U.S. patent application number 10/347141 was filed with the patent office on 2003-11-20 for methods of testing for agonists and antagonists of cell surface receptors.
This patent application is currently assigned to Duke University. Invention is credited to Caron, Marc G., Dohlman, Henrik G., King, Klim, Lefkowitz, Robert J..
Application Number | 20030215887 10/347141 |
Document ID | / |
Family ID | 24326283 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215887 |
Kind Code |
A1 |
King, Klim ; et al. |
November 20, 2003 |
Methods of testing for agonists and antagonists of cell surface
receptors
Abstract
Disclosed is a transformed yeast cell containing a first
heterologous DNA sequence which codes for a mammalian G protein
coupled receptor and a second heterologous DNA sequence which codes
for a mammalian G protein a subunit (mammalian G.sub..alpha.). The
first and second heterologous DNA sequences are capable of
expression in the cell, but the cell is incapable of expressing an
endogenous G protein .alpha.-subunit (yeast G.sub..alpha.). The
cells are useful for screening compounds which affect the rate of
dissociation of G.sub..alpha. from G.sub..beta..tau. in a cell.
Also disclosed is a novel DNA expression vector useful for making
cells as described above. The vector contains a first segment
comprising at least a fragment of the extreme amino-terminal coding
sequence of a yeast G protein coupled receptor. A second segment is
positioned downstream from the first segment (and in correct
reading frame therewith), with the second segment comprising a DNA
sequence encoding a heterologous G protein coupled receptor.
Inventors: |
King, Klim; (Durham, NC)
; Dohlman, Henrik G.; (Berkeley, CA) ; Caron, Marc
G.; (Durham, NC) ; Lefkowitz, Robert J.;
(Durham, NC) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Duke University
|
Family ID: |
24326283 |
Appl. No.: |
10/347141 |
Filed: |
January 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10347141 |
Jan 17, 2003 |
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08623284 |
Mar 28, 1996 |
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08623284 |
Mar 28, 1996 |
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08441291 |
May 15, 1995 |
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5739029 |
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08441291 |
May 15, 1995 |
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08071355 |
Jun 3, 1993 |
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5482835 |
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08071355 |
Jun 3, 1993 |
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07581714 |
Sep 13, 1990 |
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Current U.S.
Class: |
435/7.2 ;
435/254.2 |
Current CPC
Class: |
C12Q 1/6897 20130101;
C12N 15/81 20130101; C07K 2319/705 20130101; C07K 2319/32 20130101;
G01N 33/566 20130101; G01N 2333/726 20130101; C07K 2319/02
20130101; C07K 14/39 20130101; G01N 2500/00 20130101; C07K 14/4722
20130101; C07K 2319/00 20130101; C07K 14/70571 20130101; C07K
2319/61 20130101 |
Class at
Publication: |
435/7.2 ;
435/254.2 |
International
Class: |
G01N 033/53; G01N
033/567; C12N 001/18 |
Goverment Interests
[0002] This invention was made with government support under NIH
grants HL16037 and GM21841. The government may have certain rights
to this invention.
Claims
That which is claimed is:
1. A transformed yeast cell containing a first heterologous DNA
sequence which codes for a mammalian G protein coupled receptor and
a second heterologous DNA sequence which codes for a mammalian G
protein a subunit (mammalian G.sub..alpha.), wherein said first and
second heterologous DNA sequences are capable of expression in said
cell, and wherein said cell is incapable of expressing an
endogenous G protein .alpha.-subunit (yeast G.sub..alpha.).
2. A transformed yeast cell according to claim 1, wherein said
first heterologous DNA sequence is carried by a plasmid.
3. A transformed yeast cell according to claim 1, wherein said
second heterologous DNA sequence is carried by a plasmid.
4. A transformed yeast cell according to claim 1, wherein said
mammalian G protein .alpha. subunit is selected from the group
consisting of G.sub.s .alpha. subunits, G.sub.i .alpha. subunits,
G.sub.o .alpha. subunits, G.sub.z .alpha. subunits, and transducin
.alpha. subunits.
5. A transformed yeast cell according to claim 1 which expresses a
complex of the G protein .beta. subunit and the G protein .tau.
subunit (G.sub..beta..tau.).
6. A transformed yeast cell according to claim 5 which expresses
endogenous G.sub..beta..tau..
7. A transformed yeast cell according to claim 1, wherein said
first heterologous DNA sequence codes for a mammalian G
protein-coupled receptor selected from the group consisting of
dopamine receptors, muscarinic cholinergic receptors,
.alpha.-adrenergic receptors, .beta.-adrenergic receptors, opiate
receptors, cannabinoid receptors, and serotonin receptors.
8. A transformed yeast cell according to claim 1 further comprising
a third heterologous DNA sequence, wherein said third heterologous
DNA sequence comprises a pheromone-responsive promotor and an
indicator gene positioned downstream from said pheromone-responsive
promoter and operatively associated therewith.
9. A transformed yeast cell according to claim 8, wherein said
pheromone responsive promoter is selected from the group consisting
of the BAR1 gene promoter and the FUS1 gene promoter, and wherein
said indicator gene is selected from the group consisting of the
HIS3 gene and the LacZ gene.
10. A method of testing a compound for the ability to affect the
rate of dissociation of G.sub..alpha. from G.sub..beta..tau. in a
cell, comprising: providing a transformed yeast cell containing a
first heterologous DNA sequence which codes for a mammalian G
protein coupled receptor and a second heterologous DNA sequence
which codes for a mammalian G.sub..alpha., wherein said first and
second heterologous DNA sequences are capable of expression in said
cell, wherein said cell is incapable of expressing endogenous
G.sub..alpha., and wherein said cell expresses G.sub..beta..tau.;
contacting said compound to said cell; and detecting the rate of
dissociation of G.sub.a from G.sub..beta..tau. in said cell.
11. A method according to claim 10, wherein said yeast cells are
provided in an aqueous solution and said contacting step is carried
out by adding said compound to said aqueous solution.
12. A method according to claim 10, wherein said mammalian G
protein a subunit is selected from the group consisting of G.sub.s
.alpha. subunits, G.sub.i .alpha. subunits, G.sub.o .alpha.
subunits, G.sub.z .alpha. subunits, and transducin .alpha.
subunits.
