A HYBRID CELLULAR RECEPTOR
FIELD OF INVENTION
The present invention relates to a DNA construct which is hybrid of DNA sequences coding for different cellular recep tors, a hybrid receptor encoded by the DNA construct, an methods of identifying ligand-binding sites on cellula receptors and screening for ligand candidates by means of th hybrid receptor.
BACKGROUND OF THE INVENTION
In multicellular organisms, certain important cell to cel interactions involving the transmission of extracellula signals are mediated by the interaction of ligands wit cellular receptors which are typically located on cell sur faces. Such receptors are composed of an extracellular domai (including monomeric and dimeric forms thereof) which is capable of specifically recognizing and binding a particula ligand and which may have highly glycosylated and protease- resistant structure, a transmembrane domain which is respon¬ sible for anchoring the receptor in the cell membrane and which consists of a hydrophobic sequence of some 25 amino acids, and a cytoplasmic domain which is responsible for generating a cellular signal as a response to the binding of the ligand to the extracellular domain; the cytoplasmic domain may define an enzymatic activity which is triggered by ligand binding.
Thus, when a ligand (for instance a hormone or growth factor) binds to a receptor present on the cell surface, this may lead to receptor aggregation in coated pits or to conformational changes in the receptor which in turn trigger secondary cellular responses, such as the development of an enzymatic activity, that result in the stimulation or inhibition of intracellular processes. It is generally recognized that
irregularities in ligand-receptor interactions are a contri¬ buting cause of a variety of diseases and abnormal conditions.
Ligands may be grouped in two classes; agonists which stimulate receptor activity and antagonists which inhibit the effects elicited by the agonists, e.g. by inhibiting ligand binding to the receptor. The development of agonists which, although similar in function to the native ligand, differ from it in structure or composition is potentially of considerable medical importance. Agonists which are smaller than the native ligand molecule are envisaged to be particularly useful for a variety of reasons. They may have an increased bioavailability due to their smaller size which may be important for drug formulation, for instance when the agonists are intended for topical application. Agonists may also exhibit slightly different biological activities and/or different potencies which may be useful for treating specific conditions. Agonists which are smaller or which have a simpler chemical structure than the native ligand may be simpler and therefore less expensive to produce and may furthermore be formulated into more convenient dosage forms (e.g. to be taken orally) . The identification of antagonists which specifically block, for instance, growth factor receptors is also of great potential utility. Antago¬ nists which block receptors against the action of the native ligand may be used as therapeutic agents for conditions including arteriosclerosis, tumours, fibroplasia and keloid formation.
The development of new ligands which are useful for therapeutic purposes has mostly consisted in designing new compounds by chemical modification, synthesis or screening for ligand candidates by complicated and expensive screening procedures. The process of designing a new ligand typically begins with changing the structure of the native ligand. If this is a reasonably simple molecule such as a prostaglandin, this is not an unduly difficult procedure. It is, however, considerably more difficult to develop a molecule which is functionally
equivalent to the native ligand when this is a complex sub stance such as a peptide hormone or a growth factor.
Ligand candidates are currently screened by radioligand bindin methods (cf. Lefkowitz et al., Bioche . Biophys. Res. Commun. 560, 1974, pp. 703-709; Aurbach et al., Science 186, 1974, pp. 1223-1225; Atlas et al., Proc. Natl. Acad. Sci. USA 7_1, 1974, pp. 4246-4248) . Ligand candidates may be screened directly b binding the radiolabeled compounds to responsive cells, to the membrane fraction of disrupted cells or to solubilized recep- 0 tors. Alternatively, ligand candidates may be screened by their ability to compete with a known labeled ligand for cell surface receptors.
These known procedures suffer from the drawback that the membrane fractions or solubilized receptors may not be readily available. Similarly, responsive cell lines have to be iso¬ lated. Membrane fractions have to be isolated by gentle procedures which are not commercially viable. It is very difficult to maintain a high level of biological activity and biochemical purity of receptors when these are purified by conventional protein purification methods. As receptors are proteins which are anchored in cell membranes, they require cumbersome purification procedures, including the use of detergents and other solvents which interfere with their biological activity. It is seldom possible to obtain the release of soluble and active receptors by means of enzymatic treatment of cells. The use of membrane preparations for ligand screening typically results in a low reproducibility due to the variability of the membrane preparations.
A number of naturally occurring receptors have previously been identified. Thus, Rubin et al., J. Im un. 135. 1985, pp. 3172- 3177, describe the release of large quantities of the inter- leukin-2 receptor (IL-2-R) into the culture medium of activated T-cells. Bailon et al., Bio/Technology 5. 1987, pp. 1195-1198,
describe the use of a matrix-bound IL-2-R to purify recombinant interleukin-2.
It has also been suggested to produce cellular receptors by recombinant DNA techniques. Thus, the insulin receptor (Ellis et al., J. Cell Biol. 150. 1987, p. 14a), the HIV-1 envelope glycoprotein receptor CD4 (Smith et al.. Science 238, 1987, pp. 1704-1707) and the epidermal growth factor receptor (Livneh et al., J. Biol. Chem 261. 1986, pp. 12490-12497) have been secreted from mammalian cells using truncated cDNAs encoding portions of the extracellular domains.
European Patent Application, Publication No. 244 221 discloses receptors which are hybrids between the extracellular ligand- binding domain of one receptor fused to a heterologous reporter polypeptide which may be the cytoplasmic domain of another receptor. The contemplated use of the hybrid receptor is to screen ligand candidates for binding to the receptor.
B. Kobilka et al. Science 240. 3 June 1988, pp. 1310-1316, disclose chimeras between α2 and β2 adrenergic receptors constructed by replacing DNA sequences encoding various parts of the membrane-spanning domain of one of the receptors by the DNA sequences encoding corresponding parts of the other receptor and expressing the sequences in Xenopus laevis oocytes. The purpose of these constructions is to determine which of the hydrophobic domains confers ligand binding. The receptors disclosed by B. Kobilka et al. belong to the class of G-protein coupled membrane-spanning receptors, the major proportion of which is found in the lipid bilayer of the cell membrane in the form of α-helices, and they therefore differ considerably from the receptors of current interest in their molecular organisation and the manner in which ligand binding and subsequent signal generation and transmission takes place.
