WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 5 :
`C12N 15/12, CO7K 13/00
`C12N 5/10, GOIN 33/74
`
`GLP-1 receptor for screening for agonists of GLP-1 activity.
`
`(11) International Publication Number:
`
`WO 93/19175
`
`(43) International Publication Date:
`
`30 September 1993 (30.09.93)
`
`Published
`With internationalsearch report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(21) International Application Number:
`
`PCT/EP93/00697
`
`(22) International Filing Date:
`
`23 March 1993 (23.03.93)
`
`(30) Priority data:
`398/92
`
`25 March 1992 (25.03.92)
`
`DK
`
`(71)(72) Applicant and Inventor: THORENS, Bernard [CH/
`CH]; 70, Grand-Chemin, CH-1066 Epalinges (CH).
`
`(74) Agent: NOVO NORDISKA/S; Patent Department, Novo
`Allé, DK-2880 Bagsvaerd (DK).
`
`(81) Designated States: AU, BB, BG, BR, CA, CZ,FI, HU, JP,
`KP, KR, KZ, LK, MG, MN, MW, NO, NZ, PL, RO,
`RU, SD, SK, UA, US, VN, European patent (AT, BE,
`CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA,
`GN, ML, MR, NE, SN, TD, TG).
`
`(54) Title: RECEPTOR FOR THE GLUCAGON-LIKE-PEPTIDE-1 (GLP-1)
`
`25
`
`
`
`
`
` —a3GLP—1bound(fmol) oS
`
`(57) Abstract
`
`0 20 40 60 80 100120
`Bound (pM)
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`4
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`6
`GLP-1 (nM)
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`8
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`10
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`Thepresent invention relates to a recombinant glucagon-like peptide-1 (GLP-1)-receptor, to a DNA construct which com-
`prises a DNA sequence encoding a GLP-1 receptor, to methods of screening for agonists of GLP-1 activity, and to the use of the
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Vict Nam
`
`France:
`Gabon.
`United Kingdom
`Guinea
`Grecee
`Hungary
`Ireland
`Ttaly
`Japan
`Democratic People’s Republic
`of Korea
`Republic of Karea
`Kazakhstan
`Licuhtenstein
`Sri Lanka
`Luxembourg
`Monaco
`Madagascar
`Mali
`Mongolia
`
`Austria
`Australia
`Barbados
`Belgium.
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Canada
`Central African Republic
`Congo
`Switerland
`Cte d'Ivoire
`Camervon
`Cauchoslovakia
`Cech Republic
`Germany
`Denmark
`Spain
`Finland
`
`Mauritania
`Malawi
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovak Republic
`Senegal
`Sovict Union
`Chad
`Togo
`Ukraine
`United States of America
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`PCT/EP93/00697
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`RECEPTOR FOR THE GLUCAGON-LIKE-PEPTIDE-1 (GLP-1)
`
`FIELD OF THE INVENTION
`
`vi
`
`The present invention relates to a recombinant glucagon-like
`peptide-1 (GLP-1) receptor, to a DNA construct which comprises
`a DNA sequence encoding a GLP-1
`receptor,
`to methods of
`screening for agonists of GLP-1 activity, and to the use of the
`GLP-1 receptor for screening for agonists of GLP-1 activity.
`
`BACKGROUND OF THE INVENTION
`
`As used in the present specification the designation GLP-1
`comprises GLP-1(7-37) as well as GLP-1(7-36) amide.
`
`1 oa
`
`Glucose-induced insulin secretion is modulated by a number of
`hormones
`and neurotransmitters.
`In particular,
`two gut
`hormones,
`glucagon-like
`peptide-1
`(GLP-1)
`and
`gastric
`inhibitory peptide (GIP) potentiate the effect of glucose on
`insulin secretion and are thus called gluco-incretins (Dupre,
`in The Endocrine Pancreas, E. Samois Ed.