13. A method according to claim 10, wherein said yeast cell
expresses endogenous G.sub..beta..tau..
14. A method according to claim 10, wherein said first heterologous
DNA sequence codes for a mammalian G protein-coupled receptor
selected from the group consisting of dopamine receptors,
muscarinic cholinergic receptors, .alpha.-adrenergic receptors,
.beta.-adrenergic receptors, opiate receptors, cannabinoid
receptors, and serotonin receptors.
15. A method according to claim 10, said yeast cell further
comprising a third heterologous DNA sequence, wherein said third
heterologous DNA sequence comprises a pheromone-responsive promotor
and an indicator gene positioned downstream from said
pheromone-responsive promoter and operatively associated therewith;
and wherein said detecting step is carried out by monitoring the
expression of said indicator gene in said cell.
16. A DNA expression vector capable of expressing a transmembrane
protein into the cell membrane of yeast cells, comprising: a first
segment comprising at least a fragment of the extreme
amino-terminal coding sequence of a yeast G protein coupled
receptor; and a second segment downstream from said first segment
and in correct reading frame therewith, said second segment
comprising a DNA sequence encoding a heterologous G protein coupled
receptor.
17. A DNA expression vector according to claim 16, wherein a
fragment of the extreme amino-terminal coding sequence of said
heterologous G protein coupled receptor is absent.
18. A DNA expression vector according to claim 16, wherein said
first and second segments are operatively associated with a
promoter operative in a yeast cell.
19. A DNA expression vector according to claim 18, wherein said
promoter is the GAL1 promoter.
20. A DNA expression vector according to claim 16, wherein said
first segment comprises at least a fragment of the extreme
amino-terminal coding sequence of a yeast phereomone receptor.
21. A DNA expression vector according to claim 16, wherein said
first segment comprises at least a fragment of the extreme
amino-terminal coding sequence of a yeast phereomone receptor
selected from the group consisting of the STE2 gene and the STE3
gene.
22. A DNA expression vector according to claim 16, further
comprising at least a fragment of the 5'-untranslated region of a
yeast G protein coupled receptor gene positioned upstream from said
first segment and operatively associated therewith.
23. A DNA expression vector according to claim 16, further
comprising at least a fragment of the 5'-untranslated region of a
yeast phereomone receptor gene positioned upstream from said first
segment and operatively associated therewith.
24. A DNA expression vector according to claim 23, wherein said
yeast pheromone receptor gene is selected from the group consisting
of the STE2 gene and the STE3 gene.
25. A DNA expression vector according to claim 16, said vector
comprising a plasmid.
26. A DNA expression vector according to claim 16, said second
segment comprising a DNA sequence encoding a mammalian G protein
coupled receptor.
27. A DNA expression vector according to claim 16, said second
segment comprising a DNA sequence encoding a mammalian G
protein-coupled receptor selected from the group consisting of
dopamine receptors, muscarinic cholinergic receptors,
.alpha.-adrenergic receptors, .beta.-adrenergic receptors, opiate
receptors, cannabinoid receptors, and serotonin receptors.
28. A yeast cell carrying a DNA expression vector according to
claim 16.
29. A method for screening a plurality of compounds to identify a
compound that agonizes or antagonizes a G protein-coupled
receptor-mediated activity by detecting intracellular transduction
of a signal generated upon interaction of the compound with the G
protein-coupled receptor, comprising: comparing the amount of
expression of an indicator gene product in a recombinant eukaryotic
cell in the presence of the compound with the amount of expression
of an indicator gene product in the absence of the compound; and
selecting the compound from the plurality of compounds that changes
the amount of expression of the indicator gene product in the
recombinant cell in the presence of the compound compared to the
amount of expression of an indicator gene product in the absence of
the compound whereby the compound that modulates G protein-coupled
receptor mediated activity is identified, wherein: the recombinant
cell contains an indicator gene construct and expresses the G
protein-coupled receptor; and the indicator gene construct
includes: (a) a transcriptional control element that is responsive
to the intracellular signal that is generated by the interaction of
the compound with the G protein-coupled receptor; and (b) an
indicator gene that encodes a detectable product and that is in
operative association with the transcriptional control element.
30. The method of claim 29, wherein said compound is an agonist or
antagonist of said G protein-coupled receptor.
31. The method of claim 29, wherein the amount of expression of an
indicator gene product is measured by measuring the amount of
indicator gene protein that is produced.
32. The method of claim 30, wherein said compound is an
antagonist.
33. The method of claim 30, further comprising, prior to comparing
the difference in the amount of expression of the indicator gene
product, contacting the recombinant cell with an agonist that
activates said cell surface protein, whereby expression of said
indicator gene product is induced.
34. The method of claim 29, wherein the G protein-coupled receptor
is selected from the group consisting of muscarinic receptors,
adrenergic receptors, dopamine receptors, and serotonin
receptors.
35. The method of claim 29, wherein the transcriptional control
element includes a promoter sequence which is responsive to the
intracellular signal.
36. A method for screening a plurality of compounds to identify a
compound that agonizes or antagonizes a G protein-coupled
receptor-mediated activity by detecting intracellular transduction
of a signal generated upon interaction of the compound with the G
protein-coupled receptor, comprising: comparing the amount of
expression of an indicator gene product in a first recombinant
eukaryotic cell in the presence of the compound with the amount of
expression of an indication gene product in the absence of the
compound, or with the amount of expression in a second recombinant
cell; and selecting a compound from the plurality of compounds that
changes the amount of expression of the indicator gene product in
the first recombinant cell in the presence of the compound compared
to the amount of expression in the absence of the compound, or
compared to the amount of expression in the second recombinant
cell, whereby a compound that modulates G protein-coupled
receptor-mediated activity is identified wherein: the first
recombinant eukaryotic cell contains an indicator gene construct
and expresses the G protein-coupled receptor; the second
recombinant cell is identical to the first recombinant cell, except
that it does not express the G protein-coupled receptor; and the
indicator gene construct includes: (a) a transcriptional control
element that is responsive to the intracellular signal that is
generated by the interaction of the compound with the G
protein-coupled receptor; and (b) an indicator gene that encodes a
detectable product and that is in operative association with the
transcriptional control element.