I. Lax et al. , The EMBO J. 8.(2), 1989, pp. 421-427, disclose chimeras between chicken and human EGF receptors constructed by
replacing DNA sequences encoding various parts of one of th receptors by DNA sequences encoding the corresponding parts o the other receptor, and expressed on the surface of mammalia cells. One such chimeric receptor containing a specifi sequence of the human receptor inserted in the chicken recepto was isolated. This chimera had binding properties which wer similar to those of the native human EGF receptor. Based o these results, the authors conclude that this particula sequence contains a ligand-binding domain.
An object of the present invention is to provide hybri receptor polypeptides which are modified in their extracellula domains in such a way that the identification of the ligand binding site or sites is thereby facilitated.
Another object of the invention is to provide a method o identifying ligand-binding sites.
A further object of the invention is to provide methods o using the hybrid polypeptides to screen for substances whic bind to the hybrid polypeptides and to develop ligand sub stances with desirable binding properties.
SUMMARY OF THE INVENTION
The present invention relates to a hybrid DNA construct which comprises a first DNA sequence encoding part of the extra¬ cellular domain of a first cellular receptor and a second DNA sequence encoding part of the extracellular domain of a second cellular receptor which is specific for a different ligand than the first cellular receptor. The invention further relates to a hybrid polypeptide encoded by the DNA construct and com¬ prising part of the extracellular domain of a first cellular receptor and part of the extracellular domain of a second cellular receptor which is specific for a different ligand than the first cellular receptor.
In the present context, the expression "specific for a dif¬ ferent ligand" is intended to indicate that the second receptor specifically binds a ligand which, in a given organism, has a different biological function and/or activity than the ligand binding to the first receptor. The expression is intended to distinguish the present hybrid receptors from those described by, for instance, I. Lax et al., op. cit. , which are hybrids of receptors specific for essentially the same ligand derived from two different organisms.
The hybrid polypeptide of the invention is useful in a method of identifying the ligand-binding site on a cellular receptor, wherein the hybrid polypeptide is separately incubated with a ligand specific for the first cellular receptor and with a ligand specific for the second cellular receptor, and any binding between the polypeptide and either ligand is detected, thereby indicating the sequence of either one of the native receptors within which the ligand-binding site is located. In the present context, the term "ligand-binding site" is intended to indicate a region within the extracellular domain which exhibits ligand-binding properties.
In the method of the invention of identifying the ligand- binding site of the first or second cellular receptor, it may often be an advantage to produce the DNA construct by initially combining one or more exons of the first DNA sequence with one or more exons of the second DNA sequence. Once a hybrid polypeptide has been produced which is shown to be capable of binding either the ligand specific for the first receptor or the ligand specific for the second receptor with a higher affinity and which therefore contains the sequence of the native receptor containing the ligand-binding site, the location thereof may be more exactly identified by repeating the method of the invention with a series of hybrid receptor polypeptides which contain gradually shorter fragments of the appropriate native receptor, selecting for polypeptides which exhibit the required ligand-binding properties.
The present invention further relates to a polypeptide fragment of the extracellular domain of a cellular receptor, which fragment comprises an amino acid sequence conferring ligand specificity to said receptor. This polypeptide fragment may be identified by the method indicated above. The term "amino acid sequence conferring ligand specificity" is understoood to mean that the sequence has a structure which specifically recognizes a certain ligand resulting in high-affinity binding of that particular ligand by the receptor. The receptor fragment may also contain other parts of the ligand-binding site.
The hybrid receptor polypeptide of the invention or the shorter polypeptide fragment comprising the ligand-binding site of one of the receptors making up the hybrid may be used in a method of screening for a ligand or a functional equivalent thereof, or for a ligand antagonist. This method comprises
(a) incubating (i) a hybrid polypeptide comprising part of the extracellular domain of a first cellular receptor and part of the extracellular domain of a second cellular receptor on which a ligand-binding site has been identified according to the method indicated above, or (ii) a polypeptide fragment of the extracellular domain of a cellular receptor, which fragment comprises an amino acid sequence conferring ligand specificity to said receptor, with a sample suspected of containing a ligand or a functional equivalent thereof capable of binding to said polypeptide or suspected of containing a ligand antago¬ nist, and
(b) identifying any ligand or functional equivalent thereof by detecting any binding to said ligand-binding site or detecting any ligand antagonist by detecting any inhibition of binding to said ligand-binding site.
In the present context, the term "ligand" may be defined as a substance which, in nature, is capable of binding to a particu-
lar cellular receptor. Preferred ligands are those which act in a similar way as the natural ligand for the receptor in question (e.g. a hormone, growth factor, cytokine or cell adhesion molecule) . The term "functional equivalent" is intended to include any substance which is similarly capable of binding to that cellular receptor. The functional equivalent may be a natural substance, a modified natural substance (e.g. modified by mutagenesis or chemically modified) or may be chemically synthesized. The term "ligand antagonist" is intended to indicate a substance which is capable of inhibiting the binding of the ligand to the receptor and/or its biological function(s) .
The hybrid polypeptide of the invention on which a ligand- binding site has been identified according to the method indicated above or the polypeptide fragment comprising an amino acid sequence conferring ligand specificity to the receptor may further be used for establishing the three-dimensional structure of the ligand-binding site or a part thereof con¬ ferring ligand specificity and designing a ligand analogue or a functional equivalent thereof with a three-dimensional structure which is substantially complementary to the three- dimensional structure of said ligand-binding site or a part thereof conferring ligand specificity.
In the present context, the term "ligand analogue" is intended to indicate a polypeptide which is derived from the native ligand by the addition of one or more amino acids at either of or both the C-terminal or N-terminal end of the native ligand, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at one or more sites in the amino acid sequence or insertion of one or more amino acids at one or several sites in the native sequence. The analogue has retained the ability of the native ligand to bind to the receptor.
DETAILED DISCLOSURE OF THE INVENTION
In particular, the DNA construct of the present invention i one in which the first DNA sequence (encoding part of th extracellular domain of the first receptor) encodes an exon o a fragment thereof, or in which the second DNA sequenc (encoding part of the extracellular domain of the secon receptor) encodes an exon or a fragment thereof. In order to b useful for the present purposes, it is preferred that eithe the first or the second DNA sequence encodes a ligand-bindin site of the first or the second receptor.
