US 5,474,898 AGrant
Octopamine Receptor
Issue Date:1995-12-12
•5 Claims
•18 Drawing Sheets
Abstract
The present invention pertains in general to invertebrate octopamine receptor proteins and to polynucleotides encoding such receptors. The present invention also relates to insect for example, Drosophila octopamine receptors that are recombinantly expressed in mammalian cells where the receptor mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile that is unique but closely related to mammalian adrenergic receptors. The present invention further relates to drug screening methods for the development of specific human pharmacological drugs and insecticides targeted for the octopamine receptor system.
Metadata
Assignee
- The United States of America as represented by the Department of Health and Human Services
Inventors
- John C. Venter
- Claire M. Fraser
- William R. McCombie
Application Information
Application Number:US 1943383
Filing Date:1994-02-08
Priority Date:1991-03-28
Art Unit:182
Classifications
IPC:
C07K 14705
Field of Search:
4355306350
Patent Drawings (18 sheets)
Description
FIELD OF THE INVENTION
The present invention pertains, in general, to invertebrate octopamine receptor proteins and to polynucleotides encoding such receptors. The present invention relates in particular, to invertebrate receptors that are recombinantly expressed in mammalian cells. The receptor proteins of the invention mediate the attenuation of adenylate cyclase activity and exhibit a pharmacological profile that is unique but closely related to mammalian adrenergic receptors.
The present invention further relates to drug screening methods for the development of specific human pharmacological drugs and insecticides targeted for the octopamine receptor system.
BACKGROUND INFORMATION
Octopamine, a biogenic amine, plays a major role as a transmitter, hormone, and neuromodulator in invertebrates (see P. D. Evans, Adv. Insect Physiol. 15:317-473 (1980), for review). Through the use of pharmacological and biochemical assays, specific receptors for octopamine have been identified in many invertebrate phyla, including arthropods and molluscs (J. A. Nathanson and P. Greengard, Science. 180:308-310 (1973); J. Axelrod and J. M. Saavedra, Nature 265:501-504 (1977); A. J. Harmer and A. S. Horn, Mol. Pharmacol. 13:512-520 (1977); R. J. Walker and G. A. Kerkut, Comp. Biochem. 61C:261-266 (1978); S. Konishi and E. A. Kravits, J. Physiol. 279:215-229 (1978); B. Battelle and E. A. Kravits, J. Pharmacol. Exp. Ther. 205:438-448 (1978); J. A. Nathanson, Science. 203: 65-68 (1979); R. P. Bodnaryk, Insect Biochem. 9: 155-162 (1979); B. Battelle et al., Experientia 35:778 (1979); E. A. Kravits et al. , J. Expo Biol. 89:159-175 (1980); P. D. Evans, J. Physiol. 318:99-122 (1981) and P. D. Evans, J. Exp. Bio. 129:239-250 (1987); M. S. Livingston et al., J. Neurobiol. 12: 27-54 (1981); A. Uzzan and Y. Dudai, J. Neurochem. 38:1542-1550 (1982); R. M. Harris-Warrick and E. A. Kravitz, J. Neurosci. 4: 1976-1993 (1984); P. D. Evans et al., J. Pharm. Pharmacol. 40:855-861 (1988)). Pharmacological studies in insects have provided evidence for the existence of multiple subtypes of octopamine receptors (P. D. Evans, Insect. Physiol. 15:317-473 (1980) , P. D. Evans, J. Physiol. 318:99-122 (1981), P. D. Evans, J. Exp. Biol. 129:239-250 (1987); P. D. Evans et al., J. Pharm. Pharmacol. 40:855-861 (1988)). In addition, there are reports indicating that octopamine receptors, pharmacologically distinct from adrenergic and dopamine receptors, exist in the mammalian central nervous system (T. P. Hicks and H. McLennan, Brain Res. 157:402-406 (1978), T. P. Hicks and H. McLennan, Br. J. Pharmacol. 64:485-491 (1978b); W. P. C. Dao and R. J. Walker, Experientia 36: 584-585 (1980)) .
Since octopamine receptors are selectively blocked by .alpha.-adrenergic antagonists and activated by .alpha.-adrenergic agonists, there has been speculation that octopamine receptors are closely related to vertebrate adrenergic receptors (P. D. Evans, Adv. Insect Physiol. 15:317-473 (1980), P. D. Evans, J. Physiol. 318:99-122 (1981), P. D. Evans, J. Exp. Biol. 129:239-250 (1987); J. C. Venter et al., Biochem. Pharmacol. 38:1197-1208 (1988)). However, there have been no structural data available to evaluate this hypothesis. Studies of octopamine receptors have also been complicated by the lack of specificity of available ligands both among the octopamine receptor subtypes and between octopamine receptors and receptors for other biogenic amines.
The present inventions are products, processes and compositions that relate, at least in part, to an octopamine receptor cDNA and to the protein product encoded therein. Sequence analysis of the cDNA reveals that the octopamine receptor is a member of the adrenergic/muscarinic/opsin gene superfamily of receptors (J. C. Venter et al., Prog. Neurobiol. 30:105-169 (1988; J. C. Venter et al., Biochem. Pharmacol. 38:1197-1208 (1989)). Permanent expression of this cDNA in mammalian cells provides the opportunity to study an octopamine receptor in isolation and thus, allows for the unambiguous description of a single receptor type. It also allows for a rapid and effective assay for the rational design in testing of insecticides targeted against the receptor.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a segment of a DNA molecule which codes for an octopamine receptor protein derived from invertebrates, for example, Drosophila, and the encoded proteins. It is another object of the present invention to provide a recombinant DNA molecule comprising a vector and a DNA segment which codes for the invertebrate octopamine receptor.