`(Raven Press, New
`York,
`(1991), 253 - 281) and Ebert and Creutzfeld,
`(Diabetes
`Metab. Rev. 3,
`(1987)). Glucagon-like peptide-1 is a gluco-
`incretin both in rat
`and
`in man
`(Dupre
`and Ebert
`and
`Creutzfeld, vide su ra, and Kreymann et al.
`(Lancet 2 (1987),
`1300)). It is part of the preproglucagon molecule (Bell et al.
`Nature 304 (1983), 368) which is proteolytically processed in
`intestinal L. cells to GLP-1(1-37) and GLP-1(7-36)amide or GLP-
`1(7-37)
`(Mojsov et al.
`(J.Biol.Chem. 261 (1986), 11880)
`and
`Habener et al.: The Endocrine Pancreas E. Samois Ed.
`(Raven
`Press, New York (1991), 53 - 71). Only the truncated forms of
`GLP-1 are biologically active and both have identical effects
`on insulin secretion in beta cells (Mojsov et al. J.Clin.Invest
`79 (1987), 616) and Weir et al.
`(Diabetes 38 (1989), 338). They
`are the most potent gluco-incretins so far described and are
`
`20
`
`25
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`CONFIF
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`active at concentrations as low as one to ten picomolar. The
`stimulatory effect of these gluco-incretin hormones requires
`the presence of glucose at or above the normal physiological
`concentration of about 5 mM and is mediated by activation of
`adenylate cyclase and a rise in the intracellular concentration
`of cyclic AMP
`(Drucker et al. Proc.Natl.Acad.Sci. USA 84
`(1987), 3434) and Géke et al.
`(Am.J.Physiol. 257 (1989), G397).
`GLP-1
`has
`also
`a
`stimulatory effect
`on
`insulin gene
`transcription (Drucker et al. Proc.Natl.Acad.Sci. USA 84
`(1987), 3434). In a rat model of non-insulin-dependent diabetes
`mellitus
`(NIDDM)
`is associated with a reduced stimulatory
`effect of GLP-1 on glucose-induced insulin secretion (Suzuki et
`al. Diabetes 39 (1990), 1320).
`In man,
`in one study, GLP-1
`levels were elevated in NIDDM patients both in the basal state
`and after glucose ingestion; however, following a glucose load
`there was
`only
`a very small
`rise
`in plasma
`insulin
`concentration (@rskov et al. J.Clin.Invest. 87 (1991), 415).
`A recent study (Nathan et al. Diabetes Care 15 (1992), 270)
`showed that GLP-1
`infusion could ameliorate postprandial
`insulin secretion and glucose disposal in NIDDM patients. Thus,
`as a further step in understanding the complex modulation of
`insulin secretion by gut hormones and its dysfunction in
`diabetes, we isolated and characterized a complementary DNA for
`the beta cell GLP-1 receptor and showed that it is part of a
`new family of G-coupled receptors.
`
`DESCRIPTION OF THE INVENTION
`
`The present invention relates to a recombinant glucagon-like
`peptide-1 (GLP-1) receptor.
`
`3
`
`So
`
`the invention relates to a GLP-1 receptor
`More preferably,
`which comprises the amino acid sequence shown in SEQ ID No. 1,
`or an analogue thereof binding GLP-1 with an affinity constant,
`Kp, below 100 mM, preferably below 10 nM.
`In the present
`
`aN
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`&
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`PCT/EP93/00697
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`intended to indicate a
`is
`the term "analogue"
`context,
`naturally occurring variant (including one expressed in other
`animal species,
`in particular human) of
`the receptor or a
`"derivative" i.e. a polypeptide which is derived from the
`native GLP-1 receptor by suitably modifying the DNA sequence
`coding for the variant, resulting in the addition of one or
`more amino acids at either or both the C- and N-terminal ends
`of the native amino acid sequence, substitution of one or more
`amino acids at one or more sites in the native amino acid
`sequence, deletion of one or more amino acids at either or both
`ends of the native sequence or at one or more sites within the
`native sequence, or insertion of one or more amino acids in the
`native sequence.
