Promotor for functional characterization of G-protein...

Details Promotor for functional characterization of G-protein... Promotor for functional characterization of G-protein...

2001-11-07

2003-08-05

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S320100, C536S024100

Reexamination Certificate

active

06602699

ABSTRACT:

The present invention relates to a promoter, to DNA fragments containing said promoter and to the use thereof, in particular in methods for identifying substances exhibiting an activating or inhibiting action on G protein-coupled receptors.
GPCRs (G protein-coupled receptors) form a gene family of structurally and functionally linked transmembrane proteins. GPCRs are target molecules of great importance for medical research and the development of pharmacological active substances and occupy a key position in a multiplicity of pathologies (Stadel et al., 1997). The classic example of the central position of GPCRs in the pharmacological treatment of asthma is &bgr;
2
AR (&bgr;
2
-adrenergic receptor) or the involvement of a Kaposi sarcoma (KS)-associated herpesvirus GPCR in KS pathogenesis (Geras-Raaka et al., 1998). This class of receptors bind a broad spectrum of ligands such as, for example, protein hormones, chemokines, peptides or divalent cations. The identification of activators and inhibitors of G protein-coupled receptors is thus one of the most promising approaches toward a better knowledge and treatment of diseases (Wilson et al., 1998).
Many components of the GPCR signal transduction pathway have orthologous components in the GPCR signal transduction pathway of bakers' yeast,
Saccharomyces cerevisiae.
Stimulation with mating factor (pheromone &agr; or pheromone a) activates, for example, the pheromone-dependent mitogen-activated protein kinase cascade (denoted MAPK cascade hereinbelow) (Frederickson, 1999). This property of yeast can be utilized for assaying mammalian GPCRs, in particular human GPCRs, in a yeast model, in order to search for activators or inhibitors of the relevant GPCR or the corresponding signal transduction pathway, in particular since GPCRs of foreign species can be functionally expressed in yeast, resulting via the yeast MAPK cascade in a measurable cellular response when the introduced GPCR is activated by a specific ligand and this signal is passed on to the yeast MAPK cascade. The search for activators or inhibitors of GPCRs to which, up until now, no functions have been ascribed, the “orphan GPCRs”, is of particular interest.
The pheromones &agr; and a act in
S. cerevisiae
via the endogenous G protein-coupled receptors Ste2p und Ste3p (Gustin et al., 1998). In this connection, pheromone &agr; acts directly on the pheromone-dependent MAPK cascade, thereby regulating the expression of particular
S. cerevisiae
genes. Whether a substance acts in an activating or inhibiting way on a GPCR heterologously expressed in
S. cerevisiae
, may be detected by expressing the
S. cerevisiae
genes which are regulated by the MAPK cascade (marker genes). The promoters of such marker genes may be used, for example in connection with a suitable reporter system, for identifying GPCR activators or inhibitors.
Pheromone-regulated genes may be identified either by using transposon mutagenesis (Ross-Macdonald et al., 1999) or by using DNA microchips, which allows expression analysis of all mRNAs of a cell at a particular time (Wodicka et al., 1997).
Up until now, functional assays for identifying inhibitors or activators of GPCRs have been carried out mainly in mammalian cells (Wilson et al. (1998) British Journal of Pharmacology 125, 1387-1392; Geras-Raaka et al. (1998) J. Exp. Med. 188 No.2, 405-408). The use of promoters of the FUS1 and FUS2 genes for functional assays in
S. cerevisiae
has been described (Cisnowski et al. (1999) Nature 17, 878-883; Frederickson (1999) Nature Biotechnology A, 852-853). FUS1 is a gene whose expression in wild-type cells of the yeast
Saccharomyces cerevisae
is increased, after activation with pheromone &agr;. In U.S. Pat. No. 5,063,153, FUS1 and FUS2 promoters are used in order to express in large amounts a structural gene which codes for a protein of interest.
It was an object of the present invention to identify a different
S. cerevisiae
promoter which can be activated by pheromone &agr; and which can be used to achieve strong expression of the regulated gene.
The invention relates to the promoter of the
S. cerevisiae
YNL279w gene. The invention relates to the promoter having the sequence SEQ ID NO. 4.
The invention also relates to a recombinant DNA fragment containing the
Saccharomyces cerevisiae
YNL 279w gene promoter which, when functionally linked to a structural gene, regulates transcription thereof and to a recombinant DNA fragment containing a promoter having the DNA sequence nucleotides 1 to 505 of SEQ ID NO:4. The invention relates to a recombinant DNA fragment which contains the YNL 279w promoter and a structural gene and in which the promoter is functionally linked to the structural gene. The structural gene codes, for example, for a receptor gene or for a protein which is intended to be produced in large quantities in
S. cerevisiae.
The invention relates to a DNA vector and a recombinant
S. cerevisiae
cell, which contain such a recombinant DNA fragment. Such a recombinant
S. cerevisiae
cell preferably expresses no or only small quantities of functionally active endogenous receptors Ste2p and Ste3p.
The invention also relates to methods for functional characterization of GPCRs and for screening of GPCR inhibitors and/or activators and to methods for preparing proteins in
S. cerevisiae.
For example, methods for identifying activators and/or inhibitors of G protein-coupled receptors, wherein
a) a recombinant
S. cerevisiae
cell is produced, which contains a reporter gene under the control of the promoter of the YNL 279 w gene and which expresses a heterologous G protein-coupled receptor;
b) the cell is incubated with a substance to be studied, and
c) the change in reporter gene transcription is determined.
Methods for identifying constitutively active mutants of G protein-coupled receptors, wherein
a) a recombinant
S. cerevisiae
cell is produced, which contains one or more reporter genes under the control of the promoter of the YNL279w gene
b) and which expresses a mutated heterologous G protein-coupled receptor, the modification resulting in a constitutively active G protein-coupled receptor,
c) the cell is incubated with an activating or inhibiting substance, and
d) the change in reporter gene transcription is determined.
In the recombinant
S. cerevisiae
cell, the appropriate endogenous GPCRs, Ste2p and/or Ste3p, are preferably not expressed, for example because these genes have been deleted. This deletion may be carried out as described, for example, in Broach Thorner (1996) Nature, 384, 14-16.
Another embodiment relates to methods for preparing heterologous proteins in
S. cerevisiae,
wherein the structural gene of the heterologous protein to be expressed is functionally linked to the promoter of the YNL279w gene and is expressed under the control thereof.


