CA2334215A1 - Transformed cell lines which express heterologous g-protein-coupled receptors - Google Patents

Abstract

According to the invention, transformed cell lines, for example of Ustilago maydis, are used for detecting interactions between GPC-receptors (receptors with seven transmembrane sites, which interact with guanyl nucleotide-bindin g, regulatory proteins) or GPC-receptor-controlled signal transmission systems and test substances (ligands, modulators), or for searching for substances which are capable of interacting with receptors or signal transmission syste ms of this type. The transformed cell lines have a GPC-receptor-controlled sign al transduction path with positive feedback and are transformed in such a way that they express a heterologous GPC- receptor. The transformed cell lines also contain a reporter gene, the expression of which can be detected using measuring techniques and which is controlled by a promotor. Said promoter ca n be induced by stimulating a GPC-receptor, and is endogenous. The reporter ge ne is endogenous or heterologous.

Description

TRANSFORMED C~;LL LINES, WHIGH EXPRESS HETEROLOGOUS
G-PROTEIN-COUPLED RECEPTORS
Field of the Invention The invention concerns transformed cell lines in accordance with the generic term of the first, independent claim,, which cell lines express heterologous G-protein-coupled receptors (GPC-receptors) and which cell lines are suitable for detecting interactions between the GPC-receptors or the signal transmission systems controlled by the GPC-receptors with substance, (ligands, modulators), resp., for finding substances, which interact with the receptors or with the signal transmission systems. The invention further concerns vectors for producing the cell lines and the use of these cell lines for detecting 1 o the named interactions and for finding substances acting on the receptors or on the signal transmission systems (screening assays).
Prior Art G-protein-coupled receptors (GPC-receptors) are receptors with seven transmembrane domains, which in conjunction with heterotrimeric guanyl-nucleotide-binding regulatory proteins (G-proteins) form signal transduction systems for the transmission of many extra-cellular signals [H.Ci. Dohlmann, J. Thorner, M. Carom and R. J.
Lefkowitz (1991) Annu. Rev. Biochem., 60, 653-688]. Signal transduction systems of this kind occur in a broad spectrum of organisms, starting with simple fungi and extending right to the human being.
An example of a receptor of this kind is the somatostatin receptor, which represents a prototype of the GPC-receptors in mammalian cells. Somatostatin has far-reaching modulatory effects in the central nervous system and in the peripheral tissue and acts on a range of receptor subtype.
to The signal transmission through a signal transmission system with GPC-receptor and G-protein has the following general characteristics: Heterotrimeric G-proteins work as signal transmitters between a transmembrane receptor molecule (GPC-receptor) and an enzyme designated as an effector, which produces a secondary messenger substance.
Adenylate cyclase, phospholipaseC and ion channels are examples of well investigated effectors in mammalian systems.
G-proteins consist of a guanyl-nucleotide-binding a-subunit, a ~3-subunit and a y-subunit (Ga-, G(3-, Cry-subunit) [M. I. Simon, M. P. Strathmann and N. Gautam (1991) Science, 2 0 252, 802-808] . G-proteins exist in two differing forms, depending on whether GDP or GTP is bound to the a-sulbunit. If GDP is bound, the G protein occurs as a heterotrimeric a/iy-complex. Through the binding of GTP to the G-protein, the a-subunit dissociates and leaves behind a [i~y-complex. Association of a Ga[i~y-complex with an activated GPC-receptor in the cell membrane leads to an increase of the exchange rate of GTP
for bound GDP. In consequence, the dissociation rate of the bound Ga-subunit from the G(i~y-complex increases. The free a-subunit and the G(3~y-complex can transmit signals to cellular effectors of different signal transmission paths.
The GPC-receptors represent important target molecules for therapeutic compounds. The 3 o human genome probably contains about 5000 different GPC-receptor genes, on which the new therapeutic compounds can act as ligands. For the investigation of such interactions between ligands and receptors and also of modulators and corresponding signal transmission systems, screening test systems are utilized, in which in accordance with prior art, biochemical ligand-binding studies, reporter systems in mammalian cells or reporter systems in yeast cells are used.
Yeast cells utilized in test systems of this kind are transformed in such a manner, that they express heterologous GPC-receptors. In the publication WO-92/05244 (US-5739029), such yeast cE;lls are described. They contain a first heterologous DNA-sequence, which expresses a heterologous GPC-receptor, and a second heterologous DNA-sequence, which expresses an a-subunit of a mammalian G-protein.
1 o The endogenous GPC-receptors in yeast cells facilitate identification of different cell types via extra-cellular pfptides, so-called pheromones. The pheromone-activated signal transmission path inducE;s a development programme, which leads to the fusion of haploid a- and a-cells and to the formation of diploid a/a-cells [M. Whiteway and B.
Errede (1993) in: Signal Transduction, Prokaryotic and Simple Eukaryotic Systems, ed.
J. Kurjan and B. L. Taylor, Academic Press, pp. 189-237]. Cells of the cross-breed type a secrete a-factor, which in a-cells binds to the a-factor receptor (Ste2), and cells of the cross-breed type a secrete a-factor, which in a-cells binds to the specific a-factor receptor (Ste3). Both Ste2 as well as Ste3 belong to the family of the GPC-receptors.
After the binding of the pheromone to the corresponding receptor, the pheromone receptor 2 o probably changes its conformation. This leads to the dissociation of the Ga-subunit (Gpal) from the G(3~y-complex (Ste4, StelB) and therefore to the activation of the G
protein. Interestingly - and in contrast to the function of G-proteins in mammalian systems - the Ga-subunit has a negative influence on the signal transmission, while the G(3~y-sub-unit passes on the pheromone signal.
A further, fundamental difference between GPC-receptor-controlled signal transduction in mammals and in yeast consists of the fact, that up until now no effector enzyme has been discovered in yeast: cells, which generates a secondary messenger substance as a reaction to receptor stimulation. Probably the (3'y-complex directly passes the pheromone 3 o signal on to the Ste20-protein kinase, which in turn activates a protein kinase cascade, consisting of Stell, Ste7" Fus3 and Kssl, and the "structure"-protein Ste5 [I.
Herskowitz (1995) Cell, 80, 187-197_x.