In a favoured embodiment of the invention, the hybrid polypep tide is in soluble form. In order to provide the polypeptide i soluble form, the DNA construct encoding it is truncated in o substantially deleted of DNA sequences encoding the trans- membrane and cytoplasmic domains of either of the parent receptors. This facilitates the production of the hybrid receptor polypeptide since it will be secreted from cells containing said DNA construct and may readily be isolated from the culture medium of said cells rather than by the more cumbersome process of extraction from cell membranes.
Furthermore, a higher purity of the resulting polypeptide is more easily achieved when it is in soluble form which means that assays for ligand binding will tend to be more reliable as they are far less likely to be adversely affected by unspecific binding of the ligand to be tested to other cell surface proteins. Also, soluble polypeptides may more readily be used for ligand screening in existing standard assays. This implies that the results obtained in such assays will tend to be more consistent and more easily readable than assays based on the use of cells carrying the hybrid receptors on their surface. Apart from being used to facilitate screening procedures, the soluble hybrid polypeptides may also be used for preparing crystals which are suitable for X-ray crystallographic ana¬ lyses.
In a particularly preferred embodiment of the present in¬ vention, the DNA construct is one in which the DNA sequences coding for the first and second receptors exhibit a high degree of identity/homology in the organization and/or structure of the exons encoded by the sequences. This is advantageous as it is possible to systematically substitute specific fragments from one receptor for the corresponding fragments from the other receptor. Working from the hypothesis that the ligand- binding sites of such receptors may be located in approximately the same region of the extracellular domain, it is reasonable to assume that- binding of one of the ligands not only indicates the presence of a ligand-binding site in the region of the extracellular domain derived from one of the receptors, but also suggests the presence of a ligand-binding site in the corresponding region of the extracellular domain of the other receptor.
In this embodiment of the DNA construct of the invention, a DNA sequence encoding one or more exons, or a fragment thereof, of the extracellular domain of the first receptor may be replaced by a DNA sequence encoding the corresponding exon or exons, or fragment thereof, of the extracellular domain of the second receptor. Alternatively, a DNA sequence encoding one or more exons, or a fragment thereof, of the extracellular domain of the second receptor may be replaced by a DNA sequence encoding the corresponding exon or exons, or fragment thereof, of the extracellular domain of the first receptor.
As indicated above, the parent receptors are cell surface receptors, in particular receptors for hormones, growth factors, cytokines or cell adhesion molecules. Thus, the receptors may be selected from the group consisting of the insulin, insulin-like growth factor (IGF) , platelet-derived growth factor (PDGF) , epidermal growth factor (EGF) , trans¬ forming growth factor (TGF) , including TGF-α and TGF-β, growth hormone and prolactin receptors.
In the above-described preferred embodiment of the DNA con¬ struct of the invention, hybrids are constructed from receptors which exhibit the aforementioned homology in the overall organization of their DNA.
Consequently, when the first receptor is the insulin receptor, the second receptor may suitably be the IGF receptor or, conversely, the first receptor may be the IGF receptor and the second receptor may be the insulin receptor, the insulin and IGF receptors having been found to exhibit a high degree of identity/homology in their DNA seqijence and overall organiza¬ tion. More specifically, in the DNA construct of the invention, the DNA sequence coding for exon 2, or a fragment thereof, of the insulin receptor may be replaced by the DNA sequence coding for exon 2, or a fragment thereof, of the IGF receptor. Alternatively, the DNA sequence coding for exon 3, or a fragment thereof, of the insulin receptor may be replaced by the DNA sequence coding for exon 3, or a fragment thereof, of the IGF receptor. In a further alternative embodiment, the DNA sequence coding for exons 2 and 3, or a fragment thereof, of the insulin receptor may be replaced by the DNA sequence coding for exons 2 and 3, or a fragment thereof, of the IGF receptor.
In another embodiment of the DNA construct of the invention, the DNA sequence coding for exon 2, or a fragment thereof, of the IGF receptor may be replaced by the DNA sequence coding for exon 2, or a fragment thereof, of the insulin receptor. Alternatively, the DNA sequence coding for exon 3, or a fragment thereof, of the IGF receptor may be replaced by the DNA sequence coding for exon 3, or a fragment thereof, of the insulin receptor. As a further alternative, the DNA sequence coding for exons 2 and 3, or a fragment thereof, of the IGF receptor may be replaced by the DNA sequence coding for exons 2 and 3, or a fragment thereof, of the insulin receptor.
Similarly, in the DNA construct of the invention, a DNA sequence coding for exon 1 (preferably combined with exon 2 or a fragment thereof) or one or more of exons 4-11 or fragments thereof of the insulin receptor may be replaced by the corre- sponding DNA sequence from the IGF receptor, or vice versa.
An example of a useful DNA construct of the invention is one which has the DNA sequence shown in Fig. 5 or Fig. 6, or a suitable modification thereof. Suitable modifications of the DNA sequence may comprise nucleotide substitutions which do not cive rise to another amino acid sequence of the hybrid polypeptide, but which facilitate the production of the polypeptide, or nucleotide substitutions which do give rise to a different amino acid sequence of the hybrid polypeptide. Other possible modifications may be insertion of one or more nucleotides into the sequence, addition of one or more nucleo¬ tides at either end of the sequence and deletion of one or more nucleotides at either end of or within the sequence.
When the first receptor is the PDGF receptor, the second receptor may suitably be the EGF receptor or vice versa. When the first receptor is the EGF receptor, the second receptor may also be the TGF (α or β) receptor, and vice versa. When the first receptor is the growth hormone receptor, the second receptor may suitably be the prolactin receptor or vice versa.
The DNA construct of the invention encoding the hybrid receptor polypeptide of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3, 1984, pp. 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
13
The DNA construct of the invention may also be of geno ic o cDNA origin, for instance obtained by preparing a genomic o cDNA library and screening for DNA sequences coding for all o part of the polypeptide of the invention by hybridization usin synthetic oligonucleotide probes in accordance with standar techniques (cf. Sambrook et al., Molecular Cloning: A Labora tory Manual. 2nd Ed., Cold Spring Harbor, 1989). In this case a genomic or cDNA sequence encoding part of the extracellula domain of either receptor may be modified at a site correspond ing to the site(s) at which it is desired to introduce amin acid substitutions, e.g. by site-directed mutagenesis usin synthetic oligonucleotides encoding the desired amino aci sequence for homologous recombination in accordance with well known procedures.