37. A method for identifying extracellular signals that agonize or
antagonize cell surface protein-mediated activity, comprising:
comparing the difference in the amount of expression of an
indicator gene product in a recombinant cell in the presence of the
signal with the amount of expression in the absence of said signal,
or with the amount of expression in the absence of the cell surface
protein, wherein: the recombinant cell contains an indicator gene
construct and expresses the cell surface protein; and expression of
the indicator gene product is under the control of at least one
transcriptional control element whose activity is regulated by the
cell surface protein.
38. The method of claim 37, wherein said protein is a cell surface
receptor.
39. The method of claim 38, wherein said signal is an agonist or
antagonist of said cell surface receptor.
40. The method of claim 39, wherein the concentration of said
agonist or antagonist is less than 100 .mu.M.
41. The method of claim 39, wherein the concentration of said
agonist or antagonist is less than 10 .mu.M.
42. The method of claim 39, wherein the concentration of said
agonist or antagonist is less than 1 .mu.M.
43. The method of claim 39, wherein the concentration of said
agonist or antagonist is less than 0.1 .mu.M.
44. The method of claim 39, wherein the concentration of said
agonist or antagonist is less than 10 nM.
45. The method of claim 37, wherein the amount of expression is
determined by measuring the amount of indicator gene product that
is produced.
46. The method of claim 39, wherein said signal is an
antagonist.
47. The method of claim 46, further comprising, prior to or
simultaneously with comparing the difference in the amount of
expression of the indicator gene product, contacting the
recombinant cell with an agonist that activates said cell surface
protein, whereby expression of said indicator gene is induced.
48. The method of claim 37, wherein the cell surface protein is a
cell surface receptor selected from: muscarinic receptors,
adrenergic receptors, dopamine receptors, serotonin receptors.
49. The method of claim 37, wherein the transcriptional control
element includes a promoter sequence which is responsive to
intracellular signals.
50. A recombinant cell, comprising: DNA that encodes a heterologous
cell surface protein whose activity is modulated by extracellular
signals; and a reporter gene construct containing a reporter gene
in operative linkage with one or more transcriptional control
element that is regulated by said cell surface protein; wherein
said cell surface protein is a G-protein coupled receptor.
51. The recombinant cell of claim 50, wherein said cell surface
protein is a cell surface receptor selected from: muscarinic
receptors, adrenergic receptors, dopamine receptors, serotonin
receptors.
52. A method for identifying compounds that agonize or antagonize
cell surface protein-mediated activity by detecting intracellular
transduction of a signal generated upon interaction of the compound
with a cell surface protein, comprising: comparing the amount of
expression of an indicator gene product in a first recombinant cell
in the presence of the compound with the amount of indicator gene
product in a second recombinant cell; and selecting compounds that
change the amount of expression of an indicator gene product in the
first recombinant cell in the presence of the compound compared to
the amount of expression in the absence of the compound, or
compared to the amount of expression in the second recombinant
cell, wherein: the first recombinant cell contains an indicator
gene construct and expresses the cell surface protein; the second
recombinant cell is identical to the first recombinant cell, except
that it does not express the cell surface protein; and the
indicator gene construct contains: (a) a transcriptional control
element that is responsive to the intracellular signal that is
generated by the interaction of an agonist with the cell surface
protein; and (b) an indicator gene that encodes a detectable
product and that is in operative association with the
transcriptional control element.
53. The method of claim 52, wherein the compound is an agonist or
antagonist of said cell surface receptor.
54. The method of claim 52, wherein the level of expression is
determined by measuring the amount of indicator gene product that
is produced.
55. The method of claim 52, further comprising, prior to or
simultaneously with comparing the difference in the amount of
indicator gene product, contacting the recombinant cell with an
agonist that activates said cell surface protein, whereby
expression of said indicator gene product is induced.
56. The method of claim 55, wherein said compound is an
antagonist.
57. The method of claim 47, wherein said compound is an
antagonist.
58. The method of claim 37, wherein the cell is preincubated with
the extracellular signal prior to comparing the difference in
expression.
59. The method of claim 52, wherein the cell is preincubated with
the compound prior to comparing the difference in expression.
60. The method of any one of claims 37 and 52, wherein said cell
surface protein is a G-protein coupled receptor.
61. The method of claim 36, wherein said compound is an agonist or
antagonist of said G protein-coupled receptor.
62. The method of claim 36, wherein the amount of expression of an
indicator gene product is measured by measuring the amount of
indicator gene protein that is produced.
63. The method of claim 36, wherein said compound is an
antagonist.
64. The method of claim 36, further comprising, prior to comparing
the difference in the amount of expression of the indicator gene
product, contacting the recombinant cell with an agonist that
activates said cell surface protein, whereby expression of said
indicator gene product is induced.
65. The method of claim 36, wherein the G protein-coupled receptor
is selected from the group consisting of muscarinic receptors,
adrenergic receptors, dopamine receptors, and serotonin
receptors.
66. An assay for screening a plurality of compounds to determine if
a compound so screened has cell surface receptor agonist or
antagonist activity, which assay comprises (a) challenging a
recombinant eukaryotic cell with the compound, wherein the cell
recombinantly expresses the G protein-coupled receptor; (b)
determining the level of indicator gene product recombinantly
expressed in the cell; (c) comparing the amount of expression of an
indicator gene product in the eukaryotic cell with the amount of
expression of an indicator gene product in the eukaryotic cell in
the absence of the compound, and (d) identifying a compound from
the plurality of compounds that exhibits said cell surface receptor
agonist or antagonist activity, wherein the indicator gene is under
transcriptional control of a transcriptional control element
selected to be responsive to an intracellular signal that is
generated by the interaction of a compound with G protein-coupled
receptor.
67. The assay of claim 66, wherein said compound is an agonist or
antagonist of said G protein-coupled receptor.
68. The assay of claim 66, wherein the amount of expression is
measured by measuring the amount of indicator gene protein that is
produced.
69. The assay of claim 67, wherein said compound is an
antagonist.
70. The assay of claim 67, further comprising, prior to comparing
the difference in the amount of expression of the indicator gene
product, contacting the recombinant cell with an agonist that
activates said cell surface protein, whereby expression of said
indicator gene product is induced.
71. The assay of claim 66, wherein said cell surface protein is a
G-protein coupled receptor.
72. The assay of claim 66, wherein the cell surface receptor is
selected from the group consisting of muscarinic receptors,
adrenergic receptors, dopamine receptors, and serotonin
receptors.