It is a further object of the present invention to provide a host cell stably transformed with the recombinant DNA molecule provided by this invention in a manner allowing expression of the octopamine receptor protein.
It is another object of the present invention to provide methods for screening potential pharmaceuticals and insecticides that are reactive to octopamine receptors and other related biogenic amine receptors. Such methods include a radiolabel binding and competition displacement assay and a metabolic assay that measures agonists and antagonists of octopamine mediated attenuation of adenylate cyclase.
Various other objects and advantages of the present invention will become obvious from the drawings and the detail description of the invention.
In one embodiment, the present invention relates to a DNA segment encoding all, or a unique portion, of an invertebrate octopamine receptor. An example of an invertebrate may be the insect, Drosophila.
In another embodiment, the present invention relates to a substantially pure form of Drosophila octopamine receptor.
In a further embodiment, the present invention relates to a recombinantly produced protein having all, or a unique portion, of the amino acid sequence FIGS. 3A-3J given in SEQ ID NO: 2.
In a further embodiment, the present invention relates to a recombinant DNA molecule comprising a DNA segment encoding all, or a unique portion, of an invertebrate octopamine receptor and a vector.
In yet a further embodiment, the present invention relates to a host cell stably transformed with the above recombinant DNA molecule in a manner allowing expression of invertebrate octopamine receptor protein encoded in the recombinant DNA molecule.
In another embodiment, the present invention relates to drug screening methods for detecting agonists and antagonists of octopamine receptors comprising the steps of contacting CHO-K1 cells that have been stably transformed and express recombinant octopamine receptor with the labeled ligand yohimbine in the presence or the absence of a specific agonist or antagonist, and measuring the amount of bound labeled ligand.
In yet another embodiment, the present invention relates to a method of screening drugs and insecticides that recognize biogenic amine receptors comprising the steps of incubating forskolin and a potential drug or insecticide in non-transfected and octopamine receptor transfected CHO-K1 cells and comparing the change in cAMP concentrations to control cells that have been incubated with forskolin alone.
The entire contents of all publications mentioned herein are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1F shows the restriction map, sequencing strategy, and nucleotide (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the Drosophila octopamine receptor cDNA clone.
The nucleotide sequence is numbered from the first ATG of the coding region. Selected restriction sites are shown above the diagram. In the sequencing strategy diagram at the top of the figure, each arrow represents an individual clone that was sequenced at least once. The sequence has been submitted to GenBank and assigned the accession number M26181.
FIGS. 2A and 2B shows hydropathy analysis of Drosophila octopamine, receptor and the human .alpha..sub.2 -adrenergic receptor, respectively.
Hydropathy plots of Kyte and Doolittle (1982) were calculated using a window of 20 residues for both the Drosophila octopamine receptor and the human .alpha..sub.2 -adrenergic receptor (C. M. Fraser, J.Biol. Chem. 264:11754-11761 (1989a)). The predicted secondary structure of both receptors contained seven stretches of hydrophobic amino acids of sufficient length to span the bilayer, similar to the secondary structure of other members of the adrenergic/muscarinic/opsin gene superfamily. The Drosophila receptor also contains additional hydrophobic residues at its amino terminus that may play a role as a putative signal sequence, a feature observed in many membrane proteins (G. Blobel and B. Dobberstein, J. Cell Biol. 67:852-862 (1975); T. Onai et al., FEBS Lett. 255:219-225 (1989)).
FIGS. 3A-3J shows a comparison of the deduced amino acid sequences of the Drosophila octopamine, receptor with other members of the gene superfamily.
Amino acid sequences were aligned using the GAP Program of the University of Wisconsin Genetics Computer Group (J. Devereux et al., Nucl. Acids Res. 12:387-395 (1984)), with gaps introduced to maximize homology followed by minor manual alignments. Conserved amino acid residues, as defined by Dayhoff et al. (1978), were considered to be identical. The amino acid sequences shown represent those of the Drosophila octopamine, receptor (Octdrs; SEQ ID NO: 2), hamster .alpha..sub.1 -adrenergic receptor; SEQ ID NO: 3 (A1ham), human .alpha..sub.2 B and .alpha..sub.2 A-adrenergic receptors (A2b1hum and A2hum; SEQ ID NOS: 4 and 5, respectively), human and turkey .beta..sub.1 -adrenergic receptors (B1hum and B1tur; SEQ ID NOS: 6 and 7, respectively), hamster and rat .beta..sub.2 -adrenergic receptors (B2hum and B2rat; SEQ ID NOS: 8 and 9, respectively), rat D2 dopamine receptor (D2rat; SEQ ID NO: 10), rat and Drosophila muscarinic receptors (M 2rat and Mdrs; SEQ ID NOS: 11 and 12), and rat 5HT1c receptor (S1crat; SEQ ID NO: 13). Homology is indicated in the consensus line (SEQ ID NO: 14) below the receptor sequences. Putative transmembrane regions are labeled. Conserved sites for N-linked glycosylation are indicated by an asterisk. Data are from the present study and from other works (R. Dixon et al., Nature 321:75-79 (1986); Y. Yarden et al., Proc. Natl. Acad. Sci. USA (1986); F. Z. Chung et al., FEBS Lett. 211:200-206 (1987); T. Frielle et al., Proc. Natl. Acad. Sci. USA 84:7920-7924 (1987); J. Gocayne et al., Proc. Natl. Acad. Sci. USA 84:8296-8300 (1987); S. Cotecchia et al., Proc. Natl. Acad. Sci. USA 85:7159-7163 (1988); J. W. Regan et al., Proc. Natl. Acad. Sci. USA 85:6301-6305 (1988); D. Julius et al., Science 241:558-563 (1988); T. Onai et al., FEBS Lett. 255:219-225 (1989); C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989a); J. R. Bunzow et al., Nature 336:783-787 (1988)).