Belonging to the cellular consequences of the pheromone stimulation in yeast are the transcriptional induction of a whole range of genes and the arrest of the cell cycle. These pheromone-induced genes encode for products, which are required for the biosynthesis of the pheromones (MFAl, :MFA2, MFal, MFa2, STE6, STE13), for the production of the receptors STE2 and STE;3, for the pheromone signal transmission (GPAl, FUS3), for cell cycle arrest (FART, CLN2, CLN3), for morphological changes and cell fusion (FUSl, FUS2, CHSl) and for pheromone desensitization (SST2, BARD.
Pheromone-controlled transcription is facilitated by the sequence-specific DNA-binding 1 o protein Stel2. The transcription of the STE12 gene is not inducible through pheromone.
It is suspected that the functionally redundant MAP-kinase -homologues Fus3 and Kssl of the pheromone signal transduction path activate the transcription factor Stel2 through specific phosphorylation. Pheromone-inducible genes have cis-acting DNA-sequences in their promotor region, the so-called "Pheromone Response Element" (PRE). The presence of PRE-sequences in the promotor region of a gene in yeast, however, does not suffice for the transcription of this gene to be pheromone-inducible. Thus, e.g., the STEl2-gene has several I'REs, but the expression of STEl2 is not pheromone-inducible.
2 o Basic Features of the Invention The invention sets itself the objective of creating transformed cell lines (human, animal or vegetable as well as also fungal cells), which cells express heterologous GPC-receptors. These transformed cell lines shall be suitable for detecting interactions between 2 s substances (ligands, modulators) and GPC-receptors or GPC-receptor-controlled signal transmission systems, resp., for finding in corresponding screening assays, substances which interact with the receptors or the signal transmission systems. In order to be suitable for this purpose, the transformed cells shall manifest a high sensitivity to interactions of this kind.
This objective is achieved by the transformed cell lines, as they are defined in the claims.
The cell lines in accordance with the invention (human, animal or vegetable as well as also fungal cells) have a signal transmission system with GPC-receptors, which signal transmission system is acaivatable through a ligand and manifests a positive feedback.
The mechanism of the po sitive feedback consists of the fact, that transcription of the gene encoding for the transcription factor which is activated by the GPC-receptor, is itself s inducible through receptor stimulation.
The cell lines in accordance with the invention may have an endogeneous GPC-receptor signal transmission system with positive feedback or a positive feedback mechanism can be built into an existing GPC-receptor signal transmission chain by means of 1 o recombinant DNA-technology. Through the positive feedback mechanism, the natural or correspondingly modified signal transduction system manifests a significantly higher sensitivity in comparison with known, cellular systems, used for detecting the mentioned interactions. This means that cell lines having a positive feedback mechanism react more sensitively to an activation with the GPC-receptors than known cellular systems used for 1 s such detection, irrespective of whether the receptors are the endogenous ones or heterologous receptors introduced through transformation.
The maize blight fungus Ustilago maydis represents an example of a cellular test system for GPC-receptors, in which a positive feedback mechanism exists in the natural 2 o condition in the GPC-receptor-controlled signal transmission chain. This positive feedback in the pheromone-activatable signal transduction for Ustilago maydis is described by H. A. Hartmann, R. Kahmann and M. Bolker [EMBO J., 15, 1632-1641 (1996)]. The basidiomycet Ustilago maydis is utilized as eukaryontic model organism. In its pathogenic form it causes the maize blight on its host plant maize and for this reason 2 s also serves as model system for the study of pathogenic fungi. The genetic constitution of U. maydis can be relatively easily modified [F. Banuette (1995) Annu. Rev.
Genet., 29, 179-208] .
It has to be assumed, that also other cell lines, in particular fugi of the Ustilago family or 3 o the basidiomycous cells in general manifest such GPC-receptor-activatable signal transduction paths with positive feedback and therefore like Ustilago offer themselves for the mentioned test reactions in their natural condition.