Finally, the DNA construct may be of mixed synthetic an genomic, mixed synthetic and cDNA or mixed genomic and cDN origin prepared by ligating fragments of synthetic, genomic o cDNA origin (as appropriate) , the fragments corresponding t various parts of the entire DNA construct, in accordance wit standard techniques. The DNA construct may also be prepared b polymerase chain reaction using specific primers, for instanc as described in US 4,683,202.
In a further aspect, the present invention relates to a recombinant expression vector which comprises a DNA construct encoding the hybrid polypeptide of the invention. The ex¬ pression vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously repli- eating vector, i.e. a vector which exists as an extrachromoso- al entity, the replication of which is independent of chromo¬ somal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is inte¬ grated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence encoding the hybrid polypeptide of the invention should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the hybrid polypeptide of the invention in mammalian cells are the SV 40 promoter (Subramani et al., Mol. Cell Biol. X, 1981, pp. 854-864) , the MT-1 (metallothionein gene) promoter (Pal- miter et al.. Science 222, 1983, pp. 809-814) or the adenovirus 2 major late promoter. Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255, 1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. APPI. Gen. 1 , 1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPIl (US 4, 599, 311) or ADH2- 4c (Russell et al., Nature 304, 1983, pp. 652-654) promoters. Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO i . 4., 1985, pp. 2093-2099) or the tpiA promoter.
The DNA sequence encoding the hybrid polypeptide of the invention should also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPIl (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit. ) promoters. The vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs) .
The recombinant expression vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the
host cell in question. An examples of such a sequence (when the host cell is a mammalian cell) is the SV 40 origin of replica¬ tion. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomy- cin, hygromycin or methotrexate.
The procedures used to ligate the DNA sequences coding for the hybrid polypeptide of the invention, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf. , for in¬ stance, Sambrook et al., op.cit. ) .
In a further aspect, the present invention relates to a cell which contains the recombinant expression vector described above. The host cell may be any cell which is capable of producing the hybrid receptor polypeptide and is preferably a eukaryotic cell, in particular a mammalian cell. Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650) , BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159. 1982, pp. 601-621; Southern and Berg, J. Mol. AppI. Genet. 1. 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79. 1982, pp. 422-426; Wigler et al.. Cell 14., 1978, p. 725; Corsaro and Pearson, Somatic Cell Genetics 2. 1981, p. 603, Graham and van der Eb, Virology 52, 1973, p. 456; and Neumann et al., EMBO J. 1, 1982, pp. 841- 845.
Alternatively, fungal cells (including yeast cells) may be used as host cells of the invention. Examples of suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharo- yces spp., in particular strains of Saccharomyces cerevisiae. Examples of other fungal cells are cells of filamentous fungi.
e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger. The use of Asper¬ gillus spp. for the expression of proteins is described in, e.g. , EP 272 277.
In a still further aspect, the present invention relates to a process for producing a hybrid polypeptide according to the invention, which comprises culturing a cell as described above in a suitable nutrient medium under conditions which are conducive to the expression of the polypeptide, and recovering the polypeptide from the culture. The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared accord- ing to published recipes (e.g. in catalogues of the American Type Culture Collection) .
If the hybrid polypeptide produced by the cells is one which is deleted of or truncated in the transmembrane and cytoplasmic domains of the parent receptors, it will be secreted to the growth medium and may be recovered from the medium by con¬ ventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purifica- tion by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
If the hybrid polypeptide has retained the transmembrane and (possibly) the cytoplasmic domain of one of the parent recep¬ tors, it will be anchored in the membrane of the host cell and may either be purified therefrom by conventional purification procedures, or the cells carrying the hybrid receptors may be used as such in the screening assay. As noted above, however, it is preferred that the hybrid polypeptide is in soluble form.
In the method according to the invention of identifying the ligand-binding site on a cellular receptor, as described above, the ligand specific for the first receptor and the ligand specific for the second receptor may each be immobilized to a solid support with which the hybrid receptor polypeptide is then contacted. In this case, the hybrid receptor may be provided with a label. Alternatively, the hybrid polypeptide may be immobilized on a solid support and each of the ligands may then be contacted herewith. In this case, each ligand is provided with a label.
The solid support employed in the method of the invention preferably comprises a polymer. The support may in itself be composed of the polymer or may be composed of a matrix coated with the polymer. The matrix may be of any suitable material such as glass, paper or plastic. The polymer may be selected from the group consisting of a plastic (e.g. latex, a poly¬ styrene, polyvinylchloride, polyurethane, polyacrylamide, polyvinylalcohol, nylon, polyvinylacetate, and any suitable copolymer thereof) , cellulose (e.g. various types of paper, such as nitrocellulose paper and the like) , a silicon polymer
(e.g. siloxane) , a polysaccharide (e.g. agarose or dextran) , an ion exchange resin (e.g. conventional anion or cation exchange resins) , a polypeptide such as polylysine, or a ceramic material such as glass (e.g. controlled pore glass) .
The physical shape of the solid support is not critical, although some shapes may be more convenient than others for the present purpose. Thus, the solid support may be in the shape of a plate, e.g. a thin layer or icrotiter plate, or a film, strip, membrane (e.g. a nylon membrane or a cellulose filter) or solid particles (e.g. latex beads) .
The label substance with which the ligands or the hybrid polypeptide are labelled is preferably selected from the group consisting of enzymes, fluorescent or chemiluminescent sub-
stances, chromophores, radioactive isotopes and complexing agents.
Examples of enzymes useful as label substances are peroxidases (such as horseradish peroxidase) , phosphatases (such as acid or alkaline phosphatase) , β-galactosidase, urease, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucoamy- lase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase, β-glucosidase, proteases, pyruvate decarboxy- lase, esterases, etc.
Enzymes arc net in themselves detectable but must be combined with a substrate to catalyse a reaction the end product of which is detectable. Examples of substrates which may be employed in the method according to the invention include hydrogen peroxide, p-nitrophenylphosphate, lactose, urea, choline ester, β-D-glucose, carbon dioxide, starch, malate, glucose-6-phosphate. The substrate may be combined with a chromophore which may be either a donor or acceptor.