FIG. 4 shows the effect of biogenic amines on cAMP concentrations CHO-K1 cells expressing the Drosophila octopamine.sub.1 receptor.
Transfected cells (3.times.10.sup.5 cells per dish) were washed once with PBS and incubated in PBS containing 1 mM 3-isobutyl-1-methylxanthine (Sigma) at 37.degree. C. for 20 min. Agonists, at the indicated final concentrations, were added to the cultures along with 10 .mu.M forskolin (Sigma), and cultures were incubated at 37.degree. C. for an additional 20 min. Incubations were terminated by the addition of 6% trichloroacetic acid. Trichloroacetic acid extracts were extracted four times with distilled water-saturated ether, and cMAP concentrations were determined using a cAMP radioimmunoassay kit (New England Nuclear). Data are expressed as percent forskolin-stimulated cAMP production. In the indicated experiments, cell cultures were incubated with Bordetella pertussis islet activating protein (200 ng/ml; list biological lab) at 37.degree. C. for 18 hr in normal culture medium prior to the assay of cAMP. Solid and open squares represent data obtained with octopamine in transfected and control cells, respectively. Solid circles represent data with epinephrine in transfected cells. Open triangles and open circles represent data with octopamine in transfected cells in the presence of 10 .mu.M yohimbine or in cells pretreated with pertussis toxin, respectively. Data represent the average .+-.SEM of at least three separate experiments performed in duplicate.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to segments of DNA which encode an octopamine receptor for invertebrates. In particular, the present invention relates to a DNA segment that encodes the octopamine receptor for the invertebrate, Drosophila. The DNA sequence analysis shown in FIG. 1A-1F and amino acid sequence analysis shown in FIG. 3A-3J demonstrate that the octopamine receptor is a member of the adrenergic/muscarinic/opsin gene superfamily of receptors.
The invention also relates to the invertebrate octopamine receptor proteins, for example, Drosophila octopamine receptors encoded by octopamine receptor DNA segments.
The octopamine protein found in invertebrates such as Drosophila has a calculated molecular weight, as determined by DNA sequence analysis of about 64,700 daltons. In another embodiment, the octopamine receptor has the amino acid sequence as set forth in FIGS. 3A-3J. The amino acid sequence of the Drosophila protein receptor displays significant homology with mammalian .alpha. and .beta. adrenergic receptors, rat and Drosophila muscarinic acetylcholine receptor and rat D.sub.2 dopamine and 5HT receptors.
In another embodiment, the octopamine protein encoded by the Drosophila octopamine receptor gene is glycosylated, displaying conserved sites for N-linked glycosylation within the amino terminal domains similar to .alpha.- and .beta.-adrenergic receptors described above. The octopamine receptor protein of the present invention is substantially free of proteins with which it is normally associated and can be bound to a solid support such as, for example, agarose, sepharose, plastic, nylon membrane or nitrocellulose paper.
The present invention also relates to a recombinant DNA molecule comprising a vector and the above-described DNA segment which encodes the invertebrate Drosophila octopamine receptor or unique portion thereof. Possible vectors include plasmids, for example, pSVL expression vector, and other vectors known in the art that either transiently or stably transform host cells in a manner which allows expression of the octopamine receptor proteins. Examples of appropriate eukaryotic cells include, for example, Chinese hamster kidney cells. Other eukaryotic cells such as Baculovirus and Schneider cells or prokaryotic expression systems could be adapted for the production of Drosophila octopamine proteins.
The present invention further relates to drug screening methods that may be useful in developing specific insecticides and human pharmaceutical drugs targeted against the octopamine receptor system. In a binding assay provided herein, octopamine receptor genes isolated from a specific DNA library are inserted into a plasmid expression vector and used to transfect a Chinese hamster kidney cell called CHO-K1 cells. These cells stably integrate the foreign DNA into the genome. Cells that express the insect octopamine receptor and process receptor into the mammalian cell membrane are cloned, expanded and frozen down for further use as membrane preparations in a ligand binding assay. In the assay disclosed herein, the membrane bound octopamine receptor preparations are incubated with labeled yohimbine, an .alpha.2-adrenergic receptor antagonist. Suitable labels include for example, radiolabels such as .sup.3 H. Transfected CHO-K1 cell membranes displace saturable, high affinity binding of labeled ligand yohimbine, with a calculated equilibrium dissociation constant described herein. No specific labeled ligand is observed bound to membranes prepared from control, untransfected CHO-K1 cells. The binding of potential drug agonists and antagonists to the insect octopamine receptor are examined in competition displacement studies with labeled yohimbine. Thus, in one method taught by the present invention, the membrane preparations are incubated with a fixed concentration of labeled yohimbine in the presence of increasing concentrations of either agonist or antagonist. After the incubations are terminated, the reaction mixture containing labeled ligand bound to receptor membranes is calculated by measuring label. Analogs of octopamine (agonists) display a stronger affinity for the octopamine receptor and displace labeled ligand whereas antagonists show a lower affinity for octopamine receptors and do not displace bound labeled ligand.