_ CA 02334215 2000-12-04 According to the invention, cell lines having in a natural or genetically modified state a GPC-receptor-activatable signal transmission system, in which the GPC-receptor-induced transcription of target genes is amplified by positive feedback are used for detecting interactions between specific GPC-receptors or corresponding signal transmission s systems and test substances. The cells further have an endogenous or heterologous reporter gene, the expression of which is controlled by a promotor being inducible by activation of the receptor, whereby the expression of the reporter gene can be detected and quantified by measuring techniques (e.g., essential growth enzyme causing measurable cell growth, or other enzymes, which in biochemical reactions lead to 1 o measurable effects).
In the cells of the cell lines in accordance with the invention, the heterologous GPC-receptor can associate with endogenous G-protein or with heterologous G-protein, in particular with a heterologous a-subunit of the G-protein. In addition, the cell can contain is a mutation of the gene inhibiting the endogenous a-subunit responsible for the GPC-receptor-controlled signal. transmission and therewith facilitating interaction between the heterologous receptor andl the heterologous G-protein.
2 o Brief Description of the Figures Figure 1 illustrates the comparison between a signal transmission system with positive feedback (on the left), as is present in the cell lines in accordance with the invention (e.g., Ustilago maydis), and a signal transmission system without 2 s positive feedback (on the right), as, for example, is present in yeast cells.
Detailed Description of the Invention s o The term "heterologous" is used in this description in reference to the corresponding cell lines and therefore refers to DNA-sequences, proteins and other materials, which are brought into the corresponding cell line from other organisms, or to combinations, which do not occur in the corresponding cell lines in their natural condition.

_ CA 02334215 2000-12-04 The terms "upstream" and "downstream" are used in the following to refer to the direction of transcription and translation. A sequence, which is transcribed or translated prior to another sequence, is designated as "upstream" of the other sequence.
Methods and materials, with the help of which the cell lines in accordance with the invention are produced, ~~re described in detail for the example Ustilago maydis. This, however, shall in no manner whatsoever restrict the cell lines in accordance with the invention to this species. For other cell lines, the methods and materials are to be 1 o correspondingly applied, which for one skilled in the art is possible without any problem.
For Ustilago maydis in it~~ natural state a signal transmission system enables identification and fusion of compatible cell types (al and a2), in a similar way as described for yeast further up. This signal transmission system is controlled by pheromones and by the s 5 corresponding pheromone receptors [J. Kronstad and C. Staben (1997) Annu.
Rev.
Genet., 31, 245-276]. CE;lls of Ustilago maydis of the cross-breed type al secrete the peptide pheromone Mfal, which in a2-cells binds to the specific Mfal-receptor (Prat).
Cells of the cross-breed type a2 secrete the Mfa2-pheromone, which only acts in al-cells expressing Mfa2-receptor (Pral). The pheromone receptors Pral and Prat are GPC-2 o receptors.
Activation of the receptors Pral and Pra2 by binding the corresponding pheromone probably leads to the dissociation of a hetero-trimeric G-protein, of which up until now only the a-subunit Gpa3 is known [E. Regenfelder, T. Spellig, A. Hartmann, S.
Lauen-25 stein, M. Bolker and R. Kahmann (1997) EMBO J., 16, 1934-1942]. Gpa3 - in contrast to the functional homologue in yeast (Gpal) - has a positive influence on the pheromone signal transmission. Interestingly, the cellular effector of Gpa3 appears to be an adenylcyclase (Uacl), bE;cause mutations in the gpa3-gene on the one hand render the pheromone signal transmission impossible and on the other hand lead to a filament-like 3 o growth, which resembles the growth of adenylcyclase-deficient mutants.
Furthermore, the filament-like growth of gpa3-mutants can be reversed by the addition of cyclic AMP, the secondary messenger substance, which is produced by the adenylcyclase [R.
Kahmann and C. Basse (1997) Trends in Plant Sci., 2, 366-368; S. Gold, G.
Duncan, K.

