Scgl Xho-SalI fragment from pPGKH-Scgl in the SalI site) were
constructed by placing the designated plasmids in strain LY260.
Overnight liquid cultures of these strains, which are capable of
expressing only SCG1/GPA1, in SC-Dextrose (2%) lacking ura and trp
were transferred to SC-Lactate (2%) lacking ura, trp, and
subsequently to SC-Galactose (2%) lacking ura and trp medium. Cells
(2.times.10.sup.5) are then plated in 30 ml SC-Galactose (2%)
lacking ura, trp, and his medium, the indicated amounts of selected
compounds applied to paper disks situated on the surface of the
agar plate, and incubated at 30.degree. C. for 3-5 days. Halos of
growth are observed around disks saturated with varying
concentrations of S-14, demonstrating that a productive signal can
be transduced through an interaction between SSTR2 and the yeast
SCG1/GPAL protein (FIG. 15). These observations demonstrate that a
simple and broadly applicable bioassay may be established in which
any member of the class of G protein-coupled receptors may be
expressed in appropriately modified yeast strains and made to
couple to the endogenous G.alpha. protein.
[0107] Plasmid pLP86 is constructed by replacing the EcoRI fragment
in pLP71 with the EcoRI fragment encoding SCG1 from pPGKH-SCG1
[Kang, Y.-S., Kane, J., Kurjan, J., Stadel, J. M., and Tipper, D.
J. (1990) Mol. Cell. Biol. 10. 2582-2590], forming plasmid pLP75.
The XhoI/SalI fragment encoding SCGL is transferred to the SalI
site in pRS424 (Stratagene), forming plasmid pLP86.
EXAMPLE 6
Mutations in SST2 Enhance the Sensitivity of the Responses to
Somatostatin
[0108] Mutations in the SST2 gene result in supersensitivity of
otherwise wild type cells to mating pheromone. The effect of this
mutation on levels of AT resistance expressed in farl, FUS1-HIS3
strains is examined (FIG. 16). Cells (5.times.10.sup.5) from
overnight cultures of LY230 [a derivative of YPH50 (Stratagene)
MATa SST2 ura3-52 lys2-801 ade2 trpl.DELTA.63 his3.DELTA.200
leu2.DELTA.1 farl.DELTA.FUS1-HIS3] and LY238 (a modification of
LY230 sst2.DELTA.ADE2) in SCD-ura, trp are plated on SCD-ura, trp,
his, containing 10 mM AT and incubated at 30.degree. C. for 2 days.
Increased AT resistance in response to mating factor is exhibited
by LY238 (sst2). Growth of LY238 is observed around disks
containing 10 pmol of mating pheromone, while LY230 (SST2) required
1 mmol of mating factor for significant growth to be observed.
Thus, introduction of sst2 into these strains raises the
sensitivity of the bioassay by at least 100-fold, opening the
possibility of increasing the sensitivity of other G
protein-coupled receptor bioassays as well.
[0109] Yeast strains that express SSTR2 and bearing a defective
sst2 gene exhibit much greater growth around disks containing
various concentrations of somatostatin than is exhibited by strains
containing a functional SST2 gene. Overnight cultures of strains
LY268 (sst2, containing pLP82), LY266 (sst2, containing pLP83),
LY288 [a derivative of YPH500 (Stratagene) MATa SST2 ura3-52
lys2-801 ade2 trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1
farl.DELTA.LYS2 scgl.DELTA.hisG fusl.DELTA.FUS1-HIS3 bearing the
SSTR2 expression plasmid and the SCG1-G.alpha..sub.i2 expression
plasmid, pLP82] and LY290 (a modification of LY288 that contains
pLP83) in SC Dextrose (2%) lacking ura, trp were transferred to SC
Lactate medium (2%) lacking ura and trp, and subsequently to SC
Galactose (2%) medium lacking ura and trp. Cells (2.times.10.sup.5)
are then plated in 30 ml SC Galactose (2%) plates lacking ura, trp
and his, the indicated amounts of selected compounds applied to
paper disks situated on the surface of the agar plate, and
incubated at 30.degree. C. for 3-5 days. Halos of growth are
observed around disks saturated with varying concentrations of S-14
in both sst2A and SST2 strains (FIG. 17), however, the amount of
growth exhibited by strain LY268 and LY266 (sst2) is significantly
greater than that observed for strains LY288 and LY290 (SST2).
Furthermore, the down-regulatory effect of SST2 is more pronounced
in those strains that express solely the wild type SCG1/GPA1
protein, consistent with the expectation that SST2 interacts more
faithfully with the native protein than the SCG1-G.alpha..sub.i2
chimeric protein.
EXAMPLE 7
Functional Expression of a Rat Cholecystokinin (CCK.sub.B) Receptor
in Yeast
[0110] Cholecystokinin (CCK) is a major intestinal hormone that
plays an important role in regulating pancreatic secretion and bile
ejection (1). CCK is also one of the most widely distributed of
brain neuropeptides (2). CCK promotes its effects through the
action of cell surface receptors which can be classified using
pharmacological criteria into two subtypes, CCK.sub.A and CCK.sub.B
(3). Molecular cloning efforts have identified cDNAs encoding G
protein-coupled CCK.sub.A (4) and CCK, (5-8) receptors. Recently,
compounds with selective CCK, receptor antagonist properties having
potent anxiolytic activity have been identified (9). Functional
expression of CCK, receptors in yeast should permit rapid screening
for new compounds with CCK.sub.B antagonist properties and
facilitate molecular characterization of structural aspects of the
CCK.sub.B receptor required for rational design of new CCK,
ligands.
Materials and Methods
[0111] Plasmid constructions. All molecular biological
manipulations were, performed according to standard procedures
(10). The rat CCK, receptor was cloned from rat brain cDNA by PCR
using oligonucleotide primers that introduce BglII sites at 5' and
3' ends (5-AAAAGATCTAAAATGGACCTGCTCAAGCTG, 31 AAAAGATCTTCAGCCAGG
CCCCAGTGTGCT). The CCK.sub.B receptor expression plasmid, pJH20,
was constructed by inserting the BglII-digested PCR fragment in the
correct orientation into BamHI cut pMP3 (11). The G.alpha. protein
expression plasmids used in this study were constructed by
replacing DNA sequences encoding the 47 carboxy-terminal amino
acids of GPA1 in pLP83 (11) with those of, G.alpha..sub.s (pLP122),
G.alpha..sub.i2 (pLP121).
[0112] Strain; constructions. Yeast strains were constructed, and
growth media and culture conditions formulated according to
standard, procedures (12). DNA-mediated transformation of yeast was
carried out using the lithium acetate method. The yeast strains
used as the basis for all experiments described in this report were
constructed by sequential insertional deletion using recombinant
alleles. Yeast strains that express the rat CCK.sub.B receptor were
constructed by sequential DNA-mediated transformation of LY296
(MATa ura3-52 trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1 ade2-101
lys2-801 gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3 sst2.DELTA.ADE2,
ref. 7) with pJH20 followed by the G.alpha. protein expression
plasmids described above.
[0113] Radiolabeled agonist saturation binding assays. Crude yeast
membrane extracts from late log phase cultures were prepared by
glass-bead lysis and centrifugation at 40,000.times.g following a
published procedure (13). The protein content of crude membrane
fractions was measured using the Biorad protein assay kit according
to manufacturers instructions. Radioligand binding assays were
conducted according to Strnad et al (14) using .sup.3H-CCK-8
(Amersham) in the presence of 150 mM NaCl. Non-specific binding was
defined as that observed in the presence of 1 .mu.M CCK-4.
Negligible specific binding was observed in membrane fractions made
from cells lacking CCK.sub.B receptor (data not shown).
[0114] Bioassay. Functional assay of CCK, receptor expressed in
yeast was accomplished using modification of a standard procedure
(11). Yeast strains were grown overnight in 2 ml synthetic complete
liquid medium containing glucose (2%) and lacking uracil and
tryptophan (SCD-ura-trp) medium, washed to remove residual glucose
and grown overnight in 5 ml SC Galactose (2%)-ura-trp liquid
medium. Molten (50.degree. C.) SC Galactose (2%)-ura-trp-his agar
medium (30 ml, adjusted to pH 6.8 by addition of concentrated KOH
or NH.sub.4OH prior to autoclaving) was inoculated with the
overnight culture (2.times.10.sup.4 cells/ml) and poured into
square (9.times.9 cm) petri plates. Sterile filter disks were
placed on the surface of the solidified agar and saturated with 10
.mu.l of DMSO containing the indicated amounts of the designated
compounds. Plates were incubated at 30.degree. C. for 3 days.
Cholecystokinins (CCK-4, CCK-8), somatostatin (S-14), and
met-enkephalin were from Bachem. Oxymetazoline, isoproterenol, and
carbachol were from Sigma.
