WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau.
`
`PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER TFJE PATENT COOPERATION TREATY (PC1)
`(51) International Patent Qasslfication 5 :
`Cl2N 15/12, C07K 13/00
`C12N 5/10, GOIN 33/74
`
`Al
`
`(11) International Publicalion Number:
`
`WO 93/19175
`
`· (43) International Publication Date:
`
`30 September !_?93 (30.09.93)
`
`(21) International Application Number:
`
`(22) International Filing Date:
`
`23 March 1993 (23.03.93)
`
`PCT/ EP93/00697 Published
`With intemational search report
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`J
`
`(30) Priority data:
`398/92
`
`25 March 1992 (25.03.92)
`
`DK
`
`(71)(72) Applicant and Inventor: THO RENS, Bernard [CH/
`CH]; 70, Grand-Chemin, CH-1066 Epalioges (CH).
`
`(74) Agent: NOVO NORDISK A/S; Patent Department, Novo
`Alie, 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
`
`20
`
`--
`0 s ~ -"t1 15
`-
`
`i::
`~
`0
`.0 10
`1
`0...
`...:i
`c., 5
`
`•
`
`0. 20
`
`•
`0.15 ■
`~ 0.10
`CD
`0 .05
`
`•
`
`nM
`
`0.00
`0 20 40 6080 100120
`Bound (pM)
`
`..,J~,-
`
`0
`
`(S7) Abstract
`
`2
`
`4
`6
`GLP-1 (nM)
`
`8
`
`10
`
`The present invention relates to a recombinant glucagon-like peptide-I (GLP- 1) receptor, to a DNA construct which com(cid:173)
`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
`GLP-1 receptor for screening for agonists of GLP-1 activity.
`
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`Page 1 of 50
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`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States pany to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`8.8
`8£
`BF
`BC
`BJ
`BR
`CA
`CF
`cc
`CH
`Cl
`CM
`cs
`CZ
`DE
`OK
`ES
`Fl
`
`Au,trla
`Australia
`Barbados
`Belgium
`8url:in3 F:iso
`8ulgari11
`Benin
`Br:i,ll
`CanaJu
`C:cnmil African Republic
`C.'-Ongo
`Swiw:rland
`C'ote d 'Ivoire
`C'un,1;ruon
`O.cchu:1lovakia
`( 3.cch Republic
`Ocr many
`Denmark
`Spain
`Finland
`
`FR
`CA
`CB
`CN
`CR
`HU
`IE
`IT
`JP
`KP
`
`KR
`KZ
`LI
`LK
`LU
`MC
`MC
`Ml.
`MN
`
`l'rane<:
`Gabon
`UmlcJ Kingdom
`G uinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Democratic People', Republic
`or Korea
`Rcpuhlic or Korea
`Kn:,.nkhstan
`Licch tcnsu,in
`Sri 1'1nl:a
`J.u,1;mbourg
`Mun.u:o
`Madagascar
`Mali
`Mongolia
`
`MR
`MW
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`so
`SE
`SK
`SN
`SU
`TO
`TC
`UA
`us
`VN
`
`Maurit.inia
`Malawi
`Netherlands
`Norway
`New :Z.:.,land
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovak Republic
`Senegal
`Soviet Union
`C:haJ
`Togp
`Ukraine
`United States or America
`Viet Nam
`
`I
`~ -
`
`.}
`
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`W093/19175
`
`PCT /EP93/00697
`
`1
`
`RECEPTOR FOR THE GLUCAGON-LIKE-PEPTIDE-1 (GLP-1)
`
`FIELD OF 'l'BE INVENTION
`
`The present invention relates to a recombinant glucagon-like
`peptide-1 (GLP-1) receptor, to a DNA construct which comprises
`s 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 tha designation GLP-1
`10 comprises ~LP-1 (7-37) as well as GLP-1 (7-36) amide.
`
`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
`15 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. ~, (1987)). Glucagon-like peptide-1 is a gluco(cid:173)
`incretin both
`in rat and
`in man
`(Dupre and Ebert and
`20 Creutzfeld, vide supra, and Kreymann et al. (Lancet 1 (1987),
`1300)). It is part of the preproglucagon molecule (Bell et al.