`
`1
`
`uw
`
`invention relates to a DNA
`the present
`In another aspect,
`construct which comprises a DNA sequence encoding the GLP-1
`receptor of the invention, as well as a recombinant expression
`- vector carrying the DNA construct and a cell containing said
`recombinant expression vector.
`
`In one embodiment of the invention, the GLP-1 receptor molecule
`may be provided in solubilised and/or reconstituted form.
`
`2
`
`0
`
`In the present context "solubilised" is intended to indicate a
`receptor as
`present
`in
`detergent-solubilised membrane
`preparations. "Reconstituted"
`is
`intended to indicate
`a
`receptor solubilised in the prescence of essential cofactors,
`€.g. G-protein.
`In this embodiment the receptor may be in a
`reconstituted micellar form.
`
`The DNA construct of the invention encoding the GLP-1 receptor
`preferably comprises the DNA sequence shown in SEQ ID No. 1, or
`at
`least a DNA sequence coding for a
`functional analogue
`thereof binding GLP-1 with an affinity below 100 nM, preferably
`below 10 nM or a suitable modification thereof. Examples of
`suitable modifications of
`the DNA sequence are nucleotide
`
`2
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`substitutions which do not give rise to another amino acid
`sequence of the GLP-1 receptor, but which may correspond to the
`codon usage of the host organism into which the DNA construct
`is introduced or nucleotide substitutions which do give rise to
`a different amino acid sequence and therefore, possibly,
`a
`different protein structure without, however,
`impairing the
`properties of the native variant. Other examples of possible
`modifications are insertion of one or several nucleotides into
`
`w
`
`the sequence, addition of one or several nucleotides at either
`end of the sequence, or deletion of one or several nucleotides
`
`Qo
`
`at either end or within the sequence.
`
`Another example of a DNA construct of the invention is one
`which encodes a GLP-1 receptor variant particularly suitable
`
`for solubilisation and reconstitution.
`
`The DNA construct of the invention encoding the present GLP-1
`receptor may be prepared synthetically by established standard
`methods, e.g.
`the phosphoamidite method described by Beaucage
`
`and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or
`
`the method described by Matthes et al., EMBO Journal 3 (1984),
`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.
`
`The DNA construct of the invention may also be of genomic or
`cDNA origin, for instance obtained by preparing a genomic or
`cDNA library and screening for DNA sequences coding for all or
`part of the GLP-1 receptor of the invention by hybridization
`using synthetic oligonucleotide probes
`in accordance with
`standard techniques (cf. Sambrook et al., Molecular Cloning: A
`
`Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). In this
`case, a genomic or cDNA sequence encoding the GLP-1 receptor
`may be modified at a site corresponding to the site(s) at which
`it is desired to introduce amino acid substitutions, e.g. by
`
`2
`
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`2
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`3
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`site-directed mutagenesis using synthetic oligonucleotides
`encoding the desired amino acid sequence
`for homologous
`recombination in accordance with well-known procedures.
`
`ut
`
`the DNA construct may be of mixed synthetic and
`Finally,
`genomic, mixed synthetic and cDNA or mixed genomic and cDNA
`_origin prepared by ligating fragments of synthetic, genomic or
`cDNA origin (as appropriate),
`the fragments corresponding to
`various parts of the entire DNA construct,
`in accordance with
`standard techniques. The DNA construct may also be prepared by
`polymerase chain reaction using specific primers, for instance
`as described in US 4,683,202 or Saiki et al., Science 239
`(1988), 487 - 491.
`
`1 wm
`
`2°o
`
`3
`
`30
`
`The recombinant expression veétor into which the DNA construct
`of
`the invention is inserté& 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 replicating vector, i.e. a vector which exists as
`an extrachromosomal entity,
`the replication of which
`is
`independent of
`chromosomal
`replication,
`e.g.