Another aspect of the present invention relates to another drug screening method that identifies new compounds with biogenic amine (agonists) or antagonist activity for biogenic receptors by measuring adenylate cyclase concentrations in transfected CHO-K1 cells expressing octopamine receptors. In one embodiment, potential drugs are tested for their ability to attenuate forskolin-stimulated adenylate cyclase activity. Forskolin is added to control or octopamine receptor transfected CHO-K1 cells that stimulates increased intracellular cAMP levels in transfected cells over basal CHO-K1 levels. In the presence of forskolin, known concentrations of octopamine are added and the attenuation of forskolin stimulated cyclase AMP is calculated. A potential antagonist is determined by the competitive reversal of octopamine mediated attenuation of forskolin stimulated cyclic AMP accumulation when the drug is added to the assayed transfected cells. A potential agonist drug will have no effect of forskolin stimulated cyclic AMP level in either transfected or control CHO-K1 cells.
The present invention further relates to methods of isolating other neurogenic and biogenic receptors using the octopamine receptor DNA segment described in the present invention. One skilled in the art will appreciate that the invention includes methods for isolating related octopamine receptors in other biogenic amine receptors using human genomic DNA libraries and a DNA segment encoding the octopamine receptor or a unique portion thereof as a probe in known cloning and nucleotide sequencing methods. One skilled in the art will also appreciate that such related receptors could be used in the methods described above for the design of screening of insesticides and drugs.
Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.
EXAMPLES
The following materials and procedures are referred to in the Examples that follow.
Screening of a Drosophila Genomic Library
Using a human brain .beta..sub.2 -adrenergic receptor as a hybridization probe, a Drosophila genomic library was screened under low stringency conditions (hybridization with 30% formamide, 6.times. SSC, 5.times. Denhardt's, 50 mM NaPO.sub.4 [pH 7.4], 50 .mu.g/ml sheared salmon sperm DNA for 18 hr at 49.degree. C.; wash with 6.times. SSC, 0.1% SDS for 20 min at room temperature followed by 3.times. SSC for 20 min at room temperature). Three .lambda.gt11 clones were isolated, and a 367 bp BglII fragment of one of these clones was used to screen a cDNA library.
Screening of a Drosophila cDNA Library
The Drosophila head cDNA library was made from poly(A).sup.+ mRNA isolated from the heads of the Oregon-R strain of D. melanogaster. A 367 bp BglII fragment of the Drosophila genomic clone (DGSA1641; see C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989)) was radiolabeled by random priming for screening of the Drosophila cDNA library. The library was transferred to Hybond-N filters (Amersham) and screened with the .sup.32 P-labeled probe under the following hybridization conditions; 30% formamide, 6.times. SSC, 5.times. Denhardt's 50 mM sodium phosphate (pH 7.4), 50 .mu.g/ml sheared salmon sperm DNA at 42.degree. C. for 18 hr. Filters were washed in 2.times. SSC, 0.1% SDS and then in 0.5.times. SSC at 55.degree. C. for 30 min each. Seven positive clones were isolated from the 6000,000 pfu screened; 6 of these displayed identical restriction maps.
Sequence Analysis of Drosophila cDNA Clones
A 3.3 kb EcoRI cDNA insert from 1 of the 6 identical clones that hybridized on Southern blots with the DGSA1641 probe at high stringency (0.5.times. SSC at 55.degree. C. for 30 min) was isolated. Blunt ends were created with Klenow fragment of DNA polymerase, and the insert was subcloned in both orientations into the HincII site in the multiple cloning region of pVZ1 (A. Henikoff and M. K. Eghtedarzadeh, Genetics 17:711-725 (1987)). Exonuclease III digestion was performed at 37.degree. C., and aliquots were removed every 30 s as described by Henikoff (1987). Single-stranded DNA was purified and prepared for sequence analysis using an Applied Biosystems 370A Automated DNA Sequencer (J. Gocayne et al., Proc. Natl. Acad. Sci. USA 4:8296-8300 (1987)).