Fluorescent substances which may be used as labels for the present purpose may be selected from 4-methyl umbelliferyl-D- galactopyranoside, 4-methyl umbelliferyl phosphate and 3-(p- hydroxyphenyl) propionic acid. These substances are detectable in themselves by means of a fluorescence spectrophotometer.
Chemiluminescent substances which may be employed for the present purpose include isoluminol/EDTA/hydrogen peroxide, peroxidase eosin/EDTA and luciferase and a substrate therefor. These substances may be detected in themselves by means of a spectrophotometer.
Chromophores which may be employed for the present purpose may be selected from 5-aminosalicylic acid, 2,2'-azino-di-(3- ethylbenzothiazoline)-6-sulphonic acid, o-phenylenediamine, o- diaminicidine, 3-methyl-2-benzothiazoline hydrazone, 3- (dimethylamino)benzoic acid, o-toluidine, 3,3', 5,5'-tetra-
methylbenzidine, o-nitrophenyl-β-D-galactoside and p-nitro phenyl phosphate. These substances may be detected in them selves by means of a spectrophotometer and used for th determination of colour, e.g. colour intensity or colou change.
Radioactive isotopes which may be used for the present purpos may be selected from 1-125, 1-131, H-3, P-35, C-14 or S-35. Th radioactivity emitted by these isotopes may be measured in gamma-counter or a scintillation counter in a manner known pe se.
Complexing agents which may be employed for the present purpos may be selected from biotin (which complexes with avidin o streptavidin) , avidin (which complexes with biotin) , Protein (which complexes with immunoglobulins) and lectins (complexing with carbohydrate receptors) . As the complex is not directly detectable, it is necessary to label the substance with which the complexing agent forms a complex. The labelling may be carried out with any one of the label substances mentioned above for the labelling of the enzyme.
As a further alternative, the polypeptide may be incubated sequentially with each of the ligands in a dissolved state to form a complex if the polypeptide includes a ligand-binding site, followed by the addition of a substance which causes precipitation of high molecular weight substances, any binding of the polypeptide to either of the ligands being detectable as the presence of a precipitated polypeptide-ligand complex. In this embodiment, either the polypeptide or the ligands are provided with a label. The label substance may be selected from any of the substances indicated above. The substance used to precipitate high molecular weight substances may, for instance, be polyethylene glycol.
In a currently preferred embodiment, the method of the in¬ vention provides for the localization of the insulin-binding
site on the insulin receptor. Thus, the first receptor is the insulin receptor. The second receptor may suitably be the IGF receptor. More specifically, an amino acid sequence of the insulin receptor corresponding to exon 2, or a fragment thereof, may be replaced by an amino acid sequence of the IGF receptor corresponding to exon 2, or a fragment thereof, or an amino acid sequence of the IGF receptor corresponding to exon 2, or a fragment thereof, may be replaced by an amino acid sequence of the insulin receptor corresponding to exon 2, or a fragment thereof.
Ai -.ei uα v c jT f c-Ji -Iϊi-niO αύ-. oi-^ucnυc Ox -.lie iisui -Li i - ------- ι_ J corresponding to exon 3, or a fragment thereof, may be replaced by an amino acid sequence of the IGF receptor corresponding to exon 3, or a fragment thereof, or an amino acid sequence of the IGF receptor corresponding to exon 3, or a fragment thereof, may be replaced by an amino acid sequence of the insulin receptor corresponding to exon 3, or a fragment thereof.
As a further alternative, an amino acid sequence of the insulin receptor corresponding to exons 2 and 3, or a fragment thereof, may be replaced by an amino acid sequence of the IGF receptor corresponding to exons 2 and 3, or a fragment thereof, or an amino acid sequence of the IGF receptor corresponding to exons 2 and 3, or a fragment thereof, may be replaced by an amino acid sequence of the insulin receptor corresponding to exons 2 and 3, or a fragment thereof.
As a still further alternative, an amino acid sequence of the insulin receptor corresponding to exon 1 (or at least a fragment thereof comprising the first 7 amino acids of the α- subunit of the receptor, preferably in combination with an amino acid sequence corresponding to exon 2) or one or more of exons 4-11 or fragments thereof may be replaced by the corre¬ sponding amino acid sequence from the IGF receptor, or an amino acid sequence of the IGF receptor corresponding to exon 1 (pre¬ ferably in combination with an amino acid sequence correspond-
ing to exon 2 or a fragment thereof) or one or more of exons 4 11 or fragments thereof may be replaced by the correspondin amino acid sequence from the insulin receptor (including fragment of exon 1 comprising the first 7 amino acids of the α subunit, preferably combined with exon 2 or a fragment there of) .
Based on the structural and organizational homology betwee several known cellular receptors, it is currently assumed tha the ligand-binding site of many cellular receptors (especiall of the type mediating their function through tyrosine kinases - -ϊi- ti-rj* 4- ■»-
.1 !t Λ*---•
-■x! i i
The polypeptide fragment of the invention comprising an amin acid sequence conferring ligand specificity to the cellula receptor is therefore preferably derived from the N-termina part of the extracellular domain of the receptor, more pre ferably from exon 1, 2 and/or exon 3 of the receptor. I particular, the region of the insulin receptor conferrin insulin specificity has been found to be located within sequence of approximately the first 70 amino acids of the α subunit of the receptor (corresponding to 7 amino acids fro exon 1 and 63 amino acids from exon 2 of the receptor) . Thus in a preferred embodiment, the polypeptide fragment of th insulin receptor comprises a sequence from amino acid 1 t amino acid 68 of the insulin receptor sequence shown in Fig. 6, or a subsequence thereof comprising the region conferrin insulin specificity to the insulin receptor. Preliminar studies indicate that a sequence of the insulin receptor fro amino acid 1 to amino acid 27 or from amino acid 27 to amin acid 68 may be of particular importance for the insuli specificity of the receptor.
On the other hand, the region of the IGF-I receptor conferring IGF-I specificity to the receptor has surprisingly been foun
to be located within a sequence corresponding to exon 3 of the IGF-I receptor.
Apart from sequences conferring ligand specificity, it would appear that the insulin and IGF-I receptors share a common ligand-binding site which is capable of binding both ligands, albeit with different affinities. A major part of this common ligand-binding site seems to be encoded by exons 2 and 3 of the receptors.