`a plasmid.
`Alternatively,
`the vector may be one which, when introduced
`into a host cell, is integrated 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 GLP-1 receptor of
`the invention. should be operably connected to a suitable pro-
`moter 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 pro-
`moters for directing the transcription of the DNA encoding the
`GLP-1 receptor of the invention in mammalian celis are the SvV40
`promoter
`(Subramani et al., Mol. Cell Biol. 1 (1981), 854 +
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`wi
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`°o
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`
`5
`
`2
`
`6
`
`(Palmiter et
`(metallothionein gene) promoter
`the MT-1
`864),
`al., Science 222 (1983), 809 - 814) or the adenovirus 2 major
`late promoter. A suitable promoter for use in insect cells is
`the polyhedrin promoter
`(Vasuvedan et al.,
`FEBS Lett. 311,
`(1992) 7 - 11). Suitable promoters for use in yeast host cells
`include promoters from yeast glycolytic genes (Hitzeman et al.,
`dg. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki,
`J. Mol. Appl. Gen.
`31
`(1982),
`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 TPI1 (US 4,599,311) or ADH2-4c
`
`(Russell et al., Nature 304 (1983),
`
`652 - 654) promoters.
`
`Suitable promoters for use in filamentous fungus host cells
`are, for instance, the ADH3 promoter (McKnight et al., The EMBO
`
`J. 4 (1985), 2093 - 2099) or the tpiA promoter.
`
`we
`
`The DNA sequence encoding the GLP-1 receptor of the invention
`may 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 TPI1 (Alber and Kawasaki, op.
`
`cit.) or ADH3 (McKnight et al., op. cit.) terminators. The vec-
`tor may further comprise elements
`such as polyadenylation
`Signais
`(e.g.
`from SV40 or
`the adenovirus
`5 Elb region),
`
`transcriptional enhancer sequences (e.g. the $V40 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 enablingthe vector to replicate in the
`host cell in question. An example of such a sequence (when the
`host cell
`is a mammalian cell)
`is the SV40 origin of
`replication. The vector may also comprise a selectable marker,
`e.g. a gene the product of which complements a defect in the
`
`3
`
`Qa
`
`host cell, such as the gene coding for dihydrofolate reductase
`(DHFR) or one which confers
`resistance to a drug,
`e.g.
`neomycin, -hygromycin or methotrexate.
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`The procedures used to ligate the DNA sequences coding for the
`GLP-1 receptor of the invention,
`the promoter and the ter-
`minator, 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
`instance,
`Sambrook et al., op.cit.).
`
`uw
`
`The host cell into which the expression vector of the invention
`is introduced may be any cell which is capable of producing the
`GLP-1 receptor of the invention and is preferably a eukaryotic
`cell, such as invertebrate (insect) cells or vertebrate cells,
`e.g. Xenopus laevis oocytes or mammalian cells,
`in particular
`insect and mammalian cells. Examples of suitable mammalian cell
`lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL
`10), CHL (ATCC CCL39) 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), 601 - 621; Southern and Berg,
`J. Mol. Appl. Genet. 1 (1982), 327 - 341; Loyter et al., Proc.
`Natl. Acad. Sci. USA 79 (1982), 422 - 426; Wigler et al., Cell
`14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7
`(1981), 603, Graham and van der Eb, Virology 52 (1973), 456;
`and Neumann et al., EMBO J. 1 (1982), 841 - 845.
`
`10
`
`—_ uw
`
`2So
`
`Alternatively, fungal cells (including yeast cells) may be used
`as host cells of the invention. Examples of suitable yeasts
`cells
`include
`cells
`of
`Saccharomyces
`spp.