Expression of the Drosophila Receptor in CHO-K1 Cells
Based on analysis of open reading frames, the region of approximately 2.2 kb, as shown in FIGS. 1A-1F, was selected for expression in CHO-K1 cells. A 2.2 kb fragment as excised from pVZ1 by sequential enzyme cuts. The vector was linearized with Clal, and blunt ends were generated with DNA polymerase. The cDNA fragment was released from the vector by cleavage with AIwNI. The AIwNI site is contained within the defined open reading frame and this necessitated reconstruction of the 5' end of the gene with a synthetic oligonucleotide. This was accomplished by ligating the gene fragment and the following synthetic oligonucleotide:
5'-CTAGATGCCATCGGCAGATCAAATCAAATC-3' (SEQ ID NO: 15)
3'-TACGGTAGCCGTCTAGTT-5' (SEQ ID NO: 16)
into pSVL that had been cleaved with XbaI and SmaI. The synthetic oligonucleotide reconstructs the 5' end of the gene, destroys the AIwNI site with a silent mutation, and adds a terminal XbaI site, which facilitates insertion of the fragment into the expression vector in a directional manner. The 5' portion of the fragment reconstructed for expression was excised from pSVL and ligated into M13 to confirm the sequence of the 5' end of the construct used for expression. pSVL containing the Drosophila receptor gene was cotransfected with pMSVneo, which contains the selective marker for G418 resistance, into CHO-K1 cells (American Type Culture Collection) using the calcium phosphate precipitation technique (C. M. Fraser et al., Proc. Natl. Acad. Sci. USA C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989) , C. M. Fraser et al., Proc. Natl. Acad. Sci. USA 85:5478-5482 (1989)). Stable transfectants were obtained by growth of the cells in culture medium containing the antibiotic, Geneticin (G418, 500 .mu.g/ml; GIBCO). Colonies derived from single cells were isolated and expanded as described (C. M. Fraser et al., Proc. Natl. Acad. Sci. USA 85:5478-5482 (1988) , C. M. Fraser et al., J. Biol Chem. 264:11754-11761 (1989), C. M. Fraser et al., Proc. Natl. Acad. Sci. USA 85:5478-5482 (1989)) and those transfectants expressing the Drosophila gene were identified by Northern blot analysis of total cellular RNA.
Northern Analysis of Transfected CHO-K1 Cells Expressing the Drosophila Octopamine Receptor
RNA was isolated from five 150 mm plates of confluent cells using guanidinium thiocyanate according to the method of Chirgwin et al., (1979). Total RNA (20 .mu.g) from each line of transfected cells was separated on agarose gels containing formaldehyde as described by Maniatis et al. (1982). RNA was transferred to Hybond-N membranes (Amersham) in 25 mM NaPO4 buffer (pH 6.5), and the membrane was hybridized with the 3.3 kb Drosophila cDNA clone radiolabeled with [.sup.32 P]cytosine (0.5.times.10.sup.6 cpm/ml). The Northern blot was washed with 6.times. SSC, 0.1% SDS for 15 min at 42.degree. C. 2.times. SSC for 20 rain at 42.degree. C., 2.times. SSC for 30 min at 56.degree. C., and 0.5.times. SSC for 30 min at 56.degree. C.
Cell Membrane Preparation and Radioligand Binding Assays
Cell membranes from transfected cells were prepared as previously described (F.-Z. Chung et al., J. Bol. Chem. 263:4052-4055 (1988); C. M. Fraser et al., Proc. Natl. Acad. Sci. USA 85:5478-5482 (1988), C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989), C. M. Fraser et al., Proc. Nat;. Acad. Sci. USA 85:5478-5482 (1989)). [.sup.3 H]Yohimbine binding assays and competition displacement experiments were performed as described for the human .alpha..sub.2 -adrenergic receptor (C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989). Ligand binding data were analyzed using the LIGAND program of Munson and Rodbard (1980).
Example 1.
Octopamine Receptor Gene Cloning
The known nucleotide sequence similarities among G protein-linked receptors was exploited to search for genes related to adrenergic receptors in a Drosophila genomic library using a human brain .beta..sub.2 -adrenergic receptor cDNA clone (F.-Z. Chung et al., FEBS Let. 211:200-206 (1987)) as a hybridization probe. Using low stringency hybridization conditions, three .lambda.gt11 clones were isolated. Partial sequence analysis revealed that the genomic clones contained exons that, when translated, displayed high homology to the transmembrane domains of mammalian adrenergic receptor proteins. Using a 367 bp fragment of Drosophila genomic DNA (clone DGSA1641) that displayed homology with the putative sixth and seventh transmembrane domains of adrenergic receptors as a hybridization probe, a 3.3 kb cDNA clone from a Drosophila head cDNA library was isolated. This same 367 bp fragment of Drosophila genomic DNA was also used to isolate an .alpha..sub.2 -adrenergic receptor gene from a human genomic library (C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989)).
Example 2.
Sequencing Strategy and Protein Structure
The sequencing strategy and restriction map of the 3.3 kb Drosophila cDNA clone are shown in FIG. 1A. This strategy was based upon the generation of overlapping, unidirectional exonuclease III deletions (S. Henikoff, Meth. Enzymol. 155:156-165 (1987)). Automated DNA sequence analysis was carried out using an Applied Biosystems 370A DNA Sequencer (J. Gocayne et al., Proc. Natl. Acad. Sci. USA 84:8296-8300 (1987)). As illustrated in FIG. 1, the nucleotide sequence of this clone contains 3335 bp and has a large open reading frame that encodes a protein of 601 amino acids with a calculated molecular mass of 64,700 daltons. This cDNA clone and genomic clone DGSA1641 (originally used as a hybridization probe) contained identical sequences.