Results
[0115] Cholecystokinin binding to the rat CCK B receptor expressed
in yeast. High level functional expression of the rat CCK.sub.B
receptor in yeast was a necessary prerequisite to the development
of a useful bioassay. The rat CCK, receptor cDNA was placed under
the control of the GAL1 promoter in plasmid pJH20. This construct
also confers inducible overexpression of Gal4p, the transcriptional
activating protein for galactose-inducible genes, resulting in
significantly elevated levels of receptor protein in crude membrane
fractions compared to receptor expressed from a plasmid lacking
GAL4 sequences (data not shown). CCK.sub.B receptor sequences were
introduced into pJH20 without, modification of the protein coding
sequences. Previously, King et al. reported that replacement of the
amino-terminal domain of the .beta..sub.2-adrenergic receptor with
equivalent STE2 sequence was necessary for efficient receptor
expression in yeast (15). In contrast, functional expression of
CCK.sub.B receptor in yeast does not require addition of any yeast
sequences to the amino-terminus. Plasmids conferring expression of
chimeric G.alpha. proteins composed of amino-terminal
.beta..gamma.-interaction domain from Gpalp and carboxy-terminal
receptor interaction domains from rat G.alpha..sub.i2 (pLP121) or
G.alpha..sub.s (pLP122) under the control of the GPA1 promoter were
constructed. Yeast strains that contain expressed CCK.sub.B
receptor and chimeric G.alpha..sub.i2 (LY628) and G.alpha..sub.s
(LY631) protein were assembled by transformation of a yeast strain
(LY296) modified by deletion of genes encoding components of the
mating signal transduction pathway with CCK.sub.B receptor and
G.alpha. protein expression plasmids. Most G protein-coupled
receptors exhibit both high and low agonist-dependent affinity
states. High-affinity agonist binding is dependent on functional
association of receptor with a heterotrimeric G protein. If the
receptor does not associate with, or is uncoupled from the G
protein, agonist binding will be of low affinity and undetectable
in radiolabeled agonist saturation binding assays. In crude yeast
membrane fractions made from LY631 cells, the agonist .sup.3H-CCK-8
bound to the CCK, receptor with high affinity and in a saturable
manner (FIG. 18), demonstrating that (1) a functional
ligand-binding conformation of the CCK, receptor was expressed in
yeast, and (2) the receptor functionally associated with the
chimeric G.alpha. protein, resulting in a high-affinity agonist
binding state. The CCK, receptor expressed in yeast displayed an
affinity for .sup.3H-CCK-8 (K.sub.d=8 nM) substantially lower than
the high affinity binding state of the CCK.sub.B receptor expressed
in mammalian cells (K.sub.d=100 pM, ref. 5), perhaps due to an
inefficient interaction with the receptor interaction domain from
rat G.alpha..sub.s These binding parameters would be expected to
more closely resemble the native values in extracts from cells
containing the cognate G.alpha. protein, G.alpha.q (5-8). The total
number of .sup.3H-CCK-8 binding sites observed (Bmax=206 fmol/mg)
was consistent with values obtained for the yeast .alpha.-mating
pheromone receptor (200 fmol/mg, ref. 16). For many G
protein-coupled receptors, high affinity agonist binding is
sensitive to GTP and its analogs. GTP analogs induce dissociation
of the receptor/G-protein complex, resulting in a low affinity
agonist binding state. Addition of GppNHp (100 .mu.M), a
non-hydrolyzable GTP analog, to an agonist binding assay decreased
specific binding of .sup.3H-CCK-8 to the CCK, receptor by greater
than 50% in crude membrane fractions from LY631 cells. These
results: represent a further indication of functional coupling
between the CCK.sub.B receptor and the chimeric G.alpha. protein.
Thus, the rat CCK.sub.B receptor expressed in yeast exhibits
high-affinity agonist binding properties comparable to those
observed in mammalian tissues.
[0116] The CCK.sub.B receptor retained agonist selectivity when
expressed in yeast. A selective and sensitive-bioassay was designed
using yeast strains bearing the above described genetic
modifications and plasmids conferring expression of the CCK.sub.B
receptor and chimeric G.sub..alpha.i2 and G.sub..alpha.s proteins.
A dose-dependent growth response of LY628 and LY631 cells was
evident around applied CCK-4 (FIG. 19). The assay was selective:
the diameter of the growth zones was proportional to the reported
affinity of the ligands for the CCK, receptor
(CCK-8>gastrin>CCK-4) reflecting the ability of the bioassay
to discriminate between ligands of varying potency (5-8).
Detectable growth responses were not observed in response to a
variety of agonists selective for other G protein-coupled receptors
(somatostatin, met-enkephalin, oxymetazoline, isoproterenol,
carbachol), nor by yeast cells lacking the CCK, receptor (data not
shown). A detectable response was observed to 10 .mu.g of
CCK-4.
Discussion
[0117] Compounds that act at the CCK receptors, particularly
antagonists, may possess great therapeutic potential (3). In the
periphery, the inhibitory effects of CCK antagonists make them
excellent candidates for treatment of pancreatitis, pancreatic
cancer, biliary colic, disorders of gastric emptying, and irritable
bowel syndrome. CCK antagonists reverse the development of satiety
and might be useful in improving appetite in anorectic patients or
others that require increased food intake. Conversely, CCK agonists
might be useful appetite suppressants. CCK antagonists also
potentiate opiate analgesia and so might be appropriate for use in
the management of clinical pain. In the CNS, selective CCK
antagonists have promise as powerful anxiolytic agents (9).
Further, CCK antagonists relieve the anxiety associated with drawl
from drug use, and so might find a use in the treatment of
withdrawal from commonly abused, drugs. CCK agonists may have use
as antipsychotic agents.
REFERENCES CITED IN THIS EXAMPLE
[0118] 1. Ivy, A. C. and E. Oldberg. 1928. J. Physiol. (London) 86:
599-613. [0119] 2. Dockray, G. J. R. 1976. Immunochemical evidence
of cholecystokinin-like peptides in brain. Nature 264: 568-570.
[0120] 3. Woodruff, G. N. and J. Hughes. 1991. Cholecystokinin
antagonists. Annu. Rev. Pharmacol. Toxicol. 31: 469-501. [0121] 4.
Wank, S. A, R. Harkins, R. T. Jensen, S. Shapiro, A. de Weerth, and
T. Slattery. 1992. Purification, molecular cloning, and function
expression of the cholecystokinin receptor from rat pancreas. Proc.
Natl. Acad. Sci. USA 89: 3125-3129. [0122] 5. Kopin, A. S., Y-M.
Lee, E. W. McBride, L. J. Miller, M. Liu, H. Y. Lin, L. F.
Kolakowski, and M. Beinborn. 1992. Expression cloning and
characterization of the canine parietal cell gastrin receptor.
Proc. Natl. Acad. Sci. USA 89: 3605-3609. [0123] 6. Wank, S. A., J.
R. Pisenga, and A. de Weerth. 1992. Brain and gastrointestinal
cholecystokinin receptor family: Structure and function. Proc.
Natl. Acad. Sci. USA. 89: 8691-8695. [0124] 7. Lee, Y-M., M.
Beinborn, E. W. McBride, M. Lu, L. F. Kolakowski. and A. S. Kopin
1993. The human brain cholecystokinin-B/gastrin Receptor. J. Biol.
Chem. 268: 8164-8169. [0125] 8. Ito, M., T. Matsui, T. Taniguchi,
T. Tsukamoto, T. Murayama, N. Arima, E. Nakata, T. Chiba, and K.
Chibura. 1993. Functional characterization of a human brain
cholecystokinin-B receptor. J. Biol. Chem. 268: 18300-18305. [0126]
9. Hughes, J., P. Boden, B. Costall, A. Domeney, E. Kelly, D. C.
Horwell, J. C. Hunter, R. D. Pinnock, and G. N. Woodruff. 1990.
Development of a class of selective cholecystokinin type B receptor
antagonists having potent anxiolytic activity. Proc. Natl. Acad.
Sci. USA 87: 6728-6732. [0127] 10. Sambrook, J., E. F. Fritsch, and
T. Maniatis. 1989. Molecular Cloning, A Laboratory Handbook. Cold
Spring Harbor Press. Cold Spring Harbor, N.Y. [0128] 11. Price, L.
A., E. M. Kajkowski, J. R. Hadcock, B. A. Ozenberger, and M. E.
Pausch. 1995. Yeast cell growth in response to agonist dependent
activation of a mammalian somatostatin receptor submitted. [0129]
12. Rose, M., F. Winston, and P. Hieter. 1990. Methods in Yeast
Genetics. Cold Spring Harbor-Press. Cold Spring Harbor, N.Y. [0130]
13. Blumer, K. J., J. E. Reneke, and J. Thorner. 1988. The STE2
gene product is the ligand-binding component of the .alpha.-factor
receptor of Saccharomyces cerevisiae. J. Biol. Chem. 263:
10836-10842. [0131] 14. Strnad, J., C. M. Eppler, M. Corbett, and
J. R. Hadcock. 1993. The rat SSTR2 somatostatin receptor subtype
is, coupled to inhibition of cyclic AMP accumulation. Biochem.
Biophys. Res. Comm. 191: 968-976. [0132] 15. King, K., H. G.
Dohlman, J. Thorner, M. G. Caron, and R. J. Lefkowitz. 1990.
Control of yeast mating signal transduction by a mammalian
.beta..sub.2-adrenergic receptor and G.sub.s .alpha. subunit.
Science 250: 121-123. [0133] 16. Weiner, J. L., C. Guttierez-Steil,
and K. J. Bloomer. 1993. Disruption of receptor-G protein coupling
in yeast promotes the function of an SST2-dependent adaptation
pathway. J. Biol. Chem. 268: 8070-8077.
EXAMPLE 9
Functional Expression of a Rat Adenosine (A.sub.2a) Receptor in
Yeast
[0134] Adenosine, as well as ATP and related purinergic compounds,
function as both neurohormonal agents and autocoids regulating the
process of cell to cell communication (1). In this role, adenosine
regulates a broad range of physiological functions including heart
rate and contractility, smooth muscle tone, sedation, release of
neurotransmitters, platelet function, lipolysis, kidney and white
blood cell action. Adenosine promotes its effects through the
action of cell surface receptors which can be classified using
pharmacological criteria into three subtypes, A.sub.1, A.sub.2a and
A.sub.2b, and A.sub.3. Molecular cloning efforts have identified
cDNAs encoding G protein-coupled adenosine A.sub.1 (2-5), A.sub.2a
and A.sub.2b (6-9), and A.sub.3 receptors (10). Functional
expression of adenosine receptors in yeast should permit rapid
screening for new compounds with adenosine agonist and antagonist
properties and facilitate molecular characterization of structural
aspects of the adenosine receptors required for rational design of
new adenosine ligands.
Materials and Methods
[0135] Plasmid constructions. All molecular biological
manipulations were performed according to standard procedures (11).