`or Schizo-
`saccharomyces spp.,
`in particular strains of Saccharomyces
`cerevisiae. Examples of other fungal cells are cells of fila-
`mentous fungi, e.g. Aspergillus spp. or Neurospora spp.,
`in
`particular strains of Aspergillus oryzae or Aspergillus niger.
`The use of Aspergillus spp. for the expression of proteins is
`described in, e.g., EP 272 277.
`
`25
`
`30
`
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`
`The GLP-1 receptor according to the invention may be produced
`by a method which comprises culturing a cell as described above
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`
`in a suitable nutrient medium under conditions which are
`
`conducive to the expression of the GLP-1 receptor, and re-
`covering the GLP-1 receptor 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 he
`prepared according to published recipes (e.g.
`in catalogues of
`the American Type Culture Collection).
`
`If the GLP-1 receptor has retained the transmembrane and (pos-
`sibly) the cytoplasmic region of the native variant, it will be
`anchored in the membrane of
`the host cell,
`and the cells
`carrying the GLP-1 receptor may be used as
`such in the
`screening or diagnostic assay. Alternatively, the receptor may
`be a component of membrane preparations, e.g.
`in solubilised
`and/or reconstituted form as defined above.
`
`the present invention relates to a
`In a still further aspect,
`method of
`screening for agonists or enhancers of GLP-1
`activity,
`the method comprising incubating a GLP-1 receptor
`according to any of claims 1 - 3 with a substance suspected to
`be an agonist of GLP-1 activity and subsequently with a GLP-1
`or an analogue thereof,
`and detecting any effect
`from the
`suspected agonist on the binding of GLP-1
`to the GLP-1
`receptor. An enhancer being defined as a compound capable of
`stabilizing interaction between a high-affinity form of the
`receptor and the corresponding ligand, as described e.g.
`for
`the adenosin receptor (Bruns et al. Molecular Pharmacology 38
`
`(1990), 939).
`
`3
`
`oa
`
`An alternative method of screening for agonists of GLP-1
`activity, comprises incubating GLP-1 or an analogue thereof
`with a substance suspected to be an agonist of GLP-1 activity
`and subsequently with a GLP-1 receptor of the invention, and
`detecting any effect on the binding to the GLP-1 receptor. Such
`
`“
`
`Ad
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`a
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`agonists of GLP-1 activity will be substances stimulating
`glucose-induced insulin secretion and may be used in the
`treatment of NIDDM.
`
`ut
`
`The GLP-1 receptor may be immobilized on a solid support and
`may, as such, be used as a reagent in the screening methods of
`the invention. The GLP-1 receptor may be used in membrane-
`bound form,
`i.e. bound to whole cells or as a component of
`membrane preparations immobilised on a solid support.
`
`The solid support employed in the screening methods 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
`polystyrene,
`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, al-
`though 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 microtiter plate, or a filn,
`strip, membrane (e.g. a nylon membrane or a cellulose filter)
`or solid particles (e.g.
`latex beads or dextran or agarose
`beads). In a preferred embodiment, the solid support is in the
`form of wheat
`germ agglutinin-coated SPA beads
`(cf.
`US
`4,568,649).
`
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`Alternatively, screening for GLP-1 agonists can also be carried
`out using a cell
`line expressing the cloned GLP-1 receptor
`functionally coupled to a G-protein. In living cells, exposure
`to an agonist will
`give
`rise to an
`increase
`in the
`intracellular cAMP concentration. The cAMP concentration can
`then be measured directly. Changes in cAMP levels may also he
`monitored indirectly using appropriate cell lines in which a
`measurable signal is generated in response to an increase in
`intracellular cAMP.
`
`It is furthermore contemplated to locate the ligand-binding
`site on the GLP-1 receptor of the invention, for instance by
`preparing deletion or substitution derivatives of the native
`GLP-1 receptor (as described above) and incubating these with
`ligands known to bind the full-length GLP-1 receptor and
`detecting any binding of the ligand to the GLP-1 receptor
`deletion derivative. Once the ligand-binding site has been
`located, this may be used to aquire further information about
`the three-dimensional structure of the ligand-binding site.