Several features of this protein suggested that it belongs to the adrenergic/muscarinic/opsin, gene superfamily. Hydropathy profiles revealed seven stretches of hydrophobic amino acids of sufficient length to span the bilayer, similar to the secondary structure of other members of the gene superfamily (FIG. 2). However, unlike other members of this gene family, the Drosophila protein contains an additional sequence of hydrophobic residues at its amino terminus. The properties of this amino-terminal hydrophobic sequence suggest that it may function as a putative signal sequence, a feature observed in many membrane proteins (G. Blobel and B. Dobberstein, J. Cell. Biol. 67:852-862 (1975)). A similar region is also seen in the putative muscarinic acetylcholine receptor from Drosophila (T. Onai et al., FEBS Lett. 255:219-225 (1989)). As shown in FIGS. 3A-3J, the deduced amino acid sequence of the Drosophila protein displays significant homology with those of mammalian .alpha.- and .beta.-adrenergic receptors, rat and Drosophila muscarinic acetyl-choline receptors, and rat dopamine and 5 HT receptors (R. Dixon et al., Nature 321:75-79 (1986); Y. Yarden et al., Proc. Natl. Acad. Sci. USA 83:6795-6799 (1986); F.-Z. Chung et al., FEBS Lett. 211:200-206 (1987); T. Frielle et al., Proc. Natl. Acad. Sci. USA 84:7920-7924 (1987); J. Gocayne et al., Proc. Natl. Acad. Sci. USA 84:8296-8300 (1987); S. Cotecchia et al., Proc. Natl. Acad. Sci. USA 85:7159-7163 (1988); J. W. Regan et al., Proc. Natl. Acad. Sci. USA 85:6301-6305 (1988); D. Julius et al., J. Physiol. 318:99-122 (1988); T. Onai et al., FEBS Lett. 255:219-225 (1989); C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989a); J. R. Bunzow et al., Nature 336:783-787 (1988)). The Drosophila receptor exhibited highest homology with .alpha..sub.2 -adrenergic receptors (32% and 35% identity, increasing to 66% and 70% similarity with conserved substitutions for .alpha..sub.2 A and .alpha..sub.2 B receptors, respectively). The regions of greatest amino acid identity among these receptors reside within the putative transmembrane helices. All of these receptors display conserved sites for N-linked glycosylation within their amino-terminal domains and conserved aspartic acid residues in the second and third transmembrane helices; these have been shown by site-directed mutagenesis to be involved in agonist-induced receptor activation in .beta.-adrenergic (F.-Z. Chung et al., J. Biol. Chem. 263:4052-4055 (1988); C. M. Fraser et al., Proc. Natl. Acad. Sci. USA 85:5478-5482 (1988)) and muscarinic acetylcholine (C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989)) receptors.
Example 3
Permanent Expression of the Drosophila cDNA Clone in Mammalian Cells
To prove the identity and characterize the Drosophila receptor further, 2.2 kb cDNA fragment containing the open reading frame was ligated into the expression vector pSVL and cotransfected with C. M. Fraser et al., Proc. Natl. Acad. Sci. USA 85:5478-5482 pMSVneo into CHO-K1 cells as previously described (F.-Z. Chung et al., J. Biol. Chem. 263:4052-4055 (1988); (1988)). Clonal cell lines that grew in selective medium containing Geneticin were isolated and expanded. Cell lines expressing the Drosophila cDNA fragment were identified by Northern blot analysis of total cellular RNA using the .sup.32 P-labeled Drosophila cDNA clone as a hybridization probe. Several clonal cell lines were identified that contained detectable levels of receptor-specific mRNA and one was chosen with the highest level for detailed pharmacological and biochemical studies.
Due to the sequence homology between the Drosophila cDNA clone and genes encoding mammalian .alpha..sub.2 -adrenergic receptors (C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989)), the assay was performed with [.sup.3 H]yohimbine, an .alpha..sub.2 -adrenergic receptor antagonist. Transfected CHO-K2 cell membranes displayed saturable, high-affinity binding of the radio-ligand [.sup.3 H]yohimbine, with a calculated equilibrium dissociation constant (K.sub.d) of 6.2 nM and a B.sub.max of 1.75 pmol of receptor per mg of membrane protein (Table 1). No specific radioligand binding was observed to membranes prepared from control, untransfected CHO-K1 cells. The affinity of yohimbine for the octopamine receptor is similar to that for the human .alpha..sub.2 -adrenergic receptor expressed in CHO-K1 cells (3.5 nM; C. M. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989)). The binding of a series of agonists and antagonists to the Drosophila receptor was examined in competition displacement studies with [.sup.3 H]yohimbine (Table 1). Synephrine, the N-methylated analog of octopamine, showed the highest agonist affinity (K.sub.i =10.8.+-.2.5 .mu.M) for the Drosophila receptor, followed by the .alpha.-adrenergic agonist clonidine (K.sub.i =21.+-.7 .mu.M), octopamine (K.sub.i =29.+-.6 .mu.M), serotonin (K.sub.i =75.+-.18 .mu.M), and epinephrine (K.sub.i =139.+-.41 .mu.M). The .beta.-adrenergic agonist isoproterenol and dopamine displayed low affinities for the Drosophila receptor (K.sub.i s>1000 .mu.M). Antagonists displayed a random order of potency for inhibition of [.sup.3 H]yohimbine binding of chlorpromazine (K.sub.i =0.18+0.006 .mu.M)>mianserin (K.sub.i =1.2.+-.0.3 .mu.M)>phentolamine (K.sub.i =2.2.+-.0.7 .mu.M)>cyproheptadine (K.sub.i =2.6.+-.0.5 .mu.M)>metoclopramide (K.sub.i =4.6.+-.1.4 .mu.M)>propranolol (K.sub.i =5.3.+-.1.6 .mu.M). The rank orders of potency for agonist and antagonist displacement of [.sup.3 H]yohimbine binding to the Drosophila receptor strongly suggested that an octopamine receptor had been cloned (Evans, Insect. Physiol. 15:317-473 (1980), P. D. Evans, J. Physiol. 318:99-122 (1981), P. D. Evans, J. Exp. Biol. 129:239-250 (1987); P. D. Evans et al., J. Pharm. Pharmacol. 40:855-861 (1988)). The significantly greater receptor affinity for yohimbine and chlorpromazine as compared with metoclopramide suggested that this was an octopamine type 1 receptor subtype (see table 1; see also Evans, Insect. Physiol. 15:317-473 (1980), P. D. Evans, J. Physiol. 318:99-122 (1981), P. D. Evans, J. Exp. Biol. 129:239-250 (1987); P. D. Evans et al., J. Pharm. Pharmacol. 40:855-861 (1988)).