In the method of the invention of screening for a ligand or functional equivalent thereof, or for a ligand analogue, the hybrid receptor polypeptide or the polypeptide fragment may be provided with a label. In this case, the suspected ligand, functional equivalent or ligand antagonist may be immobilized on a solid support. Alternatively, the suspected ligand or functional equivalent thereof or ligand antagonist may be provided with a label, in which case the hybrid polypeptide or the polypeptide fragment may be immobilized on a solid support. Examples of suitable label substances and solid support materials are given above.
Once the ligand-binding site of a particular receptor has been located/identified by the method of the invention as described above, this may be used further to aquire information about the three-dimensional structure of the ligand-binding site. Such three-dimensional structures of proteins may, for instance, be established by means of protein engineering, computer model¬ ling, NMR and/or crystallographic techniques.
It is at present contemplated that the hybrid receptor polypep¬ tide or the polypeptide fragment of the invention may suitably be used for designing insulin analogues (or other drugs) with desirable binding properties. In this case, a hybrid receptor or polypeptide fragment is used which comprises part of the extracellular domain of the insulin receptor defining the insulin-binding site.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is further described with reference to th drawings, wherein
Fig. 1A-1I shows the full-length cDNA sequence of the huma insulin receptor;
Fig. 2A-2H shows the cDNA sequence of the soluble insuli receptor and the deduced amino acid sequence thereof, indicate in the conventional one-letter code;
Fig. 3 shows the assembly into the α-subunit and part of the β subunit of the IGF-I receptor (SIGF-I-R) of cDNA fragment generated by reverse transcription and PCR using specifi oligonucleotide primers; the circled numerals refer to th primers listed in Example 1;
Fig. 4A-4H shows the cDNA sequence of the soluble IGF- receptor and the deduced amino acid sequence thereof, indicate in the conventional one-letter code;
Fig. 5A-5I shows the cDNA sequence of a hybrid insulin/IGF- receptor, wherein exons 2 and 3 of the insulin receptor hav been replaced by exons 2 and 3 of the IGF-I receptor, and th dediced amino acid sequence thereof, indicated in the con ventional one-letter code; and
Fig. 6A-6H shows the cDNA sequence of a hybrid insulin/IGF- receptor, wherein a Xbal-Xhol fragment of the soluble IGF- receptor has been replaced by a BamHI-Xhol fragment of th insulin receptor. The deduced amino acid sequence of the hybri receptor is shown in the conventional one-letter code. Th first 27 amino acid constitute a signal peptide, while th first amino acid of the mature insulin receptor is marked b the number 1 above the amino acid H (for histidine) .
SUB
Fig. 7A-7H shows the cDNA sequence of a hybrid insulin/IGF-I receptor wherein exon 3 of the insulin receptor has been replaced by the corresponding sequence of the IGF-I receptor. The deduced amino acid sequence of the hybrid receptor is shown in the conventional one-letter code.
The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the present invention.
EXAMPLE 1
Construction of cDNA encoding a soluble insulin receptor
Insulin receptor cDNA was isolated from a cDNA library generated from poly(A)+ mRNA isolated from the human lympho- blastoid cell line IM9 (available from the American Type Culture Collection, Rockville, Maryland, under the cataloque number ATCC CCL 159) stimulated with 1.4 μg/ml cortisol for 20 hours, substantially according to the method described by Okayama and Berg, Mol. Cell Biol. 2. 1982, p. 161 ff. ; Okayama and Berg, Mol. Cell. Biol. 2(2), Feb. 1983, pp. 280-289; and Noma et al., Nature 319, 1986, p. 640 ff. An approximately 4000 bp clone containing the 3'end of the insulin receptor was isolated. To obtain a full-length clone, primer extension was applied on mRNA from the IM9 cells using (10 μg total mRNA) AMV reverse transcriptase (60 U, available from Pharmacia LKB Biotechnology, Sweden) with 800 ng of the 3' oligonucleotide primer 5*-CATCTCAGCAAGATCTTGTCA-3' . The second strand was synthesized as described by Okayama and Berg, op. cit. , using the 51 oliginucleotide 5'-ACCGGGAGCGCGCGCTCTGATC-3 • as primer. The cDNA was digested with the restriction endonucleases BssHII and Bglll and ligated into an appropriate vector. A 1550 bp clone containing the 5'end of the insulin receptor was isola¬ ted. The full-length insulin receptor was assembled in the mammalian expression vector Zem219b (described in DK Patent
SUBSTITUTESHEET
Application No. 3023/88) digested with Ncol and Xbal b ligating the 5' cDNA clone digested with Ncol and Bglll and th 3' cDNA clone digested with Spal and Bglll. The DNA sequence o the full length insulin receptor cDNA is shown in Fig. 1A-1F.
A cDNA encoding a soluble insulin receptor lacking the cyto plasmic domain was constructed by insertion, in an Aatll site, of a synthetic oligonucleotide with the following DNA sequenc specifying a termination codon
5'-C CCG TCA AAT ATT GCA AAA TAA T-3• 3'-TG CAG GGC AGT TTA TAA CGT TTT ATT AGA TC-5'
immediately before the codon corresponding to amino acid 918 i the mature insulin receptor sequence. The synthetic oligo¬ nucleotide was prepared on an Applied Biosystems Model 380-A DNA synthesizer using phosphoamidite chemistry on a controlled pore glass support (Beaucage and Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869) . Introduction of the termination codon in this manner resulted in a 2835 base pair (bp) cDNA (the numbering here and elsewhere is according to A. Ullrich et al., infra) encoding the extracellular 944 amino acids of the insulin receptor (including a signal peptide of 27 amino acids) . The cDNA sequence of the soluble insulin receptor is shown in Fig. 2A-2F.
The insulin receptor cDNA was sequenced by the enzymatic chain reaction method described by F. Sanger et al., Proc. Natl. Acad. Sci. USA 74, 1987, pp. 5463-5467, using T4 DNA polymerase (Sequenase Kit, USB, Cleveland, Ohio, USA) . The sequence was found to be identical with the sequence published by A. Ullrich et al.. Nature 313, 28 February, 1985, pp. 756-761, with the exception of the codons corresponding to amino acids 861, 862 and 1239 where the sequence was identical with the published gene sequence (S. Seino, Proc. Natl. Acad. Sci. USA 86. 1989, pp. 114-118) .