`Nature 1.Qi. (1983), 368) which is proteolytically processed in
`intestinal L cells to GLP-l(l-37) _and GLP-1(7-36)amide or GLP-
`1(7-37)
`(Mojsov et al. (J.Biol.Chem. 261 (1986), 11880) and
`25 .Habener et al. : The Endocrine Pancreas E. Samo is 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 1-a, .(1989), 338). They
`30 are the most potent gluco-incretins so far described and are
`
`CONFIF
`
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`2
`
`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
`s adenylate cyclase and a rise in the intracellular concentration
`of cyclic AMP
`(Drucker et al. Proc.Natl.Acad.Sci. USA 84
`(1987), 3434) and Goke et al. (Am.J.Physiol. 1.22. (1989), G397).
`GLP-1 has also a
`stimulatory effect on
`insulin gene
`transcription
`(Drucker et al. Proc.Natl.Acad.Sci. USA 84
`10 (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
`1s and after glucose ingestion; however, following a glucose load
`there was only
`a very
`small
`rise
`in plasma
`insulin
`concentration (0rskov 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
`20 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
`25 new family of G-coupled receptors.
`
`DESCRIPTION OF THE INVENTION
`
`The present invention relates to a recombinant glucagon-like
`peptide-1 (GLP-1) receptor.
`
`More pref er ably, the invention relates to a GLP-1 receptor
`30 which comprises the amino acid sequence shown in SEQ ID No. 1,
`or an analogue thereof binding GLP-1 with an affinity constant,
`In the present
`K0 , below 100 nM,. preferably below 10 nM.
`
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`

`

`"'
`..
`
`WO 93/19175
`
`PCT /EP93/00697
`
`3
`
`indicate a
`is intended to
`term "analogue"
`the
`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
`5 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
`10 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.
`
`In another aspect, th~ present invention relates to a DNA
`15 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
`20 may be provided in solubilised and/or reconstituted form.
`
`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,
`25 e.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
`30 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
`
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`4
`
`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
`s 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
`the sequence, addition of one or several nucleotides at either
`10 end of the sequence, or deletion of one or several nucleotides
`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~
`
`1s 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~ (1984),
`20 801
`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
`2s 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
`30 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
`
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`WO93/19175
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`PCT / EP93/00697
`
`5
`
`site-directed mutagenesi s using synthetic oligonucleotides
`encoding the desired amino acid sequence for homologous
`recombination in accordance with well-known procedures.
`
`Finally, the DNA constr uc t may be of mixed synthetic and
`s 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
`10 polymerase chain reaction using specific primers, for instance
`as described in us 4,683,202 or Saiki et al., Science ~
`(1988), 487 - 491.
`
`The recombinant expression veator into which the DNA construct
`of the invention is insert~a=: may be any vector which may
`,s 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
`20 independent of· chromosomal
`replication, e.g.
`a plasmid.
`the vector may be one which, when introduced
`Alternatively 1
`into a host cell, is integrated into the host cell genome and
`replicated togethe r with the chromosome (s) into which it has
`been integrated.
`
`25 In the vector, the DNA sequence encoding the GLP-1 receptor of
`the invention should be operably connected to a suitable pro(cid:173)
`moter sequence. The promoter may be any DNA sequence which
`shows transcriptional activity in the host cell of choice and
`may l;>e derived from genes encoding proteins either homologous
`30 or heterologous to the host cel l. Examples of suitable pro(cid:173)
`moters for directing the transcription of the DNA encoding the
`GLP-1 receptor of the invention in mammalian cells are the SV40
`promoter (Subramani et al., Mol . Cell Biol. i
`(1981) , 854 -
`
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`6
`
`864), the MT-1 (metallothionein gene) promoter (Palmiter et
`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,
`s (1992) 7 - 11). suitable promoters for use in yeast host cells
`include promoters from yeast glycolytic genes (Hitzeman et al.,
`J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki,
`J. Mel. Appl. Gen.
`(1982),
`419
`434 ) or alcohol
`.!
`dehydrogenase genes (Young et al., in Genetic Engineering of
`10 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), 652 - 654) promoters.
`Suitable promoters for use in filamentous fungus host cells
`are, for instance, the ADH3 promoter (McKnight et al. , The EMBO
`15 J • .i (1985), 2093 - 2099) or the tpiA promoter.