Barren and J. Kronstad (1994) Genes Dev., 8, 2805-2816]. In consequence of this, the pheromone signal transmission in Ustilago appears to be more similar to the corresponding mechanisms in mammalian systems than is the case for the pheromone-controlled signal transmission in yeast.
In the final instance, pheromone stimulation in Ustilago results in the transcriptional induction of all genes, which are at the hybrid type loci a and b, i.e., mfal, mfa2, pral, prat, lga2, rga2, bE, bV~~ [M. Urban, R. Kahmann and M. Bolker (1996) Mol.
Gen.
Genet., 251, 31-37]. All these genes possess at least one cis-acting DNA-sequence, the "Pheromone Response Element" (PRE), in their associated gene-regulatory regions. The PRE-sequences of Ustila~;o maydis are identified by the sequence-specific DNA-binding protein Prfl. Pheromone stimulation leads to the activation of Prfl, which imparts the pheromone-inducible transcription of these genes. Since the promotor of the prfl-gene also has PREs, the transcription of the prfl-gene is also activated through pheromone stimulation (H. A. Hartmann, R. Kahmann and M. Bolker (1996) EMBO J., 15, 1632-1641). Through the phf;romone-inducible transcription of the prfl-gene, a positive feedback mechanism is inherent to the pheromone signal transmission path of Ustilago.
GPC-receptor-controlled signal transmission systems with positive feedback can be 2 o identified by the fact, that the transcription of the gene, which codes for the transcription factor activated by stimulation of the GPC-receptor and as a result of this controls the GPC-receptor-controlled transcription of target genes, is itself induced by stimulation of the GPC-receptor.
2 5 The mechanism described above is schematically illustrated in Figure 1 on the left. This mechanism leads to a significantly higher sensitivity for detecting e.g.
binding of a ligand to the receptor. As already mentioned above, and as illustrated in Figure 1 on the right as a comparison, the corresponding yeast system and other cellular systems used for such detection in corresponding assays, do not possess a positive feedback mechanism. In the 3 o corresponding mechanism of yeast, expression of the GPC- receptor-activatable transcription factor (Stel:?) is not induced through receptor stimulation (refer to Fig.l).
However, it is conceivable to modify yeast strains utilized for assaying interactions between heterologous receptors and ligands, in such a manner, that they have a positive feedback mechanism. In order to achieve this, the promotor of the STE12-gene, which codes for the transcription factor activated by pheromone stimulation, is replaced by a promotor, which is pheromone-inducible (e.g., FUSl-promotor).
Various Ustilago strains and suitable expression vectors for ustilago transfection are known. Expression vectors are replicatable DNA-constructs, which are utilized to express a heterologous DNA-sequence in a host cell. The heterologous DNA-sequence to be expressed has to be equipped with suitable control sequences capable of controlling 1 o expression of the protein or protein-subunit encoded by the heterologous DNA-sequence in the intended host. Control sequences encompass a transcriptional promotor, optional cis-acting DNA-sequences in order to regulate transcription, suitable DNA-sequences, which impart an efficient initiation of translation, and DNA-sequences, which control termination of transcription and poly-adenylization of the mRNA.
Suitable vectors for the production of cell lines in accordance with the invention encompass plasmids, viruses and DNA-fragments being integratable into the host genome through genetic recombination. Suitable vectors contain control sequences, which come from species, which are functional in the intended expression host.
Ustilago vectors can contain an autonomously replicating sequence (UARS), which renders the plasmid capable of replicating in the Ustilago cell to a great number of copies, a promotor, heterologous DNA-sequences encoding the heterologous proteins to be expressed, sequences for the poly-adenylization and a selectable marker gene.
An example of a plasmid of this kind is pJW42 [J. Wang, D. W. Holden and S. A.
Leong (1988) Proc. Natl. Acad. Sci. USA, 85, 865-869]. This plasmid contains the hph-gene of Escherichia coli [L. Gritz and J. Davies (1983) Genes, 25, 179-188], which imparts resistance against the antibiotic HygromycinB and as a result of this can be utilized as a 3 o selectable marker. Other utilizable marker genes are, for example, the cbx-gene of Ustilago maydis, which. imparts resistance against the fungicide Carboxin [P.L.E.
Broomfield and J.A. Hargreaves (1992) Curr. Genet.,22, 117-121], or the natl-gene of Streptomyces noursei, which imparts resistance against the antibiotic nourseothricine [H.