`Such
`three-dimensional
`structures may,
`for
`instance,
`he
`established by means
`of protein
`engineering,
`computer
`modelling, NMR technology and/or crystallographic techniques.
`Based on the three-dimensional structure of the ligand-binding
`site,
`it may be possible to design substances which are
`
`agonists to the GLP-1 molecule.
`
`The characterization of the GLP-1 receptor is of considerabie
`physiological and pathological importance. It will help study
`a fundamental aspect of the entero-insular axis
`(Unger and
`Eisentraut, Arch.Int.Med. 123 (1969), 261):
`the potentiating
`effect of gut hormones on glucose-induced insulin secretion,
`the role of
`these hormones
`in the control
`of glucose
`homeostasis and also the possible therapeutic use of GLP-1 to
`stimulate insulin secretion in NIDDM patients (Mathan et al.
`Diabetes Care 15 (1992), 270). Investigation of the reguiated
`expression and desensitization of the receptor in the normal
`
`15
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`20
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`il
`
`state and during the development of diabetes will contribute to
`a better understanding of the modulation of insulin secretion
`in normal
`and pathological
`situations. Availability of
`antibodies against this receptor may also allow an analysis of
`wn the surface localization of this receptor and its distribution
`relative to the beta cell glucose transporter GLUT2 (Thorens et
`al. Cell 55 (1988), 281 and Orci et al. Science 245 (1989),
`295). This aspect pertains to the hypothesis that the beta cell
`membrane has
`a "regulatory" domain which contains hormone
`10 receptors (Bonner-Weir Diabetes 37 (1988), 616), and which may
`be distinct from GLUT2-containing membrane domains previously
`identified (Thorens et al. Cell 55 (1988), 281 and Orci et al.
`Science 245 (1989), 295). Finally,
`the identification of an
`additional member of this new family of G-coupled receptors
`1s will help design experiments to probe the structure-function
`relationship of these new molecules.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The present invention is further illustrated in the following
`examples with reference to the appended drawings in which
`
`iA together
`20 Fig. 1A and Fig. 1B which is a continuation of Fig.
`show the amino acid sequence of the rat GLP-1 receptor in a
`comparison with the sequence of
`the rat secretin receptor
`(SECR),
`the opossium parathyroid hormone receptor (PTHR) and
`the porcine calcitonin receptor (CTR1). The GLP-1 receptor has
`three N glycosylation sites
`in the extracellular domain
`(arrows). Four cysteines are conserved at identical places in
`the four receptor (boxes). Note the otherwise very divergent
`sequences in this part of the molecules as well as in the COOH-
`
`2
`
`Ww
`
`‘
`
`terminal cytoplasmic tail. Sequence identities are denoted by
`30 stars and homologies by dots. The location of the putative
`transmembrane domains are indicated by horizontal bars above
`the sequences.
`.
`
`_
`
`SUBSTITIITE SHEET
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`Fig.
`
`2 shows binding of 1251_¢rp-1 to cos cells transfected
`
`with the pGLPR~16 plasmid. Specific binding reaches saturation
`at
`1 to 10 nM GLP-1.
`Insert: Scatchard analysis of GLP-1
`binding.
`
`shows binding of 1257-¢LP-1 to INS-1 cells. Specific
`3
`Fig.
`binding reaches saturation at
`1
`to 10
`nM GLP-1.
`Insert:
`Scatchard analysis of GLP-1 binding.
`
`Fitting of the curves in Figs. 2 and 3 were performed with the
`LIGAND program (McPherson, Kinetic, EBDA, Ligand, Lowry. A
`Collection
`of
`radioligand
`analysis
`programs
`(Elsevier,
`
`1
`
`a
`
`Amsterdam, 1985)).