The rat A.sub.2a-adenosine receptor (-9) was cloned from rat brain
cDNA by PCR using oligonucleotide primers that introduce BamHI
sites at 5' and 3' ends (5' G A A G A T C T A A A A A A T G G G C T
C C T C G T G T A C, 3' ACATGCATGCAGATCTTCAGGAAGGGGCAAACTC). The
A.sub.2a-adenosine receptor expression plasmid, pJH21, was
constructed by inserting the BglII-digested PCR fragment in the
correct orientation into BamHI cut pMP3 (12). For constitutive
expression of the A.sub.2a-adenosine receptor in glucose-containing
medium, the expression vector, pLP100, was constructed. DNA
fragments encoding transcriptional regulatory sequences from the
ADH1 gene (&) were amplified by PCR and inserted into pRS426.
An ADH1 transcriptional terminator fragment was amplified from
yeast genomic DNA (YPH501, Stratagene) using synthetic
oligonucleotides that add 5' XhoI (TTTCTCGAGCGAATTTCTTATGATTT) and
3' KpnI (TTTGGTACCGGGCCCGGACGGATTACAACAGGT) sites. An ADH1 promoter
fragment was amplified from yeast genomic DNA using synthetic
oligonucleotides that add 5' SacI GGGAGCTCTGATGGTGGTACATAACG) and
3' BamHI (GGGGGATCCTGTATATGAGATAGTTGA) sites. The
A.sub.2a-adenosine receptor expression plasmid, pLP116, was
constructed by inserting a PCR fragment encoding the
A.sub.2a-adenosine receptor amplified using oligonucleotides that
add 5' BglII (AAAGATCTAAAATGGGCTCCTCGGTGTAC) and 3' SalI
(AAGTCGACTCAGGAA GGGGCAAACTC) sites BamHI-SalI cut LP100. The G
protein expression plasmids used in this study were constructed by
replacing DNA sequences encoding the 47 carboxy-terminal amino
acids of GPAL in pLP83 (12) with those of G. (pLP122) and
G.alpha..sub.i2 (pLP121).
[0136] Strain constructions. Yeast strains were constructed, and
growth media and culture conditions formulated according to
standard procedures (13). DNA-mediated transformation of yeast was
carried out using the lithium acetate method. The yeast strains
used as the basis for all experiments described in this report were
constructed by sequentional insertional deletion using recombinant
alleles. Yeast strains that express the rat A.sub.2a-adenosine
receptor were constructed by sequential DNA-mediated transformation
of LY296 (MATa ura3-52 trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1
ade2-101 lys2-801 gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3
sst2.DELTA.A.DELTA.E2, ref. 12) with A.sub.2a-adenosine receptor
expression plasmids followed by the G.alpha. protein expression
plasmids described above.
[0137] Radiolabeled agonist saturation binding assays. Crude yeast
membrane extracts from late log phase cultures were prepared by
glass-bead lysis and centrifugation at 40,000.times.g following a
published procedure (14). The protein content of crude membrane
fractions was measured using the Biorad protein assay kit according
to manufacturers instructions. Radioligand binding assays were
conducted according to Strnad et al. (15) using .sup.3H-NECA
(Amersham). Non-specific binding was defined as that observed in
the presence of 1 .mu.M NECA. Negligible specific binding was
observed in membrane fractions made from cells lacking
A.sub.2a-adenosine receptor (data not shown).
[0138] Bioassay. Functional assay of the A.sub.2a-adenosine
receptor expressed in yeast was accomplished using a modification
of a standard procedure (12). Yeast strains were grown overnight in
2 ml synthetic complete liquid medium containing glucose (2%) and
lacking uracil and tryptophan (SCD-ura-trp) medium, washed to
remove residual glucose and grown overnight in 5 ml SC Galactose
(2%)-ura-trp liquid medium. Molten (50.degree. C.) SC Galactose
(2%)-ura-trp-his agar medium (30 ml, adjusted to pH 6.8 by addition
of concentrated KOH or NH.sub.2OH prior to autoclaving) containing
5 mM 3-aminotriazole (Sigma) was inoculated with the overnight
culture (2.times.10.sup.4 cells/ml) and plated in square (9.times.9
cm) petri plates. For expression of the A.sub.2a-adenosine receptor
in glucose-containing medium, samples were removed from the first
overnight culture and assayed in agar medium composed as above with
glucose (2%) replacing galactose. Sterile filter disks were placed
on the surface of the solidified agar and saturated with 10 .mu.l
of DMSO containing the indicated amounts of the designated
compounds. Plates were incubated at 30.degree. C. for 3 days.
Adenosine ligands CGS-21680, NECA, and DPMA were purchased from
RBI. Somatostatin. (S-14) and met-enkephalin were from Bachem.
Oxymetazoline, isoproterenol, and carbachol were from Sigma.
Results
[0139] Adenosine agonist binding to the rat A.sub.2a-adenosine
receptor expressed in yeast. High level functional expression of
the A.sub.2a-adenosine receptor in yeast was a necessary
prerequisite to the development of a useful bioassay. In plasmid
pJH21, the rat A.sub.2a-adenosine receptor cDNA was placed under
the control of the inducible GAL1 promoter. This construct also
confers inducible overexpression of Gal4p, the transcriptional
activating protein for galactose-inducible genes, resulting in
significantly elevated levels of receptor protein in crude membrane
fractions compared to receptor expressed from a plasmid lacking
GAL4 sequences (data not shown). Plasmid pLP116 confers high level
constitutive expression of the A.sub.2a-adenosine receptor under
the control of the ADH1 promoter. In both plasmids, DNA sequences
encoding the A.sub.2a-adenosine receptor were introduced without
modification of the protein coding sequences. Previously, King et
al. reported that replacement of the amino-terminal chain of the
.beta..sub.2-adrenergic receptor with equivalent STE2 sequence was
necessary for efficient receptor expression in yeast (16). In
contrast, functional expression of the A.sub.2a-adenosine, receptor
in yeast does not require addition of any yeast sequences to the
amino-terminus. A chimeric G.alpha. protein composed of the
proposed amino-terminal .beta..gamma.-interaction domain from Gpalp
and a carboxy-terminal receptor interaction domain from rat
G.alpha..sub.s (pLP122) under the control of the GPA1 promoter was
constructed. Yeast strains that contain expressed
A.sub.2a-adenosine receptor and chimeric G.alpha. protein were
assembled by transformation of a yeast strain (LY296) modified by
deletion of genes encoding components of the mating signal
transduction pathway with A.sub.2a-adenosine receptor and Gm
protein expression plasmids.
[0140] Most G protein-coupled receptors exhibit both high and low
agonist-dependent affinity states. High-affinity agonist binding is
dependent on functional association of receptor with a
heterotrimeric G protein. If the receptor does not associate with,
or is uncoupled from the G protein, agonist binding will be of low
affinity and undetectable in radiolabeled agonist saturation
binding assays. In crude yeast membrane fractions from cells
bearing pLP116 and pLP122 (LY626), the agonist .sup.3H-NECA bound
to the A.sub.2a-adenosine receptor with high affinity and in a
saturable manner, (FIG. 20) demonstrating that (1) a functional
ligand-binding conformation of the A.sub.2a-adenosine receptor was
expressed in yeast, and (2) the receptor functionally associated
with the chimeric G.alpha. protein, resulting in a high-affinity
agonist binding state. The total number of .sup.3H-NECA binding
sites observed (B.sub.max=242 fmol/mg) exceeded values obtained for
the yeast .alpha.-mating pheromone receptor (200 fmol/mg, ref. 17).
The affinity of [.sup.3H] NECA for the A.sub.2a-adenosine receptor
in yeast membranes (Kd-8M) is equivalent to that observed in
mammalian cells indicating functional coupling between receptor and
G protein.
[0141] The A.sub.2a-adenosine receptor retained agonist selectivity
when expressed in yeast. A selective and sensitive bioassay was
designed using a yeast strain (LY595) bearing the above described
genetic modifications and plasmids conferring expression of the
A.sub.2a-adenosine receptor (pLP116) and GPAL (pLP83). A
dose-dependent growth response of LY595 cells was evident around
applied CGS-21680, an A.sub.2a-adenosine receptor selective
agonist. The growth response was significantly more robust than
that exhibited by cells responding to NECA and DPMA (FIG. 21). The
assay was selective: the diameter of the growth zones was
proportional to the reported affinity of the ligands for the
A.sub.2a-adenosine receptor (CGS-21680>NECA=DPMA) reflecting the
ability of the bioassay to discriminate between ligands of varying
potency. Detectable growth responses were not observed in response
to a variety of agonists selective for other G protein-coupled
receptors (somatostatin, met-enkephalin, oxymetazoline,
isoproterenol, carbachol), nor by yeast cells lacking the
A.sub.2a-adenosine receptor (data not shown). A detectable response
was observed to 10 .mu.g of CGS-21680.
Discussion
[0142] Multiple therapeutic opportunities exist for compounds that
modulate the function of the adenosine receptors (1). Adenosine
agonists may be useful in the treatment of epileptic seizure
episodes and in preventing neuronal damage in stroke and
neurodegenerative disorders. The antidysrhythmic action adenosine
suggests that adenosine agonists could be effective in the
treatment of complex tachycardia. A.sub.2 adenosine agonists have
potent sedative, anticonvulsant and anxiolytic activity.
A.sub.2a-adenosine selective agonists may be useful in stimulating
lipolysis in adipose tissue, making them useful as weight loss
treatments or antidiabetic agents and in the improvement of carcass
quality in agricultural animals. A.sub.1 adenosine antagonists may
be useful in treatment of acute renal dysfunction.
A.sub.3-antagonists may be useful in modulating mast cell
degranulation for treatment of inflamatory disorders, including
asthma.
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G. L. Stiles, and O. Civelli. 1992. Molecular cloning and
characterization of an adenosine receptor: The A3 adenosine
receptor. Proc. Natl. Acad. SCI. USA. 89: 7432-7436. [0153] 11.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Cloning, A Laboratory Handbook. Cold Spring Harbor Press. Cold
Spring Harbor, N.Y. [0154] 12. Price, L. A., E. M. Kajkowski, J. R.