REFERENCES:
patent: 5063154 (1991-11-01), Fink et al.
patent: WO 99/51620 (1999-10-01), None
patent: WO 00/12704 (2000-03-01), None
patent: WO 01/16378 (2001-03-01), None
Pausch M H., G-Protein-Coupled Receiptors inSacchromyces cerevisiae: High-Throughput Screening Assays for Drug Discovery;Trends In Biotechnology, Elsevier Publications, Cambridge, GB, Dec. 1, 1997; vol. 15, pp. 487-494.
XP-002217914—May 6, 1996, Retrieved from EBI Database Accession No. Z71555.
Mary J. Cismowski et al., “Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling”, Nature Biotechnology, Sep. 1999, pp. 878-883, vol. 17.
Scott Erdman et al., “Pheromone-regulated Genes Required for Yeast Mating Differentiation”, J. Cell Biol., pp. 461-83, vol. 140.
Robert M. Frederickson, “Budding actors in mammalian G-protein signaling”, Nature Biotechnology, Sep. 1999, pp. 852-853, vol. 17.
Elizabeth Geras-Raaka et al., “Human Interferon-y-inducible Protein 10 (IP-10) Inhibits Constitutive Signaling of Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor”, J. Exp. Med., Jul. 20, 1998, pp. 405-408, vol. 188, No. 2.
Michael C. Gustin et al., “MAP Kinase Pathways in the YeastSaccharomyces cerevisiae”, Microbiology

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