73. The assay of claim 66, wherein the transcriptional control
element includes a promoter sequence which is responsive to the
intracellular signal.
74. An assay for screening a plurality of compounds to determine if
a compound so screened has cell surface receptor agonist or
antagonist activity, which assay that comprises (a) challenging a
first recombinant eukaryotic cell with a compound to be screened
wherein the cell recombinantly expresses the G protein-coupled
receptor, (b) determining the level of expression of an indicator
gene product recombinantly expressed in the cell, (c) comparing the
amount of expression of an indicator gene product in the first
recombinant eukaryotic cell with the amount of expression of an
indicator gene product in a second recombinant eukaryotic cell that
is identical to the first eukaryotic cell except that the second
cell does not express the G protein-coupled receptor, and (d)
identifying a compound that exhibits said G protein-coupled
receptor agonist or antagonist activity, wherein the indicator gene
is under transcriptional control of a transcriptional control
element selected to be responsive to an intracellular signal that
is generated by the interaction of the compound with the G
protein-coupled receptor.
75. The assay of claim 74, wherein said compound is an agonist or
antagonist of G protein-coupled receptor.
76. The assay of claim 74, wherein the amount of expression is
measured by measuring the amount of indicator gene protein that is
produced.
77. The assay of claim 75, wherein said compound is an
antagonist.
78. The assay of claim 75, further comprising, prior to comparing
the difference in the amount of expression of the indicator gene
product, contacting the recombinant cell with an agonist that
activates said cell surface protein, whereby expression of said
indicator gene product is induced.
79. The assay of claim 74, wherein the G protein-coupled receptor
is selected from the group consisting of muscarinic receptors,
adrenergic receptors, dopamine receptors, and serotonin
receptors.
80. The assay of claim 74, wherein the transcriptional control
element includes a promoter sequence which is responsive to the
intracellular signal.
Description
RELATED INFORMATION
[0001] This application is a continuation of application Ser. No.
08/623,284 filed on Mar. 28, 1996, pending, which is a divisional
of application Ser. No. 08/441,291, filed May 15, 1995, now issued
as U.S. Pat. No. 5,739,029, which is a divisional of application
Ser. No. 08/071,355, filed Jun. 3, 1993, now issued as U.S. Pat.
No. 5,482,835, which is a continuation of application Ser. No.
07/581,714, filed Sep. 13, 1990, now abandoned.
FIELD OF THE INVENTION
[0003] This invention relates to yeast cells expressing
heterologous G protein coupled receptors, vectors useful for making
such cells, and methods of using the same.
BACKGROUND OF THE INVENTION
[0004] The actions of many extracellular signals (for example,
neurotransmitters, hormones, odorants, light) are mediated by
receptors with seven transmembrane domains (G protein coupled
receptors) and heterotrimeric guanine nucleotide-binding regulatory
proteins (G proteins). See H. Dohlman, M. Caron, and R. Lefkowitz,
Biochemistry 26, 2657 (1987); L. Stryer and H. Bourne, Ann. Rev.
Cell Biol. 2, 391 (1988). Such G protein-mediated signaling systems
have been identified in organisms as divergent as yeast and man.
See H. Dohlman et al., supra; L. Stryer and H. Bourne, supra; K.
Blumer and J. Thorner, Annu. Rev. Physiol. (in press). The
.beta.2-adrenergic receptor (.beta.AR) is the prototype of the
seven-transmembrane-segment class of ligand binding receptors in
mammalian cells. In response to epinephrine or norepinephrine,
.beta.AR activates a G protein, G.sub..alpha., which in turn
stimulates adenylate cyclase and cyclic adenosine monophosphate
production in the cell. See H. Dohlman et al., supra; L. Stryer and
H. Bourne, supra. 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.
See K. Blumer and J. Thorner, supra; I. Herskowitz, Microbiol. Rev.
52, 536 (1988).
[0005] The present invention is based on our continued research
into the expression of heterologous G protein coupled receptors in
yeast.
SUMMARY OF THE INVENTION
[0006] A first aspect of the present invention is a transformed
yeast cell containing a first heterologous DNA sequence which codes
for a mammalian G protein coupled receptor and a second
heterologous DNA sequence which codes for a mammalian G protein
.alpha. subunit (mammalian G.sub..alpha.). The first and second
heterologous DNA sequences are capable of expression in the cell,
but the cell is incapable of expressing an endogenous G protein
.alpha.-subunit (yeast G.sub..alpha.). The cell optionally contains
a third heterologous DNA sequence, with the third heterologous DNA
sequence comprising a pheromone-responsive promotor and an
indicator gene positioned downstream from the pheromone-responsive
promoter and operatively associated therewith.
[0007] A second aspect of the present invention is a method of
testing a compound for the ability to affect the rate of
dissociation of G.sub..alpha. from G.sub..beta..tau. in a cell. The
method comprises: providing a transformed yeast cell as described
above; contacting the compound to the cell; and then detecting the
rate of dissociation of G.sub..alpha. from G.sub..beta..tau. in the
cell. The cells may be provided in an aqueous solution, and the
contacting step carried out by adding the compound to the aqueous
solution.
[0008] A third aspect of the present invention is a DNA expression
vector capable of expressing a transmembrane protein into the cell
membrane of yeast cells. The vector contains a first segment
comprising at least a fragment of the extreme amino-terminal coding
sequence of a yeast G protein coupled receptor. A second segment is
positioned downstream from the first segment (and in correct
reading frame therewith), with the second segment comprising a DNA
sequence encoding a heterologous G protein coupled receptor.
[0009] A fourth aspect of the present invention is a yeast cell
transformed by a vector as described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 illustrates the construction of the yeast human
.beta.2 Adrenergic Receptor expression plasmid, pY.beta.AR2.
[0011] FIG. 2 illustrates h.beta.AR ligand binding to membranes
from pY.beta.AR2-transformed yeast cells.