Hadeock, B. A. Ozenberger, and M. R. Pausch. 1995. Yeast cell
growth in response to agonist dependent activation of a mammalian
somatostatin receptor. submitted. [0155] 13. Rose, M., F. Winston,
and P. Hieter. 1990. Methods in Yeast Genetics. Cold Spring Harbor
Press. Cold Spring Harbor, N.Y. [0156] 14. Blumer, K. J., J. E.
Reneke, and J. Thorner. 1988. The STE2 gene product is the
ligand-binding component of the .alpha.-factor receptor of
Saccharomyces cerevisiae. J. Biol. Chem. 263: 10836-10842. [0157]
15. Strnad, J., C. M. Eppler, M. Corbett, and J. R. Hadcock. 1993.
The rat SSTR2 somatostatin receptor subtype is coupled to
inhibition of cyclic AMP accumulation. Biochem. Biophys. Res. Comm.
191: 968-976. [0158] 16. King, R., H. G. Dohlman, J. Thorner, M. G.
Caron, and R. J. Lefkowitz. 1990. Control of yeast mating signal
transduction by a mammalian .beta..sub.2-adrenergic receptor and
G.sub.s .alpha. subunit. Science 250: 121-123. [0159] 17. Weiner,
J. L., C. Guttierez-Steil, and K. J. Blumer. 1993. Disruption of
receptor-G protein coupling in yeast promotes the function of an
SST2-dependent adaptation pathway. J. Biol. Chem. 268:
8070-8077.
EXAMPLE 8
Functional Expression of a Rat Somatostatin Subtype 5 Receptor in
Yeast
[0160] The cyclic tetradecapeptide somatostatin is a potent
inhibitor of secretion of several hormones, including growth
hormone from the pituitary, glucagon and insulin from the pancreas,
and gastrin from the gut. Somatostatin also acts as a
neurotransmitter and has been shown to have broad modulatory
effects in CNS and peripheral tissues (1). The effects of
somatostatin are transduced through binding of the hormone to
high-affinity, plasma membrane localized somatostatin (SSTR)
receptors (2). The SSTR's, encoded in five distinct subtypes
(SSTR1-5), which account in part for tissue-specific differences in
responses to somatostatin (3-10), comprise a subfamily of the
seven-transmembrane domain, G protein-coupled receptor superfamily
that mediates responses to a broad variety of extracellular
signals. Functional expression of SSTR5 in yeast should permit
rapid screening for new subtype-selective somatostatin agonists and
compounds with antagonist properties and facilitate molecular
characterization of structural aspects of the SSTR5 required for
rational design of new somatostatin ligands.
Materials and Methods
[0161] Plasmid constructions. All molecular biological
manipulations were performed according to standard procedures (11).
The rat SSTR5 (7) was cloned from rat genomic DNA by PCR using
oligonucleotide primers that introduce BglII sites at 5' and 3'
ends (5' AAAAAGATCTAAAATGGAGCCCCTCTCTCTG, 3' AGCAGATCTTCAGATC
CCAGAAGACAAC). The SSTR5 expression plasmid, pJH19, was constructed
by inserting the BglII-digested PCR fragment in the correct
orientation into BamHI cut pMP3 (12). The G.alpha. protein
expression plasmids used in this study were constructed by
replacing DNA sequences encoding the 47 carboxy-terminal amino
acids of GPAL in pLP83 (12) with those of G.alpha..sub.s (pLP122),
G.alpha..sub.i2 (pLP121).
[0162] Strain constructions. Yeast strains were constructed, and
growth media and culture conditions formulated according to
standard procedures (13). DNA-mediated transformation of yeast was
carried out using the lithium acetate method. The yeast strains
used as the basis for all experiments described in this report were
constructed by sequential insertional deletion using recombinant
alleles. Yeast strains that express SSTR5 were constructed by
sequential DNA-mediated transformation of LY296 (MATa ura3-52
trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1 ade2-101 lys2-801
gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3 sst2.DELTA.ADE2, ref. 12)
with pJH19 followed by the G.alpha. protein expression plasmids
described above.
[0163] Bioassay. Functional assay of SSTR5 expressed in yeast was
accomplished using modification of a standard-procedure (12). Yeast
strains were grown overnight in 2 ml synthetic complete liquid
medium containing glucose (2%) and lacking uracil and tryptophan
(SCD-ura-trp) medium, washed to remove residual glucose, and grown
overnight in 5 ml SC Galactose (2%)-ura-trp liquid medium. Molten
(55.degree. C.) SC Galactose (2%)-ura-trp-his agar medium (30
.mu.l, adjusted to pH 6.8 by addition of concentrated KOH or
NH.sub.4OH prior to autoclaving) was inoculated with the overnight
culture (2.times.10.sup.4 cells/ml) and plated in square (9.times.9
cm) petri plates. Sterile filter disks were placed on the surface
of the solidified agar and saturated with 10 .mu.l of sterile water
containing the indicated amounts of the designated compounds.
Plates were incubated at 30.degree. C. for 3 days. Somatostatin
(S-14, S-28), met-enkephalin, and CCK-8 were from Bachem.
Oxymetazoline, isoproterenol, and carbachol were from Sigma.
Results
[0164] Somatostatin dependent growth response of yeast cells
expressing the SSTR5. High level functional expression of the SSTR5
in yeast was a necessary prerequisite to the development of a
useful bioassay. The SSTR5 cDNA was placed under the control of the
GAL1 promoter in plasmid pJH19. This construct also confers
inducible overexpression of Gal4p, the transcriptional activating
protein for galactose-inducible genes, resulting in significantly
elevated levels of receptor protein in crude membrane fractions
compared to receptor expressed from a plasmid lacking GAL4
sequences (data not shown). SSTR5 sequences were introduced into
pJH19 without modification of the protein coding sequences.
Previously, King et al. reported that replacement of the
amino-terminal domain of the .beta..sub.2-adrenergic receptor with
equivalent STE2 sequence was necessary for efficient receptor
expression in yeast (15). In contrast, functional expression of
SSTR5 in yeast does not require addition of any yeast sequences to
the amino-terminus. A chimeric G.alpha. protein composed of the
proposed amino-terminal .beta..sub.2-interaction domain from Gpalp
and a carboxy-terminal receptor interaction domain from rat
G.alpha..sub.i2 (pLP121) under the control of the GPA1 promoter was
constructed. A yeast strain (LY620) that contains expressed SSTR5
and chimeric G.alpha. protein was assembled by transformation of a
yeast strain (LY296) modified by deletion of genes encoding
components of the mating signal transduction pathway with SSTR5
(pJH19) and G.alpha. protein expression (pLP121) plasmids. A
dose-dependent growth response of LY620 cells was evident around
applied S-14 (FIG. 22). Detectable growth responses were not
observed in response to a variety of agonists selective for other G
protein-coupled receptors (CCK-8, met-enkephalin, oxymetazoline,
isoproteranol, carbachol), nor by yeast cells lacking the SSTR5
(data not shown). A detectable response was observed to 60 nmol of
S-14.
REFERENCES CITED IN THIS EXAMPLE
[0165] 1. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J.
Rivier and R. Guillemin. 1973. Hypothalamic polypeptide that
inhibits the secretion of immunoreactive pituitary growth hormone.
Science 129: 77-79. [0166] 2. Reisine, T. and G. I. Bell 1993.
Molecular biology of somatostatin receptors. Trends Neurosci, 16:
34-38. [0167] 3. Meyerhof, W., B.-J. Paust, C. Schonrock, and D.
Richter. 1991. Cloning of a cDNA encoding a novel putative G
protein-coupled receptor expressed in specific rat brain regions.
DNA Cell Biol. 10: 689-694. [0168] 4. Bruno, J. F., Y. Xu, J. Song,
and M. Berelowitz. 1992. Molecular cloning and functional
expression of a brain specific somatostatin receptor. Proc. Natl.
Acad. Sci. USA 89: 11151-11155. [0169] 5. Kluxen, F.-W., C. Bruns,
and E. Lubbert. 1992. Expression cloning of a rat brain
somatostatin receptor. Proc. Natl. Acad. Sci. USA 89: 4618-4622.
[0170] 6. Li, X.-J., M. Forte, R. A. North, C. A. Ross, and S. H.
Snyder. 1992. Cloning and expression of a rat somatostatin receptor
enriched in brain. J. Biol. Chem. 267: 21307-21312. [0171] 7.
O'Carrol, A.-M., S. J. Lolait, M. Konig, and L. Mahan. 1992.
Molecular cloning and expression of a pituitary somatostatin
receptor with preferential affinity for somatostatin-28. Mol.
Pharmocol. 42: 939-946. [0172] 8. Yasuda, X., S. Rens-Domiano, C.
D. Breder, S. F. Law, C. B. Saper, T. Reisine and G. I. Bell. 1992.
Cloning of a novel somatostatin receptor, SSTR3, coupled to
adenylylcyclase. J. Biol. Chem. 28: 20422-20428. [0173] 9. Yamada,
Y., S. R. Post, K. Wang, E. S. Tager, G. I. Bell and S. Seino.
1992. Cloning and functional characterization of a family of human
and mouse somatostatin receptors expressed in brain,
gastrointestinal tract, and kidney. Proc. Natl. Acad. Sci. USA 89:
251-255. [0174] 10. Strnad, J., C. M. Eppler, M. Corbett, and J. R.
Hadcock. 1993. The rat SSTR2 somatostatin receptor subtype is
coupled to inhibition of cyclic AMP accumulation. Biochem. Biophys.
Res. Comm. 191: 968-976. [0175] 11. Sambrook, J., E. F. Fritsch,
and T. Maniatis. 1989. Molecular Cloning, A Laboratory Handbook.
Cold Spring Harbor Press. Cold Spring Harbor, N.Y. [0176] 12.
Price, L. A., E. M. Kajkowski, J. R. Hadcock, B. A. Ozenberger, and
M. E. Pausch. 1995. Yeast cell growth in response to agonist
dependent activation of a mammalian somatostatin receptor.
submitted. [0177] 13. Rose, M., F. Winston, and P. Hieter. 1990.