Construction of cDNA encoding a soluble IGF-I receptor
A soluble IGF-I receptor cDNA was prepared from mRNA from human term placenta by a method involving polymerase chain reaction (PCR) using specific oligonucleotide primers (cf. R.K. Saiki et al., Science 239, 29 Jan., 1988, pp. 487-491, and US 4,683,202). mRNA was prepared by standard procedures as described in Sambrook et al. , op. cit. The mRNA was reverse transcribed into cDNA with AMV reverse transcriptase (Pharmacia LKB Biotechnology, Sweden) using the appropriate specific primers or an oligo-dT-primer at a final concentration of 800 ng/3.5 μg mRNA.
IGF-I receptor cDNA fragments were amplified by PCR using the Gene Amp kit (Perkin Elmer Cetus, Norwalk, CT, USA) as recom¬ mended by the manufacturer.
In each PCR, approximately lμg of the reverse transcribed mRNA was used as template. The primers used for PCR all contained an endonuclease restriction site permitting subcloning and assembly of the soluble IGF-I receptor. The following specific primers were used for PCR
1. 5 -CCA AAT AGG ATC CAT GAA CTC TGG CTC CGG AGG-3
2. 3 -CCC GAA GTA GGC CTT AAG GTC GGT CTC GT-5 '
3. 5 -TGG GCA GCT GCA GCG CGC CTG-3 '
4. 3 -ATT GGA CCG TGG CCA TGG CCG-5'
5. 5 -TAA CCT GGC ACC GGT ACC GGC-3 ' 6. 3 -TCA AAG AGT TGC TTC GAA GAC-5'
7. 5 -AAC ACC ACG GCC GCA GAC ACC-3 '
8. 3 -TGT CCT ATA CTT TTG ATT AGA TCT GAC TAG T-5 '
The following restriction sites native to the IGF-I receptor were part of the PCR primers: PstI, Asp718, Xmalll and Hindlll. A BamHI site was incorporated in the primer 1 used to generate the 5' end of the receptor cDNA by introducing a mismatch in the sequence. The primer 8 used to generate the 3'
end of the soluble IGF-I receptor cDNA was designed by intro¬ ducing a mismatch in the sequence so as to include a termina¬ tion codon in nucleotide position 2842-2844 followed by an Xbal site. Each PCR reaction cycle comprised denaturation of the 5 template at 94*C for 1 minute, and annealing of the primers to the templates for 2 minutes at 50*C, followed by extension of the primers for 3 minutes at 72βC. This cycle was repeated 25 times, resulting in specific IGF-I receptor cDNA fragments.
The isolated cDNA fragments were digested with the endo-
10 nucleases BamHI and Xbal (New England Biolabs, MA, USA) and subcloned into the PBSII vector (Stratagene, CA, USA) by the method described by Sambrook et al., op. cit. Cells of E^. coli strain MC1061 (T.V. Huynk et al., in DNA Cloning. Vol. 1 (D.M. Glover, ed.), IRL Press Ltd., Oxford, England, 1983, pp. 56-
15110) and SCS-1 (D. Hanahan, J. Mol. Biol. 166. 1983, pp. 557- 580) were made competent according to the method described by D. Hanahan, in DNA Cloning. Vol. 1, supra. pp. 110-135, and used for transformation with the vectors indicated above. The soluble IGF-I receptor cDNA was assembled from four subcloned 0 cDNA fragments as shown in Fig. 3 (BamHI-PstI, Pstl-Asp718, Asp718-Pstl, Pstl-Xbal) according to the method described by Sambrook et al., op. cit. The cDNA fragment(s) were sequenced by the enzymatic chain termination method described by F. Sanger et al., op. cit.. using T4 DNA polymerase (Sequenase 5 Kit, USB, Cleveland, Ohio, USA) . The cDNA sequence of the soluble IGF-I receptor is shown in Fig. 4A-4E. The sequence was identical to the published sequence (A. Ullrich et al., The EMBO Journal 5(10) . 1986, pp. 2503-2512).
Construction of cDNA coding for a hybrid insulin-IGF-I receptor
0 cDNA coding for a hybrid receptor composed of the soluble insulin receptor described above wherein the cDNA sequence coding for exons 2 and 3 was replaced by the cDNA sequence coding for exons 2 and 3 from the IGF-I receptor, was prepared by PCR as described above. Primer 2 (shown above) , which
includes the insulin receptor "compatible" restriction site EcoRI, was used to generate an IGF-I receptor cDNA fragment corresponding to the DNA sequence encoding exon 1, 2 and 3 of the insulin receptor which was inserted into the EcoRI site of the insulin receptor cDNA by the method described by Sambrook et al., op. cit.. The sequence of the cDNA fragment was established as described above, and the sequence of the IGF- I receptor cDNA fragment inserted into the insulin receptor was found to be identical with the published sequence (A. Ullrich et al., op. cit.) . The cDNA sequence of the hybrid receptor is shown in Fig. 5A-5F.
EXAMPLE 2
The cDNA fragment encoding the hybrid insulin-IGF-I receptor constructed as described in example 1 was inserted in the mammalian expression vector Zem219b (described in DK Patent Application No. 3023/88) which carries a gene conferring methotrexate resistance to the host cell, and cDNA encoding the hybrid receptor was transfected into BHK cells. Resistant cells were selected with 0.4-2 μM methotrexate.