Kruger, G. Fiedler, C. Srruth and S. Baumberg(1993) Genes, 127, 127-131].
Suitable promotor sequences comprise the promotors of the hsp70-gene [D. W.
Holden, J. W. Kronstad and S. A. Leong (1989) EMBO J., 8, 1927-1934], of the glyceraldehyde-s 3-phosphate-dehydrogen~se gene [T. L. Smith and S. A. Leong (1990) Genes, 93, 111-117] and the translation-elongation-factor gene [H. A. Hartmann, R. Kahmann and M.
Bolker (1996) EMBO J., 15, 1632-1641]. Other promotors with the additional advantage of the transcriptional control through the growth conditions are the promotor of the crgl-gene [A. Bottin, J. Kamper and R. Kahmann (1996) Mol. Gen. Genet., 253, 342-352], 1 o which is induced through arabinose and repressed by glucose, and the promotor of the sidl-gene, which is negatively regulated through the iron concentration in the growth medium [Z. An, B. Mei, W. M. Yuan and S. A. Leong (1997) EMBO J., 16, 1742-1750].
In order to assure polyadenylization and mRNA-termination, it is also possible to ligate the termination sequences into the expression vectors associated with these genes, 15 downstream of the heterologous sequences.
For enabling efficient expression of heterologous GPC-receptors in Ustilago, novel expression vectors were developed. These expression vectors contain Ustilago maydis hsp70-promotor and -terminator, which facilitate transcription of the cDNAs for GPC-2 o receptors. Between the hsp70-promotor and -terminator, additional interfaces for restriction enzymes are introduced, in order to simplify cloning of DNA-sequences to be expressed, e.g., GPCR-cI)NAs.
In order to optimize the intra-cellular localization of the heterologous GPC-receptors to 2 s the plasma membrane in Ustilago, it is also possible to construct expression vectors, which contain a first sel;ment comprising Ustilago-DNA-sequences comprising at least one segment of the sequence encoding the amino-terminal of an Ustilago-GPC-receptor.
DNA-sequences encoding the pheromone receptors of Ustilago (e.g., the pral-gene, which codes for the Mfa2-pheromone receptor, and the prat-gene, which codes for the 3 o Mfal-pheromone receptor) are examples for Ustilago-genes, which code for GPC-receptors and can be utilized for constructing suitable vectors. A second segment, which lies downstream of the mentioned first segment and is in the correct reading frame with it, comprises a DNA-sequence encoding a heterologous GPC-receptor. Such adaptations of the translation initiation point can increase the expression of a heterologous protein.
The first and second segments are operatively associated with a promotor, such as, e.g., the constitutive hsp70-promotor or the inducible crgl-promotor, which are operative in Ustilago cells.
Every GPC-receptor and the corresponding DNA-sequences, which code for such receptors, can be utilized for producing the cell lines in accordance with the invention.
Examples of receptors of this kind are adrenergic receptors (a or ~), adenosine- receptors, angiotensine-receptors, bradykinine-receptors, cannabinoid-receptors, chemokine-1 o receptors, dopamine-receptors, glucagon-receptors, neurokinine-receptors, neurotensine-receptors, serotonin-recE;ptors, opiate-receptors, muscarinic-receptors, somatostatin-receptors and vasopressine-receptors. The term "receptor" used here also includes sub-types as well as their mutants and homologues and also the DNA-sequences, which code for them.
Every Ga-subunit and the corresponding DNA-sequences, which code for these Ga-subunits, can be utilized for producing the cell lines in accordance with the invention.
Examples of Ga-subunits are Gs-subunits, Go-subunits, Gq-subunits, Gi-subunits and Gz-subunits. The term "Ga-subunit" used here includes sub-types as well as their mutants 2 o and homologues and also DNA-sequences, which code for these. The functional expression of heterologous Ga-subunits in Ustilago can easily be verified, because a defect in the Ustilago Ga-subunit Gpa3 leads to a characteristic, visually observable filament-like growth, in contrast to the yeast-like growth form of Ustilago cells with an intact gpa3-gene. Heterologous Ga-subunits, which take over the function of the endogenous Ga-subunit Gpa3 in the pheromone signal transmission chain, complement the filament-like growth defect of the gpa3 mutant cells to normal, yeast-like growth and therefore can be easily identified.
Every Gj3y-subunit and the corresponding DNA-sequences, which code for these G(3y-s o subunits, can be utilized for producing the cell lines in accordance with the invention.
The term "G(3y-subunit" used here includes sub-types as well as their mutants and homologues and also DNA-sequences, which code for these.