Methods in Yeast Genetics. Cold Spring Harbor Press. Cold Spring
Harbor, N.Y. [0178] 14. Blumer, K. J., J. E. Reneke, and J.
Thorner. 1988. The STE2 gene product is the ligand-binding
component of the .alpha.-factor receptor of Saccharomyces
cerevisiae. J. Biol. Chem. 263: 10836-10842. [0179] 15. King, K.,
H. G. Dohlman, J. Thorner, M. G. Caron, and R. J. Lefkowitz. 1990.
Control of yeast mating signal transduction by a mammalian
.beta..sub.2-adrenergic receptor and G.alpha. .alpha. subunit.
Science 250: 121-123. [0180] 16. Weiner, J. L., C. Guttierez-Steil,
and K. J. Blumer. 1993. Disruption of receptor-G protein coupling
in yeast promotes the function of an SST2-dependent adaptation
pathway. J. Biol. Chem. 268: 8070-8077.
EXAMPLE 10
Functional Expression of the Porcine Somatostatin Subtype 2 (SSTR2)
Receptor in Yeast
[0181] The cyclic tetradecapeptide somatostatin is a potent
inhibitor of secretion of several hormones, including growth
hormone from the pituitary, glucagon and insulin from the pancreas,
and gastrin from the gut. Somatostatin also acts as a
neurotransmitter and has been shown to have broad modulatory
effects in CNS and peripheral tissues (1). The effects of
somatostatin are transduced through binding of the hormone to
high-affinity, plasma membrane localized somatostatin (SSTR)
receptors (2). The SSTR's, encoded in five distinct subtypes
(SSTR1-5), which account in part for tissue-specific differences in
responses to somatostatin (3-11), comprise a subfamily of the
seven-transmembrane domain, G protein-coupled receptor superfamily
that mediates responses to a broad variety of extracellular
signals. Functional expression of porcine SSTR2 in yeast should
permit rapid screening for new species and subtype-selective
somatostatin agonists and compounds with antagonist properties and
facilitate molecular characterization of structural aspects of the
porcine SSTR2 required for rational design of new somatostatin
ligands. Compounds identified in high-throughput, mechanism-based
screens represent leads for new growth-enhancing agents for use in
pigs.
Materials and Methods
[0182] Plasmid constructions. All molecular biological
manipulations were performed according to standard procedures (12).
The porcine SSTR2 (11) was cloned from a human brain cDNA library
by PCR using oligonucleotide primers that introduce BglII sites at
5' and 3' ends (5' AAAAGATCTAAAATGTCCATTCCATTTGAC, 3'
AAAAGGTACCAGATCTTCAGATACTGGTTTGGAG). The porcine SSTR2 expression
plasmid, pJH18, was constructed by inserting the BglII-digested PCR
fragment in the correct orientation into BamHI cut pMP3 (13).
[0183] Strain constructions. Yeast strains were constructed, and
growth media and culture conditions formulated according to
standard procedures (14). DNA-mediated transformation of yeast was
carried out using the lithium acetate method. The yeast strains
used as the basis for all experiments described in this report were
constructed by sequential insertional deletion using recombinant
alleles. Yeast strains that express porcine SSTR2 were constructed
by sequential DNA-mediated transformation of LY296 (MATa ura3-52
trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1 ade2-101 lys2-801
gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3 sst2.DELTA.ADE2, ref. 13)
with the chimeric G.alpha..sub.i2 protein expression plasmid, pLP82
(13), followed by pJH18 or pJH17.
[0184] Radiolabeled agonist saturation binding assays. Crude yeast
membrane extracts from late log phase cultures were prepared by
glass-bead lysis and centrifugation at 40,000.times.g following a
published procedure (15). The protein content of crude membrane
fractions was measured using the Biorad protein assay kit according
to manufacturers instructions. Radioligand binding assays were
conducted according to Strnad et al. (10) using radiolabeled
somatostatin (.sup.125I-tyr.sup.11-S-14, Amersham). Non-specific
binding was defined as that observed in the presence of 1 .mu.M
S-14. Negligible specific binding was observed in membrane
fractions made from cells lacking porcine SSTR2 (data not
shown).
[0185] Bioassay. Functional assay of the porcine SSTR2 expressed in
yeast was accomplished using modification of a standard procedure.
(13). Yeast strains were grown overnight in 2 ml synthetic complete
liquid medium containing glucose (2%) and lacking uracil and
tryptophan (SCD-ura-trp) medium, washed to remove residual glucose,
and grown overnight in 5 ml SC Galactose (2%)-ura-trp liquid
medium. Molten (55.degree. C.) SC Galactose (2%)-ura-trp-his agar
medium (30 ml, adjusted to pH 6.8 by addition of concentrated KOH
or NH.sub.4OH prior to autoclaving) was inoculated with the
overnight culture (2.times.10.sup.4 cells/ml) and plated in square
(9.times.9 cm) petri plates. Sterile filter disks were placed on
the surface of the solidified agar and saturated with 10 .mu.l of
sterile water containing the indicated amounts of the designated
compounds. Plates were incubated at 30.degree. C. for 3 days.
Somatostatin (S-14, S-28), met-enkephalin were from Bachem.
Oxymetazoline, isoproterenol, and carbachol were from Sigma. MK678
and sandostatin were prepared synthetically.
Results
[0186] Somatostatin binding to, the porcine SSTR2 expressed in
yeast. High level functional expression of the porcine SSTR2 in
yeast was a necessary prerequisite to the development of a useful
bioassay. The porcine SSTR2 cDNA was placed under the control of
the GAL1 promoter in plasmid pJH17 and 18. These constructs also
confer inducible overexpression of Gal4p, the transcriptional
activating protein for galactose-inducible genes, resulting in
significantly elevated levels of receptor protein in crude membrane
fractions compared to receptor expressed from, a plasmid lacking
GAL4 sequences (data not shown). The porcine SSTR2 sequences were
introduced into pJH18 without modification of the protein coding
sequences. Previously, King et al. reported that replacement of the
amino-terminal domain of the .beta..sub.2-adrenergic receptor with
equivalent STE2 sequence was necessary for efficient receptor
expression in yeast (16). In contrast, functional expression of
porcine SSTR2 in yeast does not require addition of any yeast
sequences to the amino-terminus. A chimeric G.alpha. protein
composed of the proposed amino-terminal .beta..gamma.-interaction
domain from Gpalp and a carboxy-terminal receptor interaction
domain from rat G.alpha..sub.i2 (pLP82) under the control of the
GPA1 promoter was constructed. Yeast strains that contain expressed
porcine SSTR2 and chimeric G.alpha. protein were assembled by
transformation of a yeast strain (LY296) modified by deletion of
genes encoding components of the mating signal transduction pathway
with porcine SSTR2 (pJH17, pJH18) and G.alpha. protein expression
(pLP82) plasmids.
[0187] Most G protein-coupled receptors exhibit both high and low
agonist-dependent affinity states. High-affinity agonist binding is
dependent on functional association of receptor with a
heterotrimeric G protein. If the receptor does not associate with,
or is uncoupled from the G protein, agonist binding will be of low
affinity and undetectable in radiolabeled agonist saturation
binding assays. In crude yeast membrane fractions from cells
bearing pJH17, the agonist .sup.125I-tyr.sup.11-S-14 bound to the
porcine SSTR2 with high affinity and in a saturable manner,
demonstrating that (1) a functional ligand-binding conformation of
the porcine SSTR2 was expressed in yeast, and (2) the receptor
functionally associated with the chimeric G.alpha. protein,
resulting in a high-affinity agonist binding state. The total
number of .sup.125I-tyr.sup.11-S-14 binding sites observed
(B.sub.max=146 fmol/mg) was consistent with values obtained for the
yeast .alpha.-mating pheromone receptor (200 fmol/mg, ref. 17).
[0188] The porcine SSTR2 retained agonist selectivity when
expressed in yeast. A selective and sensitive bioassay was designed
using a yeast strain (LY474) bearing the above described genetic
modifications and plasmids conferring expression of the porcine
SSTR2 (pJH18) and a Gpal-G.alpha..sub.i2 chimeric protein (pLP82).
A dose-dependent growth response of LY474 cells was evident around
applied S-14, MK678, and sandostatin (FIG. 23). The assay was
selective: the diameter of the growth zones was proportional to the
reported affinity of the ligands for the porcine SSTR2
(S-14=MK678>sandostatin) reflecting the ability of the bioassay
to discriminate between ligands of varying potency (18). Detectable
growth responses were not observed in response to a variety of
agonists selective for other G protein-coupled receptors
(met-enkephalin, oxymetazoline, isoproterenol, carbachol), nor by
yeast cells lacking the porcine SSTR2 (data not shown). A
detectable response was observed to as little as 60 pmol of S-14,
illustrating the exquisite sensitivity of the bioassay.
REFERENCES CITED IN THIS EXAMPLE
[0189] 1. Brazeau, P., W. Vale, R. Burgus, N. Ling, M. Butcher, J.
Rivier and R. Guillemin. 1973. Hypothalamic polypeptide that
inhibits the secretion of immunoreactive pituitary growth hormone.
Science 129: 77-79. [0190] 2. Reisine, T. and G. I. Bell 1993.
Molecular biology of somatostatin receptors. Trends Neurosci. 16:
34-38. [0191] 3. Meyerhof, W., H.-J. Paust, C. Schonrock, and D.
Richter. 1991. Cloning of a cDNA encoding a novel putative G
protein-coupled receptor expressed in specific rat brain regions.
DNA Cell Biol. 10: 689-694. [0192] 4. Bruno, J. F., Y. Xu, J. Song,
and M. Berelowitz. 1992. Molecular cloning and functional
expression of a brain specific somatostatin receptor. Proc. Natl.
Acad. Sci. USA 89: 11151-11155. [0193] 5. Kluxen, F.-W., C. Bruns,
and H. Lubbert. 1992. Expression cloning of a rat brain
somatostatin receptor. Proc. Natl. Acad. Sci. USA 89: 4618-4622.
[0194] 6. Li, X.-J., M. Forte, R. A. North, C. A. Ross, and S. S.