[0012] FIG. 3 shows a comparison of .beta.-adrenergic agonist
effects on pheromone-inducible gene activity. .alpha.-MF, 10 .mu.M
.alpha.-mating factor; (-) ISO, 50 .mu.M (-) isoproterenol; (-)
ALP, 50 .mu.M (-) alprenolol; (+) ISO, 100 .mu.M (+)
isoproterenol.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Nucleotide bases are abbreviated herein as follows:
1 A = Adenine G = Guanine C = Cytosine T = Thymine
[0014] Amino acid residues are abbreviated herein to either three
letters or a single letter as follows:
2 Ala; A = Alanine Leu; L = Leucine Arg; R = Arginine Lys; K =
Lysine Asn; N = Asparagine Met; M = Methionine Asp; D = Aspartic
acid Phe; F = Phenylalanine Cys; C = Cysteine Pro; P = Proline Gln;
Q = Glutamine Ser; S = Serine Glu; E = Glutamic acid Thr; T =
Threonine Gly; G = Glycine Trp; W = Tryptophan His; H = Histidine
Tyr; Y = Tyrosine Ile; I = Isoleucine Val; V = Valine
[0015] The term "mammalian" as used herein refers to any mammalian
species (e.g., human, mouse, rat, and monkey).
[0016] 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), or combinations thereof not naturally
found in yeast.
[0017] The terms "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.
[0018] G proteins are comprised of three subunits: a
guanyl-nucleotide binding .alpha. subunit; a .beta. subunit; and a
.tau. subunit. G proteins cycle between two forms, depending on
whether GDP or GTP is bound thereto. When GDP is bound the G
protein exists as an inactive heterotrimer, the
G.sub..alpha..beta..tau.complex. When GTP is bound the .alpha.
subunit dissociates, leaving a G.beta..tau. complex. Importantly,
when a G.sub..alpha..beta..tau. 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 dissociation of the bound the a subunit from the
G.sub..beta..tau. complex increases. This fundamental scheme of
events forms the basis for a multiplicity of different cell
signaling phenomena. See generally Stryer and Bourne, supra.
[0019] Any mammalian G protein coupled receptor, and the DNA
sequences encoding these receptors, may be employed in practicing
the present invention. Examples of such receptors include, but are
not limited to, dopamine receptors, muscarinic cholinergic
receptors, .alpha.-adrenergic receptors, .beta.-adrenergic
receptors, opiate receptors, cannabinoid receptors, and serotonin
receptors. The term receptor as used herein is intended to
encompass subtypes of the named receptors, and mutants and homologs
thereof, along with the DNA sequences encoding the same.
[0020] The human D.sub.1 dopamine receptor cDNA is reported in A.
Dearry et al., Nature 347, 72-76 (1990).
[0021] The rat D.sub.2 dopamine receptor cDNA is reported in J.
Bunzow et al., Nature 336, 783-787 (1988); see also O. Civelli, et
al., PCT Appln. WO 90/05780 (all references cited herein are to be
incorporated herein by reference).
[0022] Muscarinic cholinergic receptors (various subtypes) are
disclosed in E. Peralta et al., Nature 343, 434 (1988) and K.
Fukuda et al., Nature 327, 623 (1987).
[0023] Various subtypes of .alpha..sub.2-adrenergic receptors are
disclosed in J. Regan et al., Proc. Natl. Acad. Sci. USA 85, 6301
(1988) and in R. Lefkowitz and M. Caron, J. Biol. Chem. 263, 4993
(1988).
[0024] Serotonin receptors (various subtypes) are disclosed in S.
Peroutka, Ann. Rev. Neurosci. 11, 45 (1988).
[0025] A cannabinoid receptor is disclosed in L. Matsuda et al.,
Nature 346, 561 (1990).
[0026] Any DNA sequence which codes for a mammalian G .alpha.
subunit (G.sub..alpha.) may be used to practice the present
invention. Examples of mammalian G .alpha. subunits include G.sub.s
.alpha. subunits, G.sub.i .alpha. subunits, G.sub.o .alpha.
subunits, G.sub.z .alpha. subunits, and transducin .alpha.
subunits. See generally Stryer and Bourne, supra. G proteins and
subunits useful for practicing the present invention include
subtypes, and mutants and homologs thereof, along with the DNA
sequences encoding the same.
[0027] Heterologous DNA sequences are expressed in a host by means
of an expression 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 effecting the expression of a protein or protein subunit coded
for by the heterologous DNA sequence in the intended host.
Generally, control sequences include a transcriptional promoter, an
optional operator sequence to control transcription, a sequence
encoding suitable mRNA ribosomal binding sites, and (optionally)
sequences which control the termination of transcription and
translation.
[0028] Vectors useful for practicing the present invention include
plasmids, viruses (including phage), and integratable DNA fragments
(i.e., fragments integratable into the host genome by homologous
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.
[0029] 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.
[0030] 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. In general, the host cells are incapable of expressing
an endogenous G protein .alpha.-subunit (yeast G.sub..alpha.). The
host cells do, however, express a complex of the G protein .beta.
subunit and the G protein .tau. subunit (G.sub..beta..tau.). The
host cells may express endogenous G.sub..beta..tau., or may
optionally be engineered to express heterologous G.sub..beta..tau.
(e.g., mammalian) in the same manner as they are engineered to
express heterologous G.sub..alpha..
[0031] 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 yeast, although a number of other strains are commonly
available. See, e.g., U.S. Pat. No. 4,806,472 (Kluveromyces lactis
and expression vectors therefor); U.S. Pat. No. 4,855,231 (Pichia
pastoris and expression vectors therefor). Yeast vectors may
contain an origin of replication from the 2 micron yeast plasmid or
an autonomously replicating sequence (ARS), a promoter, DNA
encoding the heterologous DNA sequences, sequences for
polyadenylation and transcription termination, and a selection
gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282,
39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al.,
Gene 10, 157 (1980)). This plasmid contains the trpl gene, which
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example ATCC No. 44076 or
PEP4-1 (Jones, Genetics 85, 12 (1977)). The presence of the trpl
lesion in the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence
of tryptophan.
[0032] Suitable promoting sequences in yeast vectors include the
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes
(Hess et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et
al., Biochemistry 17, 4900 (1978)), 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 2, isocytochrome C, acid phosphatase,
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.
[0033] 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
mRNA.