In order to detect binding of a ligand to a heterologous GPC-receptor or in general to detect interaction between a modulator and the GPC-receptor-controlled signal transmission system in the cell lines in accordance with the invention, it is particularly appropriate to equip the cells with a third DNA-construct, which encompasses a s promoter and a reporter gene. The promoter is inducible through activation of the heterologous GPC-receptors. The reporter gene is placed downstream of the GPC-receptor-inducible promoter and is operatively associated with it. The expression of the reporter gene can be recorded with measuring technology and reflects the activation of the heterologous GPC-receptor through suitable ligands. In the exemplary Ustilago 1 o maydis, e.g., the promoter of the mfal-gene, the promoter of the mfa2-gene, the promoter of the pral-gene, the promoter of the prat-gene or the promoter of the prfl-gene can be utilized as GPC-receptor-inducible promoters. Various, endogenous or heterologous genes can be used as reporter genes. Examples for reporter genes are the pyr6-gene [J. W. Kronstad, J. Wang, S. F. Covert, D. W. Holden, G. L. McKnight and S.
15 A. Leong (1989) Genes, 79, 97-106], the pyr3-gene [A. Spanos, N. Kanuga, D.
W.
Holden and G. R. Banns (1992) Genes, 117, 73-79], the lacZ-gene, the hph-gene (hygromycine resistance) [L. Gritz and J. Davies (1983) Genes, 25, 179-188], the ble-gene (phleomycine resistance) [D. Drocourt, T. Calmels, J. P. Reynes, M. Baron and G.
Tiraby (1990) Nucl. Acids Res., 18, 4009], a GFP-gene (Green Fluorescent Protein) [T.
a o Spellig, A. Boffin and R. Kahmann (1996) Mol. Gen, Genet., 225, 503-509]
or the uidA
(GUS) gene [R. A. Jefferson, S. M. Burgess and D. Hirsh (1986) Proc. Natl.
Acad. Sci.
USA, 86, 8447-8451].
2 s Example 1:
Production of Ustilago expression vectors (pDT78 and pDT99) For enabling U. maydis to express heterologous GPC-receptors the Ustilago expression vectors pDT78 and pDT99 were constructed.
For the expression vector pDT78, the 3.1 kb HindIII fragment of the autonomously replicating Ustilago vector pCM54 [T. Tsukuda, S. Carleton, S. Fotheringham and W. K.
Holloman (1988) Mol. Celh Biol., 8, 3703-3709] was substituted by a 2 kb HindIII

fragment of the plasmid p:DWHlO [J. Wang, D.W. Holden and S. A. Leong (1988) Proc.
Natl. Acad. Sci. USA, 85, 865-869]. This 2 kb HindIII fragment contains the promotor and the transcription terminator of the U. maydis hsp70 gene, separated by a BgIII
interface. The resulting plasmid pDT48 was cut with SacI and PstI, and a 1.5 kb SacI-s PstI fragment, isolated from the plasmid pNATl (pDT65), was introduced, which contains the hatl gene of Str°eptomyces nouysei, which imparts resistance against the antibiotic nourseothricine of the streptothricine family [H. Krugel, G.
Fiedler, C. Smith and S. Baumberg (1993) Gene, 127, 127-131]. The expression of the natl gene in U.
maydis is controlled through the promotor of the U. maydis glyceraldehyde-3-phosphate z o dehydrogenase (GAPDH) gene [T.L. Smith and S.A. Leong (1990) Genes, 93, 111-117]
and the transcription terminator of the cycl gene of Saccharomyces cerevisiae [D. Dro-court, T. Calmels, J.P. R.eynes, M. Baron and G. Tiraby (1990) Nucleic Acids Res. 18, 4009]. The plasmid pD'T78 resulting from it possesses a single interface for BgIII
between the U. maydis hsp70 promotor and the U. maydis hsp70 terminator. This 15 restriction enzyme interface can be utilized for introducing a DNA-sequence to be expressed in U. maydis, e.g., a cDNA, which codes for a heterologous GPC-receptor.
The transcription of the cDNA, which specifies the heterologous GPC-receptor, is therefore imparted by tlhe transcription control sequences of the U. maydis hsp70 promotor. The pDT78 expression vector and its derivatives can be introduced in Ustilago 2 o maydis by means of known transformation methods [J. Wang, D.W. Holden and S. A.
Leong (1988) Proc. Natl. Acad. Sci. USA, 85, 865-869] and by means of adding the antibiotic nourseothricine (40 ~g/ml) into the growth medium, a selection is made for the presence of this plasmid in U. maydis cells.
2 s In order to simplify the c;loning of DNA-sequences to be expressed in Ustilago, further expression plasmids werE; constructed with the hsp70 promotor, which, however, instead of having only one restri<;tion interface (as e.g. pDT78) between the hsp70 promotor and terminator, have interfaces for several different restriction enzymes. This is illustrated here with the example of pDT99.
pDT49 is identical with the Pdt48 described above, with the exception that the 2 kb HihdIII fragment, which contains the promotor and the transcription terminator of the U.
maydis hsp70 gene, was introduced in the opposite orientation, i.e., the E.
coli lacZ