Snyder. 1992. Cloning and expression of a rat somatostatin receptor
enriched in brain. J. Biol. Chem. 267: 21307-21312. [0195] 7.
O'Carrol, A.-M., S. J. Lolait, M. Konig, and L. Mahan. 1992.
Molecular cloning and expression of a pituitary somatostatin
receptor with preferential affinity for somatostatin-28. Mol.
Pharmocol. 42: 939-946. [0196] 8. Yasuda, K., S. Rens-Domiano, C.
D. Breder, S. F. Law, C. B. Saper, T. Reisine and G. I. Bell. 1992.
Cloning of a novel somatostatin receptor, SSTR3, coupled to
adenylylcyclase. J. Biol. Chem. 28: 20422-20428. [0197] 9. Yamada,
Y., S. R. Post, K. Wang, H. S. Tager, G. I. Bell and S. Seino.
1992. Cloning and functional characterization of a family of human
and mouse somatostatin receptors expressed in brain,
gastrointestinal tract, and kidney. Proc. Natl. Acad. Sci. USA 89:
251-255. [0198] 10. Strnad, J., C. M. Eppler, M. Corbett, and J. R.
Hadcock. 1993. The rat SSTR2 somatostatin receptor subtype is
coupled to inhibition of cyclic AMP accumulation. Biochem. Biophys.
Res. Comm. 191: 968-976. [0199] 11. Matsumoto, K., Y. Yokogoshi, Y.
Fujinaka, C. Z. Xhang, and S. Saito. 1994. Molecular cloning and
sequencing, of porcine somatostatin receptor 2. Biochem. Biophys.
Res. Comm. 199: 298-305. [0200] 12. Sambrook, J., E. F. Fritsch,
and T. Maniatis. 1989. Molecular Cloning, A Laboratory Handbook.
Cold Spring Harbor Press. Cold Spring Harbor, N.Y. [0201] 13.
Price, L. A., E. M. Kajkowski, J. R. Hadcock, B. A. Ozenberger, and
M. H. Pausch. 1995. Yeast cell growth in response to agonist
dependent activation of a mammalian somatostatin receptor.
submitted. [0202] 14. Rose, M., F. Winston, and P. Hieter. 1990.
Methods in Yeast Genetics. Cold Spring Harbor Press. Cold Spring
Harbor, N.Y. [0203] 15. Blower, K. J., J. E. Reneke, and J.
Thorner. 1988. The STE2 gene product is the ligand-binding
component of the .alpha.-factor receptor of Saccharomyces
cerevisiae. J. Biol. Chem. 263: 10836-10842. [0204] 16. King, K.,
H. G. Dohlman, J. Thorner, M. G. Caron, and R. J. Lefkowitz. 1990.
Control of yeast mating signal transduction by a mammalian
.sub..beta.2-adrenergic receptor and G.sub.s .alpha. subunit.
Science 250: 121-123. [0205] 17. Weiner, J. L., C. Guttierez-Steil,
and K. J. Blumer. 1993. Disruption of receptor-G protein coupling
in yeast promotes the function of an SST2-dependent adaptation
pathway. J. Biol. Chem. 268: 8070-8077. [0206] 18. Marbach, P., W.
Bauer, and U. Briner. 1988. Structure-function relationships of
somatostatin analogs. Horm Res. 29: 54-58.
EXAMPLE 11
Deletion of MSG5 Increases the Sensitivity of Response to
Agonist
[0207] The responsiveness of a signal transduction system to a
persistent stimulus diminishes with time. This phenomenon, known as
desensitization or adaptation, is a universal characteristic of
signal response systems. Several molecular mechanisms for
adaptation have been described for the yeast mating signal
transduction pathway (1). Mutations in the SST2 gene confer defects
in adaptation and increased mating pheromone sensitivity (2, 3):
The response to applied somatostatin by yeast cells that
functionally express the rat SSTR2 is greatly increased in sst2
mutant cells (4). Mutations in others genes whose products play a
role in the adaptation response would be expected to have similar
effects. Mutations in the MSG5 gene, which encodes a putative
protein tyrosine phosphatase, cause increased sensitivity to mating
pheromone (5). In this study, deletion of MSG5 in cells that
express the rat SSTR2 greatly increases sensitivity to
somatostatin. The effect of the MSG5 mutation is additive with an
SST2 deletion mutation. The double mutant sst2 msg5 cells form the
basis of an extremely sensitive bioassay for compounds that
interact with G protein-coupled receptors and G proteins.
Materials and Methods
[0208] Strain constructions. All molecular biological manipulations
were performed according to standard procedures (6). Yeast strains
were constructed, and growth media and culture conditions
formulated according to standard procedures (7). DNA-mediated
transformation of yeast was carried out using the lithium acetate
method. The yeast strains used in these experiments were
constructed using the recombinant msg5.DELTA.LEU2 allele in pS/PDel
and multicopy YEpMSG5 (5). Yeast strains bearing altered MSG5
levels were constructed by DNA-mediated transformation of LY268
(MATa ura3-52 trplD63 his3D200 leu2.DELTA.1 ade2-101 lys2-801
gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3 sst2.DELTA.ADE2, pJH2,
pLP82) yielding MPY459 (LY268 msg5.DELTA.LEU2 sst2.DELTA.ADE2) and
MPY467 (LY268 YEpMSG5) and LY288 (LY268 SST2) yielding MPY458
(LY288 msg5.DELTA.LEU2 SST2) and MPY466 (LY288 YEpMSG5) (4).
[0209] Bioassay. Functional bioassay of the rat SSTR2 expressed in
yeast was accomplished using modification of a standard procedure
(4). Yeast strains were grown overnight in 2 ml synthetic complete
liquid medium containing glucose (2%) and lacking uracil,
tryptophan and leucine (SCD-ura-trp-leu) medium, washed to remove
residual glucose, and grown overnight in 5 ml SC Galactose
(2%)-ura-trp-leu liquid medium. Molten (55.degree. C.) SC Galactose
(2%)-ura-trp-leu-his agar medium (35 ml, adjusted to pH 6.8 by
addition of concentrated NH.sub.4OH prior to autoclaving) was
inoculated with the overnight culture (2.times.10.sup.4 cells/ml)
and plated in square (9.times.9 cm) petri plates. Sterile filter
disks were placed on the surface of the solidified agar and
saturated with 10 .mu.l of sterile water containing the indicated
amounts of the somatostatin (S-14). Plates were incubated at
30.degree. C. for 3 days. Somatostatin (S-14) was from Bachem.
Results
[0210] Deletion of MSG5 promotes increased sensitivity to ligand.
The effects of alterations in the expression of the MSG5 gene
product were assessed by comparing the growth response to S-14 by
cells that express the rat SSTR2 (FIG. 24). Mutations that abolish
MSG5 adaptation function would be expected to increase the
sensitivity of the bioassay to applied S-14, while overexpression
of MSG5 should blunt the growth response. As expected, a
dose-dependent growth response to applied S-14 was observed for
LY288 (SST2 MSG5). Consistent with expectations, deletion of the
MSG5 gene in MPY458 causes a substantial improvement in sensitivity
of the bioassay. The growth response of MPY458 is comparable to
that exhibited by LY268 (sst2.DELTA.ADE2, MSG5). The effects of
mutations in both genes was observed in the double mutant MPY459
(msg5.DELTA.LEU2 sst2.DELTA.ADE2) which showed a further
improvement in the growth response overexpression of MSG5 in SST2
(MPY466) and sst2.DELTA.ADE2 (MPY467) strains severely reduced the
growth response.
REFERENCES CITED IN THIS EXAMPLE
[0211] 1. Sprague, G. F. and J. W. Thorner. 1992. Pheromone
response and signal transduction during the mating process of
Saccharomyces cerevisiae, 657-744. In J. R. Pringle, E. W. Jones
and J. R. Broach, Gene expression, 2. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. [0212] 2. Chan, R. K. and C. A.
Otte. 1982. Isolation and genetic analysis of Saccharomyces
cerevisiae mutants supersensitive to G1 arrest by a factor and a
factor pheromones. Mol. Cell. Biol. 2: 11-20. [0213] 3. Chan, R. K.
and C. A. Otte. 1982. Physiological characterization of
Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a
factor and a factor pheromones. Mol. Cell. Biol. 2: 21-29. [0214]
4. Price, L. A., E. M. Kajkovski, J. R. Hadcock, B. A. Ozenberger,
and M. H. Pauach. 1995. Yeast cell growth in response to agonist
dependent activation of a mammalian somatostatin receptor.
submitted. [0215] 5. Doi, K., A. Gartner, G. Ammerer, B. Errede, B.
Shinkawa, K. Sugimoto, and K. Matsumoto. 1994. MSG5; a novel
protein phosphatase promotes adaptation to pheromone response in S.
cerevisiae. EMBO J. 13: 61-70. [0216] 6. Sambrook, J., E. F.
Fritsch, and T. Maniatis. 1989. Molecular Cloning, A Laboratory
Handbook. Cold Spring Harbor Press. Cold Spring Harbor, N.Y. [0217]
7. Rose, M., F. Winston, and P. Hieter. 1990. Methods in Yeast
Genetics. Cold Spring Harbor Press. Cold Spring Harbor, N.Y.
EXAMPLE 12
Functional Expression of a Human Growth Hormone Release Factor
(GRF) Receptor in Yeast
[0218] The growth hormone release factor (GRF) is a potent
stimulator of secretion of growth hormone from the pituitary (1).
The effects of GRF are transduced through binding of the hormone to
high-affinity, plasma membrane localized GRF receptors. The GRF
receptor and related secretin-class receptors comprise a subfamily
of the seven-transmembrane domain, G protein-coupled receptor
superfamily that mediates responses to a broad variety of
extracellular signals, and are distinguished by the presence of a
large amino-terminal ligand-binding domain (2). Functional
expression of the human GRF receptor (3) in yeast should permit
rapid screening for new species-selective agonists and facilitate
molecular characterization of structural aspects of the GRF
receptor required for rational design of new GRF receptor ligands.