`
`;.
`
`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. , .Qlh
`cit. ) or (for fungal hosts) the TPil (Alber and Kawasaki, .Qlh
`20 cit.) or ADH3 (McKnight et al., .Qlh cit.) terminators. The vec(cid:173)
`tor may further comprise elements such as polyadenylation
`signals (e.g.
`from SV40 or the adenovirus 5 Elb region),
`transcriptional enhancer sequences (e.g. the SV40 enhancer) and
`translational enhancer sequences
`(e.g.
`the ones encoding
`25 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 example of such a sequence (when the
`host cell is a mammalian cell)
`is the SV40 origin of
`30 replication. 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
`t o a drug, e.g.
`neomycin, .hygromycin or methotrexate.
`
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`
`7
`
`The procedures used to ligate the DNA sequences coding for the
`GLP-1 receptor of the invention, the promoter and the ter(cid:173)
`minator, respectively, and to insert them into suitable vectors
`containing the information necessary for replication, are well
`s known to persons skilled in the art (cf. ,
`for
`instance,
`Sambrook et al., op.cit.).
`
`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
`10 cell, such as invertebrate (insect) cells or vertebrate cells,
`~ Xenopus laeyis 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
`15 of transfecting mammalian cells and expressing DNA sequences
`introd~ced 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 e~ al., Cell
`20 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 1
`(1981), 603, Graham and van der Eb, Virology 52 (1973), 456;
`and Neumann et al., EMBO J. 1 (1982), 841 - 845.
`
`Alternatively, fungal cells (including yeast cells) may be used
`as host cells of the invention. Examples of suitable yeasts
`25 cells
`include cells of
`saccharomyces
`spp. or Schizo(cid:173)
`saccharomyces spp. ,
`in particular strains of Saccharomyces
`cereyisiae. Examples of other fungal cells are cells of fila(cid:173)
`mentous fungi, e.g. Aspergillus spp. or Neurospora spp., in
`particular strains of Aspergillus oryzae or Aspergillus niger.
`30 The use of Aspergillus spp. for the expression of proteins is
`described in, e.g., EP 272 277.
`
`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(cid:173)
`covering the GLP-1 receptor from the culture. The medium used
`to cul.ture the cells may be any conventional medium suitable
`s 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 according to published recipes (e.g. in catalogues of
`the American Type Culture Collection).
`
`10 If the GLP-1 receptor has retained the transmembrane and (pos(cid:173)
`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
`1s be a component of membrane preparations, e.g. in solubilised
`and/or reconstituted form as defined above.
`
`In a still further aspect, the present invention relates to a
`method of screening for agonists or enhancers of GLP-1
`activity, the method comprising incubating a GLP-1 receptor
`20 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 ef feet from the
`suspected agonist on
`the binding of GLP-1
`to the GLP-1
`receptor. An enhancer being defined as a compound capable of
`25 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).
`
`An a1ternative .method of screening for agonists of GLP-1
`30 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 r_eceptor of the invention, and
`detecting any effect on the binding to the GLP-1 receptor. such
`
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`9
`
`agonists of GLP-1 activity will be . substances stimulating
`glucose-induced insulin secretion and may be used in the
`treatment of NIDDM.
`
`fJ
`
`The GLP-1 receptor may be immobilized on a solid support and
`s may, as such, be used as a reagent in the screening methods of
`the invention. The GLP-1 receptor may be used in membrane(cid:173)
`bound form, i.e. bound to whole cells or as a component of
`membrane preparations immobilised on a solid support.
`
`The solid support emplpyed in the screening methods of the
`10 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.
`1s 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
`20 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(cid:173)
`though some shapes may be more convenient than others for the
`25 present purpose. Thus, the solid support may be in the shape of
`a plate, e.g. a thin layer or microtiter plate, or a film,
`strip, membrane (e.g. a nylon membrane or a cellulose filter)
`or solid particles (e.g. latex beads or dextran or agarose
`beads). !n a preferred embodiment, the solid support is in the
`30 form · of wheat germ agglutinin-coated SPA beads
`(cf. US
`4,568,649).
`
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`10
`
`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
`s intrace·llular cAMP concentration. The cAMP concentration can
`then be measured directly. Changes in cAMP levels may also be
`monitored indirectly using appropriate cell lines in which a
`measurable signal is generated in response to an increase in
`intracellular cAMP.