promotor of the plasmid and the introduced hsp~0 promotor impart the transcription in the opposite direction. In order to eliminate the SmaI, BamHI, XbaI, SaII and PstI
interfaces of pDT49, pDT49 was digested with SmaI and with PstI, the 3'-overhang of the PstI interface was removed with T4 DNA polymerase and the plasmid was relegated.
s Into the BgIII interface between the hsp70 promotor and terminator of the resulting plasmid pDT85 a double-stranded oligonucleotide, which contains interfaces for the restriction enzymes KpnI, EcoRI, NotI, NcoI, MZuI, StuI, SphI, BamHI and SacII
(in this sequence), was legated in such a manner, that the SacII interface came to lie closer to the hsp70 promotor than the KpnI interface. In the resulting plasmid pDT90 a 2.3 kb SacI
to fragment of the plasmid fjahCbx8 was introduced into the SacI interface, which plasmid contains a gene, which in U. maydis imparts resistance against the fungicide Carboxin [P.L.E. Broomfield and J.A. Hargreaves (1992) Curr. Genet., 22, 117-121].
pDT99 can be introduced into U. maydis cells by means of transformation [J. Wang, D.W.
Holden and S. A. Leong (1988) Proc. Natl. Acad. Sci. USA, 85, 865-869] and by means of s adding the fungicide Carboxin (2 ~g/ml) into the growth medium a selection is made for the presence of this plasmid in U. maydis cells.
Example 2:
2 o Expression of the human ~2-adrenergenic receptor into U ~ydis (pDT94) In order to express the human (32-adrenergenic receptor ((32-AR) in U. maydis, approx.
0.1 ~g DNA of the plasmid pTF3 [B.K. Kobilka, R.A.F. Dixon, T. Frielle, H.G.
Dohlman, M.A. Bolanowski, LS. Sigal, T.L. Yang-Feng, U. Francke, M.G. Carom R.J.
2 s Lefkowitz (1987) Proc. Natl. Acad. Sci. USA, 84, 46-50], which contains a cDNA of the human (32-adrenergenic receptor, was amplified with the primers 5'-CGGGATCCACAATGACCCAACCCGGCAACGGCAGCG-3' and 5'-CGGGATCCTCAGAGCAGCGAGTCATTTGTGCTACA-3' (wherein A = adenosine;
C = cytosine; G = guanine; T = thymidine) by means of the polymerase chain reaction 3 0 (PCR). In comparison with the human DNA sequence of the [32-adrenergenic receptor, in particular the environment of the translation initiation ATG codon was changed in such a manner, that it corresponds to the corresponding consensus sequence for filamentous fungi [D. J. Ballance (1991) in Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi; S.A: Leong and R.M. Berka (eds.), Dekker, New York, pp 1-29]. The resulting 1.2 kb PCR product was digested with BamHI and cloned in the BamHI interfacf: of the BamHI linearized and dephosphorylated vector pBLUESCRIPTII KS+ (;itratagene Inc.). The DNA sequence of the 1.2 kb BamHI b2-AR PCR products such cloned was verified. The 1.2 kb BamHI fragment of the resulting plamid pDT87 was then introduced into the BgIII interface of the expression vector pDT78 in such a manner, that the (32-AR sequence is transcribed in the correct orientation of the hsp70 hromotor. The resulting ~i2-AR expression plasmid pDT94 can now be introduced into LI, maydis cells by means of known transformation methods [J.
Zo Wang, D.W. Holden and S. A. Leong (1988) Proc. Natl. Acad. Sci. USA, 85, 865-869].
The biochemical proof, that the human (32-AR is expressed in U. maydis, can be supplied by means of ligand binding studies. Such ligand binding studies can be carried out on membrane fractions of U. maydis cells transformed with pDT94 using, e.g., the is radioactive marked [i2,-AR-ligand 3-[1~I]iodocyanopindolol in accordance with published protocols [H.)K. Dohlman, M.G. Carom A. DeBlasi, T. Frielle and R.J.
Lefkowitz (1990) Biochemistry, 29, 2335-2342].
The proof, that the human (32-AR expressed in U maydis is functional and interacts with 2 o the pheromone signal transmission chain of U. maydis, can be provided by introducing a pheromone-inducible reporter gene into the U. maydis cells transformed with pDT94.
The plasmid pMLTl contains the bacterial uidA gene, which codes for the enzyme ~i-glucuronidase (GUS) [R. A. Jefferson, S. M. Burgess and D. Hirsh (1986) Proc.
Natl.
Acad. Sci. USA, 86, 8447-8451] and its expression is regulated through the strongly 2 s pheromone-inducible promotor of the mfal gene. [M. Urban, R. Kahmann and M.
Bolker (1996) Mol. Gen. Genet., 251, 31-37]. In consequence of this, the binding of a [32- .AR agonist to the X32-AR expressed in U maydis can be proven by stimulating U.
maydis transform~ds, vvhich contain pDT94 and pMUl, with e.g., the (3-adrenergenic agonist isoproterenol. Tlhe stimulation of the receptor can be proven through a simple 3o biochemical test for GLfS-activity, as described, e.g., in A. Gururaj Rao and P. Flynn (1992) in GUS Protocol;.; S.R. Gallagher (ed.), Academic Press Inc., 89-99.