GRF agonists represent a new class of growth promoting agents for
use in agricultural animals and may find human therapeutic
application in the management of growth of children of short
stature.
Materials and Methods
[0219] Plasmid constructions. All molecular biological
manipulations were performed according to standard procedures (4).
The human GRF receptor (3) was cloned from a human brain cDNA
library by PCR using oligonucleotide primers that introduce BamHI
sites at 5' and 3' ends (5' ATAGGATCCAAAATGGACCGCCGGATGTGGGGG, 3'
ATATGGATCCCTAGCACATAGATGTCAG). The GRF receptor expression plasmid,
pJH25, was constructed by inserting the BamHI-digested PCR fragment
in the correct orientation into BamHI cut pMP3 (5). The
G.sub..alpha. protein expression plasmids used in this study were
constructed by replacing DNA sequences encoding the 47
carboxy-terminal amino acids of GPA1 in pLP83 (12) with those of
G.alpha..sub.s (pLP122).
[0220] Strain constructions. Yeast strains were constricted, and
growth media and culture conditions formulated according to
standard procedures (6). DNA-mediated transformation of yeast was
carried out using the lithium acetate method. The yeast strains
used as the basis for all experiments described in this report were
constructed by sequential insertional deletion using recombinant
alleles. Yeast strains that express human GRF receptor were
constructed by sequential DNA-mediated transformation of LY296
(MATa ura3-52 trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1 ade2-101
lys2-801 gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3 sst2.DELTA.ADE2,
ref. 5) with pJH25, followed by the chimeric G.sub..alpha.s protein
expression plasmid, pLP122 (5).
[0221] Bioassay. Functional assay of the human GRF receptor
expressed in yeast was accomplished using modification of a
standard procedure (5). Yeast strains were grown overnight in 2 ml
synthetic complete liquid medium containing glucose (2%) and
lacking uracil and tryptophan (SCD-ura-trp) medium, washed to
remove residual glucose, and grown overnight in 5 ml SC Galactose
(2%)-ura-trp liquid medium. Molten (55.degree. C.) SC Galactose
(2%)-ura-trp-his agar medium (30 ml, adjusted to pH 6.8 by addition
of concentrated KOH or NH.sub.4OH prior to autoclaving) was
inoculated with the overnight culture (2.times.10.sup.4 cells/ml)
and plated in square (9.times.9 cm) petri plates. Sterile filter
disks were placed on the surface of the solidified agar and
saturated with 10 .mu.l of sterile water containing the indicated
amounts of the designated compounds. Plates were incubated at
30.degree. C. for 3 days. Human GRF (hGRF (1-29)-NH.sub.2),
(D-ala.sup.2)-hGRF (1-29)--NH.sub.2, and met-enkephalin were from
Bachem. Oxymetazoline, isoproterenol, and carbachol were from
Sigma.
Results
[0222] GRF binding to the human GRF receptor expressed in yeast.
High level functional expression of the human GRF receptor in yeast
was a necessary prerequisite to the development of a useful
bioassay. The GRF receptor cDNA was placed under the control of the
GAL1 promoter in plasmid pJH25. These constructs also confer
inducible overexpression of Gal4p, the transcriptional activating
protein for galactose-inducible genes, resulting in significantly
elevated levels of receptor protein in crude membrane fractions
compared to receptor expressed from a plasmid lacking GAL4
sequences (data not shown). The GRF receptor sequences were
introduced into pJH25 without modification of the protein coding
sequences. Previously, King et al. reported that replacement of the
amino-terminal domain of the b.sub.2-adrenergic receptor with
equivalent STE2 sequence was necessary for efficient receptor
expression in yeast (9). In contrast, functional expression of GRF
receptor in yeast does not require addition of any yeast sequences
to the amino-terminus. A chimeric G.alpha. protein composed of the
proposed amino-terminal .beta..gamma.-interaction domain from Gpalp
and a carboxy-terminal receptor interaction domain from rat
G.sub.as (pLP122) under the control of the GPA1 promoter was
constructed. Yeast strains that contain expressed GRF receptor and
chimeric G.sub.a protein were assembled by transformation of a
yeast strain (LY296) modified by deletion of genes encoding
components of the mating signal transduction pathway with human GRF
receptor (pJH25) and chimeric Gpal-G.alpha..sub.s protein
expression (pLP122) plasmids.
[0223] The human GRF receptor retained agonist selectivity when
expressed in yeast. A selective and sensitive bioassay was designed
using a yeast strain (CY990) bearing the above described genetic
modifications and plasmids conferring expression of the human GRF
receptor (pJH25) and a Gpal-G.alpha..sub.s chimeric protein
(pLP122). A dose-dependent growth response of CY990 cells was
evident around an agonist analog of GRF [hGRF (1-29)-NH.sub.2, FIG.
25A] which was inhibited by coadministration of an antagonist
analog [(D-arg.sup.2)-hGRF (1-29)-NH.sub.2, FIG. 25B]. The assay
was selective: detectable growth responses were not observed in
response to a variety of agonists selective for other G
protein-coupled receptors (met-enkephalin, oxymetazoline,
isoproteranol, carbachol), nor by yeast cells lacking the GRF
receptor (data not shown). A detectable response was observed to 20
nmol of GRF, illustrating the sensitivity of the bioassay.
REFERENCES CITED IN THIS EXAMPLE
[0224] 1. Bohlen, P. F. Esch, P. Brazeau, N. Ling, and Guillemin.
1983. Isolation and characterization of the porcine hypothalamic
growth hormone releasing factor. Biochem. Biophys. Res. Comm. 116:
726-734. [0225] 2. Segre, G. V. and S. R. Goldring. 1993. Receptors
for secretin, calcitonin, parathyroid hormone (PTH)/PTH-related
peptide, vasoactive intestinal peptide, glucagonlike peptide 1,
growth hormone releasing hormone, and glucagon belong to a newly
discovered G protein linked receptor family. Trends Endo. Metab. 4:
309-314. [0226] 3. Gaylinn, B. D., J. K. Harrison, J. R. Zysk, C.
E. Lyons, K. R. Lynch, and M. O. Thorner 1993. Molecular cloning
and expression of a human anterior pituitary receptor for growth
hormone-releasing hormone. Mol. Endocrinol. 7: 77-84. [0227] 4.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
Cloning, A Laboratory Handbook. Cold Spring Harbor Press. Cold
Spring Harbor, N.Y. [0228] 5. Price, L. A., E. M. Kajkowski, J. R.
Hadcock, B. A. Ozenberger, and M. E. Pausch. 1995. Yeast cell
growth in response to agonist dependent activation of a mammalian
somatostatin receptor. submitted. [0229] 6. Rose, M., F. Winston,
and P. Hieter. 1990. Methods in Yeast Genetics. Cold Spring Harbor
Press. Cold Spring Harbor, N.Y. [0230] 7. Blumer, K. J., J. E.
Reneke, and J. Thorner. 1988. The STE2 gene product is the
ligand-binding component of the .alpha.-factor receptor of
Saccharomyces cerevisiae. J. Biol. Chem. 263: 10836-10842. [0231]
8. Strnad, J., C. M. Eppler, M. Corbett, and J. R. Hadcock. 1993.
The rat SSTR2 somatostatin receptor subtype is coupled to
inhibition of cyclic AMP accumulation. Biochem. Biophys. Res. Comm.
191: 968-976. [0232] 9. King, K., H. G. Dohlman, J. Thorner, M. G.
Caron, and R. J. Lefkowitz. 1990. Control of yeast mating signal
transduction by a mammalian .sub..beta.2-adrenergic receptor and
G.sub.s .alpha. subunit. Science 250: 121-123. [0233] 10. Weiner,
J. L., C. Guttierez-Steil, and K. J. Blumer. 1993. Disruption of
receptor-G protein coupling in yeast promotes the function of an
SST2-dependent adaptation pathway. J. Biol. Chem. 268:
8070-8077.
EXAMPLE 13
Overexpression of STE50 Enhances the Sensitivity of Yeast
Bioassay
[0234] Several molecular mechanisms for adaptation have been
described for the yeast mating signal transduction pathway (1).
Alterations to one or more of these mechanisms should serve to
enhance the sensitivity of a bioassay by altering desensitization
pathways and, therefore, prolonging the signal initiated by agonist
binding to receptor. The effects of an sst2 mutation on the
sensitivity of the yeast bioassay were described previously
(Example 6). As an alternative to the genetic modification at sst2,
overexpression of the yeast STE50 gene was predicted to have
similar effects (2), although by a different mechanism of action
(2, 3). The STE50 gene was isolated and placed under the control of
a strong constitutive promoter in a high-copy-number plasmid
resulting in significant overexpression of the gene. Yeast
engineered to respond to the mammalian hormone somatostatin through
an expressed SSTR2 somatostatin receptor were found to exhibit a
more robust response to hormone if STE50 was overexpressed.
Materials and Methods
[0235] Construction of STE50 expression plasmid. Growth of
bacterial strains and plasmid manipulations were performed by
standard methods (4). The protein coding sequences for STE50 were
amplified by polymerase chain reaction (PCR) using oligonucleotides
selected by examination of the published sequence (2). The sense
oligonucleotide (5'-GTCGACAAATCAG ATG GAG GAC GGT AAA CAG G-3')
contained the translation start codon (underlined) and a SalI
restriction site and the antisense oligonucleotide (5'-GAGCTCA TTA
GAG TCT TCC ACC GGG GG-3') contained the translation stop codon
(underlined) and a SacI restriction site. These oligonucleotides
were used as primers in a standard PCR to amplify STE50 from
Saccharomyces cerevisiae genomic DNA. The 1,100 basepair
amplification product was cloned into the pCR2 vector (Invitrogen
Corp., San Diego, Calif.) and confirmed by DNA sequencing. The
STE50 sequences were then isolated on a SalI-SacI restriction
fragment and cloned into a pADH expression vector (5), placing the
expression of STE50 under the control of the strong constitutive
.DELTA.DH1 promoter. This plasmid was designated pOZ162.