Example 4
Octopamine-Mediated Attenuation of Adenylate Cyclase Activity
Numerous reports have described an octopamine-sensitive adenylate cyclase system in insects (J. A. Nathanson and P. Greengard, Science. 180:308-310 (1973); J. Axelrod and J. M. Saavedra, Nature 265:501-504 (1977); A. J. Harmer and A. S. Horn, Mol. Pharmacol. 13:512-520 (1977); B. Battlee and E. A. Kravits, J. Pharmacol. Exp. Ther. 205:438-448 (1978); J. A. Nathanson, Science. 203:65-68 (1979); R. P. Bodnaryk, Insect Biochem. 9:155-162 (1979); A. Uzzan and Y. Dudai, J. Neurochem 38:1542-1550 (1982); P. D. Evans, J. Exp. Biol. 129:239-250 (1987)); however, none of the agonists listed in Table 1 produced an increase over basal levels of intracellular cAMP in transfected CHO-K1 cells. These agonists also had no effect on phosphoinositide hydrolysis in transfected cells. Because of the pharmacological and structural similarities of the Drosophila receptor to mammalian .alpha..sub.a -adrenergic receptors, the ability of octopamine to attenuate forskolin-stimulated adenylate cyclase activity was tested. Addition of forskolin (10 .mu.M) to control or transfected CHO-K1 cells produced an average 35-fold increase in intracellular cAMP levels over basal values. As shown in FIG. 4, octopamine produced a dose-dependent attenuation of forskolin-stimulated cAMP levels in transfected cells that was not observed in control cells. In the presence of forskolin plus 100 .mu.M octopamine, cAMP levels were only 46% of those seen with forskolin alone. Octopamine-mediated attenuation of forskolin stimulated cAMP accumulation was competitively antagonized by yohimbine (FIG. 4), whereas epinephrine had no effect on forskolin-stimulated cAMP levels in either transfected or control CHO-K1 cells (FIG. 4). Furthermore, in transfected cells pretreated with Bordetella pertussis islet activating protein, the ability of octopamine to attenuate forskolin-stimulated cAMP levels was abolished, suggesting that this response was mediated through a pertussis toxin-sensitive G protein (P. J. Casey and A. G. Gilman, J. Biol. Chem. 263:2577-2580 (1988); M. A. Lochrie and M. I. Simon, Biochemistry 27:4957-4965 (1988)), as is observed with the .alpha..sub.2 -adrenergic receptor (C. A. Fraser et al., J. Biol. Chem. 264:11754-11761 (1989)).
Example 5
Drosophila Chromosome and Tissue Localization
To determine where the octopamine receptor gene maps in the Drosophila genome, one of the genomic clones, DGSA1641, was biotinylated using a nick translation kit (Bethesda Research Laboratories) and biotin-11-dUTP (Enzo biochemical) and hybridized to Drosophila salivary gland polytene chromosomes. The clone was detected using a DNA detection kit (Bethesda Research Laboratories) following the procedures of Engel et al. (1985). The probe hybridized to a single site on the right arm of chromosome 3 at 99A10-B1. Studies are in progress to determine whether any of the known mutations that map to this site represent the octopamine receptor gene.
A Drosophila genomic Southern blot was probed with the octopamine receptor cDNA clone. Eight micrograms of genomic DNA isolated from the Canton-S wild-type strain (T. Jowett, Preparation of nucleic acids. In Drosophila: A practical Approach, D. B. Roberts, ed. (Washington, D.C.: IRL Press), pp. 275-277 (1986)) was digested with EcoRI, BamHI or HindIII and electrophoresed on a 0.6% agarose gel. Following capillary blotting, the filter was hybridized with the octopamine cDNA clone that had been .sup.32 P-labeled by the random primer method. The blot was hybridized overnight at 45.degree. C. in 5.times. SSC, 0.05M sodium phosphate buffer (pH 6.8), 0.1% SDS, 5.times. Denhardt's, and 0.2 mg/ml denatured salmon sperm DNA. The blot was washed one time in 2.times. SSC, 0.1% SDS fir 30 min at room temperature followed by two 30 min washes at successive temperatures (45.degree. C., 50.degree. C., and 65.degree. C.). The pattern of hybridization was the same regardless of wash temperature used in the 45.degree. C.-65.degree. C. range. There do not appear to be BamHI or EcoRI sites within this gene, since only single bands of 23 kb (BamHI) or 6.6 kb (EcoRI) appear in these lanes. The hybridization pattern was unchanged by washing at 45.degree. C., 50.degree. C., 55.degree. C., or 65.degree. C. Thus, under conditions used in this example DNA probe appears to be hybridizing to a single gene. This is consistent with other in situ hybridization studies, which also showed a single hybridization site. This gene does not appear to be highly polymorphic in the Canton-S wild-type strain. It remains to be determined whether lower stringency hybridization conditions can be used to isolate other pharmacologically distinct octopamine receptor genes.