`
`i
`
`uw
`
`2
`
`0
`
`Fig. 4 shows displacement of 12°51-GLp-i binding to COS cells
`transfected with the rat GLP-1 receptor cDNA. Transfected cells
`were incubated with 20 pM 125y;.gEP-1 in the presence of
`increasing concentrations of cold peptides. Each point was
`measured in duplicate and the experiments repeated three times
`for GLP-1, GIP and glucagon and once for VIP and secretin.
`
`Fig. 5 shows stimulation of cyclic AMP formation in COS cells
`transfected with the rat GLP-1 receptor cDNA. COS cells were
`transfected with the pcDNA-1 vector alone (open bars) or the
`pGLPR-1 plasmid (stripped bar) and incubated in the absence or
`
`the presence of GLP-1 at the indicated concentration.
`cAMP
`production was measured in triplicate with a radioimmunoassay
`
`(Amersham).
`
`6 shows tissue specificity of GLP-1 receptor expression
`Fig.
`assessed by Northern blotting of RNA from different tissues and
`from the INS-1 cell
`line. Ten micrograms of total RNA was
`analyzed on each lane. Two major RNA species of 2.7 and 3.6 kb
`were detected in all
`tissues
`in which the receptor was
`detected. The position of the migration of the ribosomal RNAs
`is indicated to the left of the picture.
`
`3
`
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`
`7 is a comparison of the rat GLP-1 receptor amino acid
`Fig.
`sequence (rat) and a partial amino acid sequence of the human
`GLP-1 receptor (human).
`
`wn
`
`The present invention is further illustrated in the following
`examples which is not intended to be in any way Limiting to the
`scope of the invention as claimed.
`
`EXAMPLE 1
`
`Molecular Cloning and Characterisation of the Rat Islet GLP-1
`Receptor cDNA.
`
`A rat pancreatic islet cDNA library was constructed in the
`PCDNA~1 expression vector (Rat pancreatic islets were prepared
`according to Gotoh et al.
`(Transplantation 43 (1985), 725).
`PolyA+ RNA was prepared and the cDNA library was constructed in
`the pcDNA-1 vector
`(In Vitrogen) as described by Aruffo and
`Seed (Proc.Natl.Acad.Sci. USA 84 (1987), 8573) and Lin et al.
`(Proc.Natl.Acad.Sci. USA 88 (1991), 3185). Plasmid DNA was
`prepared from pools of five to eight thousands bacterial clones
`(Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold
`Spring Harbor Laboratory, 1982) and transfected into COS cells
`(Sompayrac and Dana, Proc.Natl.Acad.Sci. USA 78 (1981), 7575).
`The presence of GLP-1 receptor expressed in COS cells was
`assessed by binding of the radioiodinated peptide
`followed by
`photographic emulsion autoradiography and screening by dark
`field microscopy (Gearing et al. EMBO J. 8 (1989), 3667). GLP-
`1(7-36)amide, as well as the other peptides, were purchased
`from Peninsula Laboratories.
`Iodination was performed by the
`iodine monochloride method (Contreras et al. Meth.Enzymol. 92
`(1983), 277), the peptide was purified by passage over Sephadex
`G-10
`followed by CM-Sepharose
`and
`specific activity was
`determined by the self displacement
`technique (Calvo et al.
`Biochem. 212 (1983), 259). A 1.6 kb cDNA clone (pGLPR-1) was
`isolated by subfractionation of an original positive pool and
`
`2°o
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`
`two
`by DNA hybridization screening,
`was used to isolate,
`additional clones from primary positive pools. These plasmids
`(pGLPR-16 and -87) had inserts of 3.0 and 2.0 kb, respectively.
`Transfection of these clones into COS cells generated high
`affinity (Kp = 0.6 nM) binding sites for GLP-1 (Fig. 2). This
`affinity is comparable to that seen for binding of GLP-1 to the
`rat insulinoma cell line INS-1 (Asfari et al. Endocrinology 130
`(1992), 167)
`(Kp = 0.12 nM; Fig. 3).