`
`10 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
`1s 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, be
`20 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 considerable
`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,
`30 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 regulated
`expression and desensitization of the receptor in the normal
`
`it
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`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
`s 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 M (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
`
`20 Fig. lA and Fig. lB which is a continuation of Fig. lA together
`show the amino acid se.quence of the rat GLP-1 receptor in a
`comparison . with the sequence of the rat secret in receptor
`(SECR), the opossium parathyroid hormone receptor (PTHR) and
`the porcine calcitonin receptor (CTRl). The GLP-1 receptor has
`2s 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 par_t of the molecules as well as in the COOH(cid:173)
`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.
`
`'\
`
`:i,
`
`SUBSTJTI ITF RI-IF~T
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`Fig. 2 shows binding' of 125I-GLP-1 to cos cells transfected
`with the pGLPR-16 plasmid. Specific binding reaches saturation
`at i
`to 10 nM GLP-1. Insert: Scatchard analysis of GLP-1
`binding.
`
`s Fig. 3 shows binding of 125I-GLP-1 to INS-1 cells. Specific
`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
`10 Collection of
`radioligand analysis programs
`(Elsevier,
`Amsterdam, 1985)).
`
`Fig. 4 shows displacement of 125I-GLP-1 binding to cos cells
`transfected with the rat GLP-1 receptor cDNA. Transfected cells
`were incubated with 20 pM 12·5r-GLP-1
`in the presence of
`1s 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
`20 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).
`
`~ Fig. 6 shows tissue specificity of GLP-1 receptor expression
`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
`30 detected. The position of the migration of the ribosomal RNAs
`is indicated to the left of the picture.
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`Fig. 7 is a comparison of the rat GLP-1 receptor amino acid
`sequence (rat) and a partial amino acid sequence of the human
`GLP-1 receptor (human).
`
`i
`
`The present invention is further ill ustrated i n t he following
`5 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.
`
`10 A rat pancreatic i slet cDNA : library was constructed in the
`pcDNA-1 expression vector (Rat pancreatic islets were prepared
`according to Gotch et al. (Transplantation il (1985), 725).
`PolyA+ RNA was prepared and the cDNA library was constructed in
`the pcDNA-1 vector (In Vitrogen) as described by Aruffo and
`15 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
`20 (Sompayrac and Dana, Proc.Natl.Acad.Sci. USA 78 (1981) , 7575 ) .
`The presence of GLP-1 receptor expressed in cos · cell s was
`assessed by binding of the radioiodinated peptide
`followed by
`photographic emulsion autoradiography and screening by dark
`field microscopy (Gearing et. al. EMBO J. j
`(1989), 3667). GLP-
`2s 1(7-36)amide, as well as the other peptides, were purchased
`fro~ 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
`30 determined by the self displacement technique (Calvo et al.
`Biochem. 212 (.1983) , 259) .. A 1. 6 kb cDNA clone (pGLPR-1) was
`isolated by subf~actionation of an original positive pool and
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`was used to isolate, by DNA hybridization screening,
`two
`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
`s affinity (Ko= 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
`(Ko = o .12 nM; Fig. 3). In both cases a single
`(1992), 167)
`high affinity binding component was detected. The binding to
`10 GLP-1 receptor transfected cos cells reached a plateau between
`1 and 10 nM. At concentrations above 10 nM a second, high
`capacity,
`low affinity, binding component was detected.
`Although specifically displacable by cold GLP-1, this binding
`was also present in COS cells transfected with the expression
`1s vector alone and was therefore not further characterized.
`
`Binding of GLP-1 to the receptor expressed in COS cells was
`displaced by cold GLP-1 with a 50 percent displacement achieved
`at o. 5 to 1 nM (Fig. 4) • Other peptide hormones of related
`structure such as secretin, gastric inhibitory peptide (GIP)
`20 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.~, (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
`30 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
`-receptor to be homologous to the secretin .receptor (Ishihara et
`s al. EMBO J. 10
`(1991), 1635)
`( 40 percent identity) ,
`the
`parathyroid hormone receptor (Juppner 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
`10 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
`1s 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,
`20 cytoplasmic, ends of each receptor are also very . different.
`These. receptors all st