Claims (15)

1. Transformed fungal cells for detecting interactions with GPC-receptors or with a GPC-receptor-controlled signal transmission system, wherein the transformed cells express a heterologous GPC-receptor as well as a promotor inducible through stimulation of the heterologous GPC-receptor and a reporter gene controlled by the promotor, characterized in that the GPC-receptor-controlled signal transmission system of the fungal cells comprises an endogenous positive feedback mechanism or a positive feedback mechanism created by means of genetic engineering.

2. Transformed fungal cells in accordance with claim 1, characterized in that the fungal cells have the GPC-receptor-controlled signal transmission system with a positive feedback mechanism in their natural condition.

3. Transformed fungal cells in accordance with claim 2, characterized in that they are cells of Ustilago maydis.

4. Transformed fungal cells in accordance with claim 1, characterized in that the positive feedback mechanism in the GPC-receptor-controlled signal transmission system is created by means of genetic re-combination.

5. Transformed fungal cells in accordance with one of claims 1 to 4, characterized in that the heterologous GPC-receptor is an .alpha.-adrenergic receptor, a .beta.-adrenergic receptor, an adenosine receptor, a bradykinine receptor, a cannabinoid receptor, a chemokine receptor, a dopamine receptor, a glucagon receptor, a neurokinine receptor, a neurotensine receptor, a serotonin receptor, an opiate receptor, a muscarinic receptor, a somatostatin receptor or a vasopressine receptor.

6. Transformed fungal cells in accordance with one of claims 1 to 5, characterized in that the cells in addition express heterologous subunits of G-proteins.

7. Transformed fungal cells in accordance with claim 6, characterized in that the cells express heterologous G.alpha.-subunits, which are Gs-, Go-, Gq-, Gi- or Gz-subunits.

8. Transformed fungal cells in accordance with claim 6, characterized in that the cells express heterologous G.beta..gamma.-subunits.

9. Transformed fungal cells in accordance with claim 3, characterized in that the promotor inducible through stimulation of the heterologous GPC-receptor is the promotor of the Ustilago maydis gene mfa1, mfa2, pra1, pra2 or prf1.

10. Transformed fungal cells in accordance with one of claims 1 to 9, characterized in that the reporter gene controlled through the promotor is the pyr6-gene, the pyr3-gene, the lacZ-gene, the hph-gene, the ble-gene, a GFP-gene or the uidA-gene.

11. Transformed fungal cells in accordance with claim 3, characterized in that the reporter gene controlled through the promotor is the pyr6-gene of Ustilago maydis or the pyr3-gene of Ustilago maydis.

12. Use of the transformed fungal cells in accordance with one of the claims 1 to 11 for detecting interactions of test substances with the heterologous GPC-receptor or with the GPC-receptor-controlled signal transmission system, wherein the test substance and the fungal cells are brought into interaction and the expression of the reporter gene is recorded by means of measuring technology.

13. Use in accordance with claim 12, characterized in that the reporter gene expresses an essential growth enzyme and that the cell growth is recorded by means of a turbidity measurement.

14. Use in accordance with claim 12, characterized in that the expression of the reporter gene is subjected to a biochemical reaction and the reaction product is recorded by means of measuring technology.

15. Use in accordance with claim 14, characterized in that the reporter gene is the uidA-gene and codes for .beta.-glucuronidase and that the expressed .beta.-glucuronidase is recorded with a suitable biochemical test.