[0034] A novel DNA expression vector described herein which is
particularly useful for carrying out the present invention contains
a first segment comprising at least a fragment of the extreme
amino-terminal coding 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 DNA sequence encoding a heterologous G protein coupled
receptor (e.g., a mammalian G protein coupled receptor). In a
preferred embodiment, this vector comprises a plasmid. In
constructing such a vector, a fragment of the extreme
amino-terminal coding sequence of the heterologous G protein
coupled receptor may be deleted. 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
phereomone receptors (e.g., the STE2 gene, which encodes the
.alpha.-factor receptor, and the STE3 gene, which encodes the
.alpha.-factor receptor). The levels of expression obtained from
these novel vectors are enhanced if at least a fragment of the
5'-untranslated region of a yeast G protein coupled receptor gene
(e.g., a yeast pheromone receptor gene; see above) is positioned
upstream from the first segment and operatively associated
therewith.
[0035] Any of a variety of means for detecting the dissociation of
G.sub..alpha. from G.sub..beta..tau. can be used in connection with
the present invention. The cells could be disrupted and the
proportion of these subunits and complexes determined physically
(i.e., by chromatography). The cells could be disrupted and the
quantity of G.sub..alpha. present assayed directly by assaying for
the enzymatic activity possessed by G.sub..alpha. in isolation
(i.e., the ability to hydrolyze GTP to GDP). Since whether GTP or
GDP is bound to the G protein depends on whether the G protein
exists as a G.sub..beta..tau. or G.sub..alpha..beta..tau. complex,
dissociation can be probed with radiolabelled GTP. As explained
below, morphological changes in the cells can be observed. A
particularly convenient method, however, is to provide in the cell
a third heterologous DNA sequence, wherein the third heterologous
DNA sequence comprises a pheromone-responsive promotor and an
indicator gene positioned downstream from the pheromone-responsive
promoter and operatively associated therewith. This sequence can be
inserted with a vector, as described in detail herein. 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 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 gene and the LacZ gene.
[0036] As noted above, transformed host cells of the present
invention express the protein or protein subunit coded for by the
heterologous DNA sequence. 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
stereospecific binding of ligands on the extracellular side of the
cell membrane and for functional interaction with G proteins on the
cytoplasmic side of the cell membrane).
[0037] The ability to control the yeast pheromone response pathway
by expression of a heterologous adrenergic receptor and its cognate
G protein .alpha.-subunit has the potential to facilitate
structural and functional characterization of mammalian G
protein-coupled receptors. By scoring for responses such as growth
arrest or .beta.-galactosidase induction, the functional properties
of mutant receptors can now be rapidly tested. Similarly, as
additional genes for putative G protein-coupled receptors are
isolated, numerous ligands can be screened to identify those with
activity toward previously unidentified receptors. See F. Libert et
al., Science 244, 569 (1989); M. S. Chee et al., Nature 344, 774
(1990). Moreover, as additional genes coding for putative G protein
.alpha.-subunits are isolated, they can be expressed in cells of
the present invention and screened with a variety of G protein
coupled receptors and ligands to characterize these subunits. These
cells can also be used to screen for compounds which affect
receptor-G protein interactions.
[0038] Cells of the present invention can be deposited in the wells
of microtiter plates in known, predetermined quantities to provide
standardized kits useful for screening compounds in accordance with
the various screening procedures described above.
[0039] 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
Construction of the Human .beta.2-Adrenergic Expression Vector
pY.beta.AR2 and Expression in Yeast
[0040] To attain high level expression of the human
.beta.2-adrenergic receptor (h.beta.AR) in yeast, a modified
h.beta.AR gene was placed under the control of the GAL1 promoter in
the multicopy vector, YEp24 (pY.beta.AR2).
[0041] FIG. 1 illustrates the construction of yeast expression
plasmid pY.beta.AR2. In pY.beta.AR2, expression of the h.beta.AR
sequence is under the control of the GAL1 promoter. FIG. 1A shows
the 5'-untranslated region and the first 63 basepairs (bp) of
coding sequence of the h.beta.AR gene in PTZNAR, B. O'Dowd et al.,
J. biol. Chem. 263, 15985 (1988), which was removed by Aat II
cleavage and replaced with a synthetic oligonucleotide
corresponding to 11 bp of noncoding and 42 bp of coding sequence
from the STE.2 gene. See N. Nakayama et al., EMBO J. 4, 2643
(1985); A. Burkholder and L. Hartwell, Nucleic Acids Res. 13, 8463
(1985). The resulting plasmid, pTZYNAR, contains the modified
h.beta.AR gene flanked by Hind III sites in noncoding sequences.
The Hind III-Hind III fragment was isolated from pTZYNAR and
inserted into pAAH5 such that the 3'-untranslated sequence of the
modified h.beta.AR gene was followed by 450 bp containing
termination sequences from the yeast ADH1 gene. See G. Ammerer,
Methods. Enzymol. 1, 192 (1983).
[0042] As illustrated in FIG. 1B, py.beta.13AR2 was constructed by
inserting the Bam HI-Bam HI fragment containing h.beta.AR and ADJ1
sequences into YEpG24. E. Wyckoff and T. Hsieh, Proc. Natl. Acad.
Sci. U.S.A. 85, 6272 (1988). Where maximum expression was sought,
cells were cotransformed with plasmid pMTL9 (from Dr. S. Johnston)
containing LAC9, a homolog of the S. cerevisiae GAL4 transactivator
protein required for GAL1-regulated transcription. J. Salmeron et
al., Mol. Cell. Biol. 9, 2950 (1989). Cells grown to late
exponential phase were induced in medium containing 3% galactose,
supplemented with about 10 .mu.M alprenolol, and grown for an
additional 36 hours. Standard methods for the maintenance of cells
were used. See F. Sherman et al., Methods in Yeast Genetics (Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986).
[0043] Maximal expression required (i) expression of a
transcriptional transactivator protein (LAC9), (ii) replacement of
the 5' untranslated and extreme NH.sub.2-terminal coding sequence
of the h.beta.AR gene with the corresponding region of the yeast
STE2 (.alpha.-factor receptor) gene, (iii) induction with galactose
when cell growth reached late exponential phase, and, (iv)
inclusion of an adrenergic ligand in the growth medium during
induction.
[0044] The plasmid pY.beta.AR2 was deposited in accordance with the
provisions of the Budapest Treaty at the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA, on Sep.
11, 1990, and has been assigned ATCC Accession No. 40891.