The resulting transfected BHK cells were grown to confluence in Dulbecco's Modified Eagle medium (Gibco) supplemented with 10% foetal calf serum (FCS) , and incubated for 18-52 hours in the same medium supplemented with 2% FCS. Hybrid receptors secreted from the BHK cells were partially purified on a Mono Q column (HR 5/5 available from Pharmacia, Sweden) followed by gel filtration on Superose 6 (HR 10/30 available from Pharmacia, Sweden) as follows. 10-15 ml of the culture medium was diluted with 1 volume of 20 mM Tris-HCl, pH 8.0, and applied on the column. Bound material was eluted with a gradient of 0-500 mM NaCl in 20 mM Tris-HCl, pH 8.0, and 1 ml fractions were collected. 200 μl fractions containing IGF-I binding activity were subsequently applied on a Superose 6 column at a flow rate of 0.25 ml/min. using 25 mM Hepes-Cl, pH 8.0, 100 mM NaCl as eluent, and fractions containing binding activity were used
directly for cross-linking. 50 μl fractions were incubated wit 0.05 μCi of 125-1 insulin (Au iodinated, available from Nov Nordisk A/S) or 125-1 IGF-I (available from Amersham) for 9 minutes at room temperature and cross-linked with disuccinimi dyl suberate (DSS) (available from Pierce) as described by J. Massague and M.P. Czech, Meth. in Enzymol. 109. pp. 179-185, i the absence or presence of 10 μg/ml insulin (Novo Nordisk) o 2 μg/ml IGF-I (Amersham) , respectively. Fractions from th Superose 6 column which, on SDS-PAGE, resulted in a band of approximately 340 kD under non-reducing conditions and a band of approximately 125 kD under reducing conditions on cross- linking with 125-I-IGF-I were used in the following binding assay. 50 μl of such fractions were incubated with 25,000 cpm 125-1 insulin or 125-1 IGF-I and increasing levels of un- labelled IGF-I (0.5 ng/ml - 1.25 μg/ml) and insulin (120 ng/ml - 25 μg/ml) in 150 μl 100 M Hepes buffer (100 mM NaCl, 10 mM MgCl2, 0.5 % bovine serum albumin), pH 8.0, for 16 hours at 4*C followed by precipitation with 20 μl 2% bovine gammaglobulin (Sigma) and 250 μl 30% PEG 8000 (Sigma) as described by M.D. Hollenberg and P. Cuatrecasas, in Methods of Receptor Research. Part II (M. Blecher, Ed.), Marcel Dekker, New York, 1976, pp. 429-477.
The results are shown in Table 1 below.
SIR sIGF-I-R SIR 23
(In the table, sIR denotes the soluble insulin receptor, sIGF- I-R denotes the soluble IGF-I receptor, and sIR 23 denotes the insulin/IGF-I receptor hybrid described in Example 1.)
It appears from Table 1 that the hybrid receptor has a high affinity for IGF-I. This implies that exons 2 and 3 of the IGF- I receptor are essential for IGF-I binding. The results also suggest that at least part of the ligand-binding site of the insulin receptor may be located within exon 1 (i.e. the amino acid sequence constituting the first 7 amino acids of the a- subunit of the receptor) , exon 2 and/or exon 3 of the insulin receptor, as the hybrid receptor lacking exons 2 and 3 of the insulin receptor has a low affinity for insulin.
EXAMPLE 3
Construction of a hybrid insulin-IGF-I receptor
Localization of the insulin-binding site of the insulin receptor
0.3 μg of the soluble IGF-I receptor prepared according to Example 1 was digested with Xhol and Xbal, and 0.3 μg of the soluble insulin receptor prepared according to Example 1 was digested with BamHI and Xhol. The two fragments were then ligated with T4 DNA ligase and sequenced as described by F. Sanger et al., op. cit. The cDNA sequence of the hybrid receptor sIGF-I-R.1-68 is shown in Fig. 6A-6E. The ligated fragment was subsequently inserted into the mammalian ex¬ pression vector Zem219b described above and transfected into BHK cells and expressed as described in Example 2.
The affinity of the hybrid receptor for insulin or IGF-I was established according to the procedure described in Example 2. The results are shown in Table 2 below.
10
9
The results indicate a significant increase in the affinity of the receptor for insulin although it retains a high affinity for IGF—I (about one order of magnitude lower than that of the native IGF-I receptor) . This demonstrates that at least a significant part of the region conferring receptor specificity for insulin is located within the BamHI-XhoI fragment of the insulin receptor (discounting the sequence encoding the signal peptide) .
EXAMPLE 4
Construction of a hybrid insulin-IGF-I receptor
Localization of the IGF-I binding site of the IGF-I receptor
cDNA coding for a hybrid receptor composed of the soluble insulin receptor described in example 1 wherein cDNA encoding exon 3 was replaced by the corresponding IGF-I cDNA, was prepared by PCR overlap extension.
Hybrid receptor cDNA fragments were amplified by PCR using the Gene Amp kit (Perkin Elmer Cetus, Norwalk, CT, USA) as recom¬ mended by the manufacturer. In each PCR approximately 0.5 μg plasmid DNA was used as template. Each PCR reaction comprised 15 cycles of denaturation of the template at 94°C for 1 minute, annealing of primers to the template at 50βC for 2 minutes and extension of primers at 72 ° C for 3 minutes.
The following specific oligonucleotide primers were used for PCR:
1. 5'-GGA GAC ATC TGT CCG GGT ACC GCG AAG GG-3'
2. 5'-GCA CAT TTT CTG GCA GTG ACT ATG A-3'
53. 5'-CTG CCA GAA AAT GTG CCC AAG CAC GTG TGG GAA G-3 • 4. 5'-TGC TCT GGC TGG AAT TCC GGA TGA AGC CC-3'
In two separate PCR reactions an insulin receptor fragment was amplified using insulin receptor cDNA as template and oligo¬ nucleotides 1 and 2 as primers and an IGF-I receptor fragment 0 was amplified using IGF-I receptor cDNA as template and oligonucleotides 3 and 4 as primers.
The insulin receptor fragment includes an Asp718I site and the exon 2 - exon 3 junction. The IGF-I fragment overlaps the exon 2 - exon 3 junction and includes an EcoRI site compatible with 5 the EcoRI site at the exon 3 - exon 4 junction of the insulin receptor.
In a third PCR reaction the products of the PCRs obtained above were combined, annealed and extended using oligonucleotides 1 and 4 as primers. The final amplified cDNA fragment including 0 the exon 2 - exon 3 junction was digested with Asp718I and EcoRI and subcloned into the pBS+ vector (Stratagene, CA, USA) .
The cDNA sequence of the fragment was verified as described in example 1.
The hybrid cDNA fragment was subsequently inserted into the 5 soluble insulin receptor as described in example 1, replacing the insulin receptor exon 3, and was transfected into BHK cells and expressed as described in example 2.
The affinity of this hybrid receptor for insulin or IGF-I was established according to the procedure described in example 2.
The results (cf. Table 2) show a significant increase in the affinity of the receptor for IGF-I while retaining a high affinity for insulin. This demonstrates that a significant part of the region conferring receptor specificity for IGF-I is located within the IGF-I receptor region corresponding to the insulin receptor exon 3.