`In both cases a single
`high affinity binding component was detected. The binding to
`GLP-1 receptor transfected COS cells reached a plateau between
`1 and 10 nM. At concentrations above 10 nM a second, high
`
`ut
`
`10
`
`binding component was detected.
`low affinity,
`capacity,
`Although specifically displacable by cold GLP-1, this binding
`was also present in COS cells transfected with the expression
`
`- vector alone and was therefore not further characterized.
`
`20
`
`2ul
`
`30
`
`Binding of GLP-1 to the receptor expressed in COS cells was
`displaced by cold GLP-1 with a 50 percent displacement achieved
`at 0.5 to 1 mM (Fig. 4). Other peptide hormones of related
`structure such as secretin, gastric inhibitory peptide (GIP)
`and vasoactive intestinal peptide (VIP)
`(Dupre in The Endocrine
`Pancreas, E. Samois Ed.
`(Raven Press, New York,
`(1991), 253 -
`
`281) and Ebert and Creutzfeld, Diabetes Metab. Rev. 3,
`(1987)
`did not displace binding. Glucagon could displace the binding
`by 50 percent but only at a concentration of one micromolar
`(Fig. 4). The addition of subnanomolar concentrations of GLP-1
`to transfected COS cells stimulated the production of cyclic
`AMP indicating that the receptor was functionally coupled to
`activation of adenylate cyclase (Fig. 5).
`
`DNA sequence analysis of the rat GLP-1 receptor cDNA revealed
`a major open reading frame coding for
`a 463
`amino acid
`polypeptide (SEQ ID No. 1). Hydrophaphy plot analysis indicated
`the presence of an amino-terminal hydrophobic region most
`probably representing a leader
`sequence. This hydrophobic
`segment is followed by a hydrophilic domain of about 120 amino
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`
`acids which contains three N-linked glycosylation sites. Seven
`hydrophobic segments are present which may form transmembrane
`domains. Search for
`sequence
`identities showed the GLP-1
`
`wn
`
`-receptor to be homologous to the secretin receptor (Ishihara et
`al.
`EMBO J. 10 (1991), 1635)
`(40 percent
`identity),
`the
`parathyroid hormone
`receptor
`(Jippner et al.
`(Science 254
`(1991),
`1024)
`(32.4 percent
`identity)
`and the calcitonin
`receptor (Lin et al. Science 254 (1991), 1022)
`(27.5 percent
`identity)
`(Fig. 1). These four receptors do not share any
`significant sequence homology with other known members of the
`G-coupled receptor family and are characterized by a relatively
`long amino
`terminal,
`probably extracellular,
`domain. The
`sequence of
`the extracellular domain
`is unique
`for
`each
`receptor, yet four cysteines are perfectly conserved (boxes in
`Fig. 1). A fifth cysteine at position 126 of the GLP-1 receptor
`is also conserved in the parathyroid and calcitonin receptors
`and at a similar location in the secretin receptor (position
`123). The highest sequence identity between the four proteins
`resides in the transmembrane domains. The carboxyl
`terminal,
`cytoplasmic, ends of each receptor are also very different.
`These receptors all stimulate the production of cyclic AMP in
`response to ligand binding (Ishihara et al. EMBO J. 10 (1991),
`1635), Jippner et al.
`(Science 254 (1991), 1024) and Lin et al:
`Science 254 (1991), 1022) and are presumably coupled to the
`cyclase via Gsa.
`In that respect, it is interesting to note
`that a sequence motif present in the third cytoplasmic loop of
`the GLP-1
`receptors
`(RLAK, present
`just before the sixth
`transmembrane domain)
`is very similar to a motif of the beta2
`adrenergic receptor
`(KALK) present at the same location and
`whose basic amino acids have been shown to