EXAMPLE 2
Binding Affinity of h.beta.AR Ligands in Yeast Transformed with
pY.beta.AR2
[0045] A primary function of cell surface receptors is to recognize
only appropriate ligands among other extracellular stimuli.
Accordingly, ligand binding affinities were determined to establish
the functional integrity of h.beta.AR expressed in yeast. As
discussed in detail below, an antagonist, .sup.125I-labeled
cyanopindolol (.sup.125I-CYP), bound in a saturable manner and with
high affinity to membranes prepared from pY.beta.AR2-transformed
yeast cells. By displacement of .sup.125I-CYP with a series of
agonists, the order of potency and stereospecificity expected for
h.beta.AR was observed.
[0046] SC261 cells (MATa ura3-52 trpl leu2 prb1-1122 pep4-3
prcl-407) (from Dr. S. Johnston) harboring pY.beta.AR2 (URA3) and
pMTL9 (LEU2) were grown in minimal glucose-free selective media to
late log phase (OD.sub.600=5.0), and then induced with the addition
of 3% galactose and 40 .mu.M alprenolol. After 36 hours, cells were
harvested and spheroplasts were prepared as described. See E.
Wyckoff and T. Hsieh, Proc. Natl. Acad. Sci. U.S.A. 85, 6272
(1988). Briefly, the spheroplasts were resuspended in 50 mM
Tris-HCl pH 7.4, 5 mM EDTA and were lysed by vortex mixing with
glass beads for three one-min periods at 4.degree. C. Crude
membranes were prepared from the lysates and binding assays with
.sup.125I-CYP were performed by methods described previously. See
H. Dohlman et al., Biochemistry 29, 2335 (1990).
[0047] FIG. 2 illustrates h.beta.AR ligand binding to membranes
from pY.beta.AR2-transformed yeast cells. (A) B.sub.max (maximum
ligand bound) and K.sub.d (ligand dissociation constant) values
were determined by varying 125.sub.I-CYP concentrations (5-400 pM).
Specific binding was defined as the amount of total binding
(circles) minus nonspecific binding measured in the presence of 10
.mu.M (-) alprenolol (squares). A K.sub.d of 93 .mu.M for
125.sub.I-CYP binding was obtained and used to calculate agonist
affinities (below). (B) Displacement of 18 pM 125.sub.I-CYP with
various concentrations of agonists was used to determine apparent
low affinity K.sub.i values (non G protein coupled, determined in
the presence of 50 .mu.M GTP) for receptor binding, squares; (-)
isoproterenol, circles; (-) epinephrine, downward-pointing
triangles; (+) isoproterenol, upward pointing triangles; (-)
norepinephrine. COMPARATIVE EXAMPLE A
Ligand Binding Affinity for h.beta.AR Expressed in Yeast and
Mammalian Cells
[0048] The binding data of FIGS. 2(A) and (B) were analyzed by
nonlinear least squares regression, see A. DeLean et al., Mol.
Pharmacol. 21, (1982), and are presented in Table I. Values given
are averages of measurements in triplicate, and are representative
of 2-3 experiments. Binding affinities in yeast were nearly
identical to those observed previously for h.beta.AR expressed in
mammalian cells.
3TABLE 1 Comparison of ligand Binding Parameters for High Level
Expression of Human .beta.-Adrenergic Receptor in Yeast and COS-7
Cells* Yeast Monkey SC261 COS-7 (pY, .beta.AR2, pMTL9) (pBC12:
.beta.,BAR) 125.sub.1-CYP: .sup.1K.sub.d 0.093 nM .+-. 0.013 0.110
nM .+-. 0.009 .sup.2B.sub.max 115 pmol/mg 24 pmol/mg
.sup.3K.sub.1(M): (-) isoproterenol 103 .+-. 26 130 .+-. 15 (+)
isoproterenol 3670 .+-. 420 4000 .+-. 184 (-) epinephrine 664 .+-.
123 360 .+-. 30 (-) norepinephrine 6000 .+-. 1383 5800 .+-. 373
*Values derived from Fig. 2 and H. Dohiman et al., Biochemistry 29,
2335 (1990).; .+-. S.E. .sup.1K.sub.d, ligand dissociation constant
.sup.2B.sub.max, maximum ligand bound .sup.3K.sub.1, inhibition
constant
EXAMPLE 3
Agonist-Dependent Activation of Mating Signal Transduction in Yeast
Expressing h.beta.AR
[0049] A second major function of a receptor is agonist-dependent
regulation of downstream components in the signal transduction
pathway. Because the pheromone-responsive effector in yeast is not
known, indirect biological assays are the most useful indicators of
receptor functionality. See K. Blumer and J. Thorner, Annu. Rev.
Physiol. in press; I. Herskowitz, Microbiol. Rev. 52, 536 (1988).
In yeast cells expressing high concentrations of h.beta.AR, no
agonist-dependent activation of the mating signal transduction
pathway could be detected by any of the typical in vivo assays; for
example, imposition of G1 arrest, induction of gene expression,
alteration of morphology (so-called "shmoo" formation) or
stimulation of mating. A likely explanation for the absence of
responsiveness is that h.beta.AR was unable to couple with the
endogenous yeast G protein.
EXAMPLE 4
Coexpression of h.beta.AR and Mammalian G.sub.s .alpha.-Subunit in
Yeast
[0050] Expression of a mammalian G.sub.s .alpha.-subunit can
correct the growth defect in yeast cells lacking the corresponding
endogenous protein encoded by the GPA1 gene. See C. Dietzel and J.
Kurjan, Cell 50, 1001 (1987). Moreover, specificity of receptor
coupling in mammalian cells is conferred by the .alpha.-subunit of
G proteins. See L. Stryer and H. Bourne, Annu. Rev. Cell Biol. 2,
391 (1988). Thus, coexpression of h.beta.AR and a mammalian G.sub.s
.alpha.-subunit (GS.alpha.) in yeast was attempted to render the
yeast responsive to adrenergic ligands. Accordingly, a cDNA
encoding rat G.sub.s.alpha. under the control of the
copper-inducible CUP1 promoter was introduced on a second plasmid,
pYSK136G.alpha.s. See C. Dietzel and J. Kurjan, Cell 50, 1001
(1987). In yeast (NNY19) coexpressing h.beta.AR and rat
G.sub.s.alpha., but containing wild-type GPA1, no adrenergic
agonist-induced shmoo formation, a characteristic morphological
change of yeast in response to mating pheromone, was observed.