[0236] Yeast strain construction. Growth and transformation of
yeast strains were performed as described by Rose et al. (6). The
SSTR2 somatostatin receptor expression strain LY268 (MATa ura3-52
trpl.DELTA.63 his3.DELTA.200 leu2.DELTA.1 ade2-101 lys2-801
gpal.DELTA.hisG farl.DELTA.LYS2 FUS1-HIS3 sst2.DELTA.ADE2, pJH2,
pLP82) was described in prior examples and by Price et al. (7).
Strain LY268 was transformed with either the STE50 expression
plasmid pOZ162 or the pADH vector. These strains are denoted CY560
or CY562, respectively.
[0237] Bioassay. Bioassay of SSTR2 somatostatin receptor expressed
in yeast was described in prior examples and by Price et al. (7).
Briefly, yeast strains were grown overnight in 2 ml synthetic
complete liquid medium containing glucose (2%) and lacking uracil,
tryptophan and leucine (SCD-ura-trp-leu), washed to remove residual
glucose, and grown overnight in 5 ml SC Galactose (2%)-ura-trp-leu
liquid medium. Molten (52.degree. C.) SC Galactose
(2%)-ura-trp-leu-his agar medium (30 ml, adjusted to pH 6.8 by
addition of KOH prior to autoclaving) was inoculated with 0.06 ml
of the overnight culture to produce a final cell density of
approximately 10.sup.5 cells/ml and poured in square (9.times.9 cm)
petri plates. Sterile filter discs were placed on the surface of
the solidified agar and saturated with 10 .mu.l of sterile water
containing the indicated amounts of somatostatin-14 (Bachem
Bioscience Inc., Philadelphia, Pa.) or a mating pheromone (Sigma,
St. Louis, Mo.). Plates were incubated at 30.degree. C. for 3
days.
Results
[0238] The effect of STE50 overexpression on the sensitivity of the
yeast bioassay was examined by comparing strains differing only in
the level of STE50 expression (FIG. 26). Bioassay plates were made
containing either the STE50 overexpression strain CY560 or the
control strain CY562. The responses of these strains to
somatostatin or yeast pheromone were examined as described in
Materials and Methods. Both strains had very weak responses to
yeast pheromone because the wild-type yeast Gm gene, GPA1, had been
disrupted and functionally replaced by a gene expressing a chimeric
Gpal/G.alpha..sub.i2 protein (7). This chimeric G.alpha. protein
does not efficiently interact with the yeast pheromone receptor.
However, the response of these cells to somatostatin is strong
(FIG. 26). As predicted, the overexpression of STE50 resulted in a
more robust response (FIG. 26a). These data demonstrate that the
overexpression of STE50 produces a hypersensitivity to ligands
acting through G protein-coupled receptors coupled to the yeast
signal transduction pathway, even if the ligand and receptor
originate from a heterologous source.
REFERENCES CITED IN THIS EXAMPLE
[0239] 1. Sprague G F, Thorner J W. Pheromone response and signal
transduction during the mating process of Saccharomyces cerevisiae.
In: The molecular and cellular biology of the yeast Saccharomyces.
E W Jones J R Pringle and J R Broach, eds. Cold Spring Harbor
Laboratory Press, 1992. [0240] 2. Ramezani-Rad M, Xu G, Hollenberg
C P 1992 STE50, a novel gene required for activation of conjugation
at an early step in mating in Saccharomyces cerevisiae. Mol Gen
Genet. 236:145-154 [0241] 3. Chan R K, Otte C A 1982 Isolation and
genetic analysis of Saccharomyces cerevisiae mutants supersensitive
to G1 arrest by a factor and a factor pheromones. Mol Cell Biol
2:11-20 [0242] 4. Maniatus T, Fritsch E F, Sambrook J. Molecular
cloning. Cold Spring Harbor Laboratory Press, 1982 [0243] 5. Martin
G A, Viskochil D, Bollag G, et al. 1990 The GAP-related domain of
the neurofibromatosis type 1 gene product interacts with ras p21.
Cell 63:843-849 [0244] 6. Rose M D, Winston F, Rieter P. Methods in
yeast genetics. Cold Spring Harbor Laboratory Press, 1990 [0245] 7.
Price L A, Kajkowski E M, Hadcock J R, Ozenberger E A, Pausch M E
1995 Yeast cell growth in response to agonist-dependent activation
of a mammalian somatostatin receptor. Submitted.
EXAMPLE 14
Identification of Compounds with Somatostatin Receptor Agonist
and/or Antagonist Properties
[0246] Novel subtype-selective compounds with somatostatin agonist
properties have significant therapeutic potential in the detection
and treatment of various types of cancer. Compounds with
somatostatin antagonist properties may be useful in promoting
growth hormone release in agricultural species. Increased growth
hormone release may lead to useful improvements in growth
performance and carcass quality. To these ends, a yeast-based
mechanism-based screening assay was developed to assay compounds
for those that possessed desirable somatostatin agonist and/or
antagonist properties.
[0247] Bioassay. A bioassay designed to detect compounds with
somatostatin agonist and/or antagonist properties was mobilized
using a yeast strain (LY364 MATa ura3-52 trpl.DELTA.63
his3.DELTA.200 leu2.DELTA.1 ade2-101 lys2-801 gpal.DELTA.hisG
farl.DELTA.LYS2 FUS1-HIS33 sst2.DELTA.ADE2, pJH2, pLP82) that
functionally expressed the rate SSTR2. The assay was accomplished
using a modification of a standard procedure. Y364 was grown
overnight in 2 ml synthetic complete liquid medium containing
glucose (2%) and lacking uracil and tryptophan (SCD-ura-trp)
medium, washed to remove residual glucose, and grown overnight in 5
ml SC Galactose (2%)-ura-trp liquid medium. Molten (55.degree. C.)
SC Galactose (2%)-ura-trp-his agar medium (150 ml, adjusted to pH
6.8 by addition of concentrated (2.times.10.sup.4 cells/ml) and
plated in square (500 cm.sup.2) petri plates For assay of
antagonists, somatostatin (20 nM S-14) was added to the molten agar
prior to pouring. Sterile filter disks were placed on the surface
of the solidified agar and saturated with 10 .mu.l of sterile water
containing candidate compounds. Plates were incubated at 30.degree.
C. for 3 days.
[0248] Results. Active compounds from a primary screen were
reassayed and the results displayed in FIG. 27. The left hand panel
displays the results of an assay for compounds with somatostatin
agonist properties. Four compounds exhibited substantial growth
promoting activity expected of compounds with somatostatin agonist
properties. The compounds found in the bottom left four positions
are varying amounts of somatostatin applied as controls. The right
hand panel displays the results of an assay for compounds with
somatostatin antagonist activity. In the antagonist bioassay,
somatostatin is added to the molten agar prior to pouring. In this
way, all cells within the plate are induced to grow in response to
somatostatin. As applied active compounds with antagonist
properties diffuse into the agar medium and come into contact with
the cells within, the growth response induced by somatostatin is
interrupted, yielding a clear zone of inhibited growth. Several
compounds exhibited detectable growth inhibiting properties.
EXAMPLE 15
[0249] Fusion of STE2 sequences to the amino terminal of SSTR2
reduces signaling efficiency in response to somatostatin.
[0250] High level functional expression in yeast of G
protein-coupled receptors in general, and the SSTR2 in particular,
was a necessary prerequisite to the development of a useful
biassay. King et al. reported that replacement of the
amino-terminal domain of the .beta..sub.2-adrenergic receptor with
equivalent STE2 sequence was necessary for efficient receptor
expression in yeast. To test this hypothesis and the effect of STE2
sequences on expression of the somatostatin receptor in yeast, the
rat SSTR2 cDNA was placed under the control of the GAL1 promoter in
plasmids pJH1 and pJH2. These constructs confer inducible
overexpression of Gal4p, the transcriptional activating protein for
galactose-inducible genes, resulting in significantly elevated
levels of receptor protein in crude membrane fractions compared to
receptor expressed from a plasmid lacking GAL4 sequences (data not
shown). In SSTR2 expression plasmid pJH1, DNA sequences encoding
the first 13 amino acids of SSTR2 were replaced with coding
sequence for the first 23 amino acids of STE2 (FIG. 11). The rate
SSTR2 sequences were introduced into pJH2 without modification of
the protein coding sequences. Yeast strains containing these
constructs (LY322: MATa ura3-52 trpl.DELTA.63 h is 3.DELTA.200
leu2.DELTA.1 ade2-101 lys2-801 gpal.DELTA.hisG farl.DELTA.LYS2
FUSI-HIS3 sst2.DELTA.ADE2, pJH1, pLP82; LY268: MATa ura3-52
trpl.DELTA.63 his3.DELTA.200 ade2-101 lys2-801 gpal.DELTA.hisG
farl.DELTA.LYS2 FUSI-HIS3 sst2.DELTA.ADE21 pJH2, pLP82) bear a
plasmid (pLp82) that confers expression of a chimeric G.alpha.
protein composed of the proposed amino-terminal
.beta..gamma.-interaction domain from Gpalp and carboxy-terminal
receptor interaction domain from rat G.sub..alpha.i2 under the
control of the GPA1 promoter. The magnitude of the response of
these strains to applied somatostatin (S-14) was measured (FIG.
28). LY268 cells exhibited a robust growth response to applied
S-14, demonstrating that rat SSTR2 does not require STE2 sequences
to be functionally expressed in yeast (FIG. 28A) The growth
response of LY268 cells was substantially greater than that
exhibited by LY322 cells (FIG. 28B). The sole difference between
these strains is the presence of STE2 sequences in pJH1 found in
LY322. Thus, replacement of the amino terminum of SSTR2 with the
equivalent segment of STE2 greatly reduces the efficiency of
signalling in response to applied somatostatin. In spite of the
observations of King et al., heterologous G protein-coupled
receptors expressed in yeast do not require amino-terminal protein
coding sequences from any yeast protein for functional expression.
Sequence CWU 1
1
* * * * *