Octopamine receptors have been found in the central and peripheral nervous systems of insects (P. D. Evans, Adv. Insect Pysiol. 15:317-473 (1980)) as well as in skeletal muscle (P. D. Evans, J. Physiol. 318:99-122 (1981), P. D. Evans, J. Exp. Biol. 129:239-250 (1987); P. D. Evans et al., J. Pharm. Pharmacol. 40:855-861 (1988)). To determine whether the cloned octopamine receptor gene showed differential expression in nervous system versus skeletal muscle and gut, a comparison was made between the intensity of hybridization of .sup.32 P-labeled cDNA to equivalent amounts of poly(A).sup.+ mRNA from Drosophila heads versus bodies. The heads are enriched in nervous system, whereas the bodies are enriched for skeletal muscle (from the flight muscles in the thorax) and for gut (in the abdomen). RNA was prepared from 1 g of heads and 2 g of bodies using the guanidinium isothiocyanate homogenization-CsCl centrifugation procedure (J. M. Chirgwin et al., Biochemistry 18:5294-5299 (1979)). Each preparation was enriched for poly(A).sup.+ mRNA by passing it through one Pharmacia oligo (dT) column according to the manufacturer's instructions. Approximately 8-9 .mu.g of mRNA was added to each lane of a formaldehyde, 0.8% agarose gel. The electroblotted filter was prehybridized for 2 hr at 42.degree. C. with 5.times. SSPE, 10.times. Denhardt's, 50% deionized formamide, 5 mg/ml denatured salmon sperm DNA and then hybridized overnight at 42.degree. C. in 5.times. SSPE, 1.times. Denhardt's, 50% formamide, 1 mg/ml denatured salmon sperm DNA, 0.1% SDS using the cDNA probe labeled with .sup.32 P by the random primer method. The blot was washed as follows; a brief rinse in 2.times. SSC, 15 min in 2.times. SSC at room temperature, 15 min in 0.1.times. SSC at room temperature, and two 30 min washes in 0.1.times. SSC at 60.degree. C. All washes contained 0.1% SDS. Heads are substantially enriched for a single 3.6 kb mRNA compared with bodies. After a 7 day exposure, there is distinct hybridization in the heads, but no signal is apparent in the bodies. Control experiments using actin as a probe showed that each lane on this Northern gel had approximately the same level of mRNA. The octopamine receptor mRNA is relatively rare, since the control actin hybridization was visible after a 1 hr exposure but the same gel required a 7 day exposure to see a signal for the receptor mRNA.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.
Claims
What is claimed is:
1. A naturally occurring invertebrate octopamine receptor protein encoded by SEQ ID NO: 1 or by a DNA sequence hybridizing to the complement of the sequence shown in SEQ ID NO: 1 under low stringency conditions, said octopamine receptor protein substantially free of proteins with which it is normally associated.
2. A recombinantly produced polypeptide comprising the amino acid sequence encoded by SEQ ID NO: 1 or a polypeptide encoded by a DNA sequence hybridizing to the complement of the sequence shown in SEQ ID NO: 1 under low stringency conditions, said sequence encoding a naturally occurring invertebrate octopamine receptor.
3. A method of testing a compound for its affinity for an invertebrate octopamine receptor, comprising: contacting the membrane of a cell transformed with a polynucleotide with a labeled .alpha..sub.2 -adrenergic receptor antagonist in the presence and absence of said compound, said polynucleotide selected from the group consisting of a first polynucleotide encoding a naturally occurring invertebrate octopamine receptor protein comprising the amino acid sequence shown in SEQ ID NO: 2 and a second polynucleotide, said second polynucleotide capable of hybridizing to the complement of said first polynucleotide under low stringency hybridization conditions and which encodes a naturally occurring invertebrate octopamine receptor protein; and measuring the amount of labeled antagonist bound to said receptor in the presence of said compound versus in the absence of said compound.
4. A method of testing a compound for its ability to recognize an octopamine receptor, comprising: incubating forskolin and said compound with control CHO-K1 cells and CHO-K1 cells, said cells transformed with the DNA sequence shown in SEQ ID NO: 1 or with a DNA sequence hybridizing to the complement of the sequence shown in SEQ ID NO: 1 under low stringency conditions and encoding a naturally occuring octopamine receptor; and measuring the increase or decrease of cAMP concentration in said control and transformed CHO-K1 cells.
5. The method of claim 4 wherein the receptor transfected CHO-K1 cells are Drosophila octopamine receptor transfected CHO-K1 cells.
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