EXAMPLE 5
Coexpression of h.beta.AR and Mammalian G.sub.s.alpha.-Subunit in
Yeast Lacking an Endogenous G Protein .alpha.-Subunit
[0051] To prevent interference by the endogenous yeast G protein
.alpha.-subunit, qpal mutant cells (strain 8c) were used.
[0052] Yeast strain 8c (MATa ura3 leu2 his3 trpl gpal::Hl53), I.
Miyajima et al., Cell 50, 1011 (1987), carrying plasmids
pYSK136G.alpha.s (TR1), C. Dietzel and J. Kurjan, Cell 50, 1001
(1987), pMTL9 (LEU2), J. Salmeron et al., Mol. Cell. Biol. 9, 2950
(1989), and pY.beta.AR2 (URA3) was maintained on glucose-free
minimal selective plates containing 3% glycerol, 2% lactic acid, 50
.mu.M CuSO.sub.4 and 3% galactose. Colonies were transferred to
similar plates containing 0.5 mM ascorbic acid and the indicated
adrenergic ligand(s). After 16-20 hours at 30.degree. C., the
colonies were transferred to similar liquid media at a density of
10.sup.6-10.sup.7 cells/ml and examined by phase contrast
microscopy.
[0053] Morphologies of yeast cells cotransformed with pY.beta.AR2,
pMTL9, and pYSK136G.alpha.s were examined after incubation with (A)
no adrenergic agent; (B) 100 .mu.M (-) isoproterenol; (C) 100 .mu.M
(-) isoproterenol and 50 .mu.M (-) alprenolol; and (D) 100 .mu.M
(+) isoproterenol. Results showed that treatment of 8c cells
coexpressing h.beta.AR and rat G.sub.s.alpha. with the
.beta.-adrenergic agonist isoproterenol indeed induced shmoo
formation, and that this effect was blocked by the specific
antagonist alprenolol.
EXAMPLE 6
Coexpression of h.beta.AR and Mammalian G.sub.s.alpha.-Subunit in
Yeast Containing a .beta.-Galactosidase Signal Sequence
[0054] The isoproterenol-induced morphological response of 8c cells
coexpressing h.beta.AR and rat G.sub.s.alpha. suggested that these
components can couple to each other and to downstream components of
the pheromone response pathway in yeast lacking the endogenous G
.alpha.-subunit. To confirm that the pheromone signaling pathway
was activated by h.beta.AR and rat G.sub.s.alpha., agonist
induction of the pheromone-responsive FUSl gene promoter was
measured in a strain of yeast derived from 8c cells (8cl) in which
a FUSl-lacZ gene fusion had been stably integrated into the genome.
See S. Nomoto et al., EMBO J. 9, 691 (1990).
[0055] Strains 8c (FIG. 3, legend) and NNYl9 (MATa ura3 leu2 his3
trpl lys2 FUS1-LacZ::LEU2) were modified by integrative
transformation with YIpFUS102 (LEU2), S. Nomoto et al., supra, and
designated 8cl and NNYl9, respectively. These strains were
transformed with pY.beta.AR2 and pYSK136G.alpha.s and maintained on
minimal selective plates containing glucose and 50 .mu.M
CuSO.sub.4. Colonies were inoculated into minimal selective media
(3% glycerol, 2% lactic acid, 50 .mu.M CuSo.sub.4), grown to early
log phase (OD.sub.600=1.0), and induced for 12 hours by addition of
3% galactose. Cells were washed and resuspended in induction media
(OD.sub.600=5.0) containing 0.5 mM ascorbic acid and the indicated
ligands. After a 4 hour incubation at 30.degree. C., cells were
harvested, resuspended into 1 ml of Z-buffer, see J. Miller,
Experiments in Molecular Genetics (Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., 1972), supplemented with 0.0075% SDS, and
.beta.-galactosidase activities were determined in 3-4 independent
experiments as described previously. See J. Miller, supra.
[0056] FIG. 3 shows a comparison of .beta.-adrenergic agonist
effects on pheromone-inducible gene activity. .alpha.-MF, 10 .mu.M
.alpha.-mating factor; (-) ISO, 50 .mu.M (-) isoproterenol; (-)
ALP, 50 .mu.M (-) alprenolol; (+) ISO, 100 .mu.M (+) isoproterenol.
In 8cl (gpal) cells coexpressing h.beta.AR and rat G.sub.s.alpha.,
a dramatic isoproterenol-stimulated induction of
.beta.-galactosidase activity was observed. Agonist stimulation was
stereoselective and was blocked by addition of a specific
antagonist. Agonist responsiveness was dependent on expression of
both h.beta.AR and rat G.sub.s.alpha., and required a strain in
which the endogenous G protein .alpha.-subunit was disrupted. The
final .beta.-galactosidase activity achieved in response to
isoproterenol in transformed 8cl cells was comparable to that
induced by .alpha.-factor in nontransformed cells that express GPA1
(NNY19), although basal .beta.-galactosidase activity in NNY19
cells was considerably lower than in 8cl cells. Taken together, our
results indicated that coexpression of h.beta.AR and rat
G.sub.s.alpha. was sufficient to place under catecholamine control
key aspects of the mating signal transduction pathway in yeast.
However, the adrenergic agonist did not stimulate mating in either
8c cells or NNY19 cells coexpressing h.beta.AR and rat
G.sub.s.alpha., in agreement with recent observations that yeast
pheromone receptors, in addition to binding pheromones, participate
in other recognition events required for mating. See A. Bender and
G. Sprague, Genetics 121, 463 (1989).
[0057] h.beta.AR stimulates adenylate cyclase in animal cells via
the action of the .alpha.-subunit of its G protein. In contrast,
mating factor receptors in yeast trigger their effector via the
action of the .beta..tau. subunits. M. Whiteway et al., Cell 56,
476 (1989). Our present results indicate that activation of
h.beta.AR in yeast leads to dissociation of mammalian
G.sub.s.alpha. from yeast .beta..tau., and it is the .beta..tau.
subunits that presumably elicit the response.
[0058] The foregoing examples are illustrative of the present
invention, and are not to be construed as limiting thereof. The
invention is defined by the following claims, with equivalents of
the claims to be included therein.
* * * * *