Patent Application: US-59554900-A

Abstract:
a new galanin receptor , galr3 , is described . also provided are nucleic acids encoding same and various assays to identify ligands particular to said receptor . ligands so identified are useful for the treatment of obesity , treatment of pain , and treatment of cognitive disorders .

Description:
“ substantially free from associated proteins ” means that the receptor is at least about 90 %, and preferably at least about 95 % free from other cell membrane proteins which are normally found in a living mammalian cell which expresses a galanin receptor . “ substantially free from associated nucleic acids ” means that the nucleic acid is at least about 90 %, and preferably at least about 95 %, free from other nucleic acids which are normally found in a living mammalian cell which naturally expresses a galanin receptor gene . “ substantially the same biological activity ” means that the receptor - galanin binding constant is within 5 - fold of the binding constant of galr3 and galanin , and preferably within 2 - fold of the binding constant of galr3 and galanin . “ stringent post - hybridizational washing conditions ” means 0 . 1 × standard saline citrate ( ssc ) at 65 ° c . “ relaxed post - hybridizational washing conditions ” means 6 × ssc at 30 ° c ., or 1 to 2 × ssc at 55 ° c . “ functional equivalent ” means that a receptor which does not have the exact same amino acid sequence of a naturally occurring galr3 protein due to alternative splicing , deletions , mutations , or additions , but retains at least 1 %, preferably 10 %, and more preferably 25 % of the biological activity of the naturally occurring receptor . such derivatives will have a significant homology with a natural galr3 and can be detected by reduced stringency hybridization with a dna sequence obtained from a galr3 . the nucleic acid encoding a functional equivalent has at least about 60 % homology at the nucleotide level to a naturally occurring receptor nucleic acid . it has been found , in accordance with this invention , that there is a third galanin receptor , which is designated galr3 . the human ( clone 3 — 3 and 3 - 2 ) and rat galr3 sequences are given in fig1 and 8 , respectively , and are referenced in the examples ; however it is to be understood that this invention specifically includes galr3 without regard to the species and , in particular , specifically includes rodent ( including rat and mouse ), rhesus , swine , chicken , cow and human . the galanin 3 receptors are highly conserved throughout species , and one of ordinary skill in the art , given the rat , human and / or mouse sequences presented herein , can easily design probes to obtain the galr3 from other species . galr3 proteins contain various functional domains , including one or more domains which anchor the receptor in the cell membrane , and at least one ligand binding domain . as with many receptor proteins , it is possible to modify many of the amino acids , particularly those which are not found in the ligand binding domain , and still retain at least a percentage of the biological activity of the original receptor . thus this invention specifically includes modified functionally equivalent galr3s which have deleted , truncated , or mutated n - terminal portions . this invention also specifically includes modified functionally equivalent galr3s which contain modifications and / or deletions in other domains , which are not accompanied by a loss of functional activity . additionally , it is possible to modify other functional domains such as those that interact with second messenger effector systems , by altering binding specificity and / or selectivity . such functionally equivalent mutant receptors are also within the scope of this invention . the proteins of this invention were found to have structural features which are typical of the 7 - transmembrane domain ( tm ) containing g - protein linked receptor superfamily ( gpc - r &# 39 ; s or 7 - tm receptors ). thus galr3 proteins make up new members of the gpc - r family of receptors . the intact galr3 of this invention was found to have the general features of gpc - r &# 39 ; s , including seven transmembrane regions , three intra - and extracellular loops , and the gpc - r protein signature sequence . the tm domains and gpc - r protein signature sequence are noted in the protein sequences of the galr3 . not all regions are required for functioning , and therefore this invention also comprises functional receptors which lack one or more non - essential domains . determination of the nucleotide sequence indicated that the galr3 belongs to the intron - containing class of gpc - r &# 39 ; s . the dna sequence encoding the putative galr3 is shown in fig1 and 3 . the human putative galr3 gene is organized similarly to human galr2 with a single intron (˜ 1 kb ) dividing the open reading into two exons with exon 1 consisting of ˜ 350 bp , and exon 2 ˜ 700 bp . based on database searching , the open reading frame protein sequence for this novel gene ( fig2 and 4 ) is most closely related to galr2 and galr1 with 58 , 75 % identity and similarity to human galr2 , and 37 , 61 % identity and similarity to rat galr1 ( fig5 ). differences in open reading frame dna sequence and the resulting deduced amino acid sequence between clone galr3 - 2 and galr3 — 3 may be allelic in nature . phylogenetic analysis of the putative galr3 protein sequence supports the notion that this gene encodes a receptor for galanin ( fig6 ). the human galr3 protein bears strong sequence identity and similarity to the rat galr3 ortholog . this invention also relates to truncated forms of galr3 , particularly those which encompass the extracellular portion of the receptor , but lack the intracellular signaling portion of the receptor , and to nucleic acids encoding these truncated forms . such truncated receptors are useful in various binding assays . thus this invention specifically includes modified functionally equivalent galr3s which have deleted , truncated , or mutated n - terminal portions . this invention also specifically includes modified functionally equivalent galr3s including receptor chimeras which contain modifications and / or deletions in other domains , which are not accompanied by a loss of functional activity . additionally , it is possible to modify other functional domains such as those that interact with second messenger effector systems , by altering binding specificity and / or selectivity . such functionally equivalent mutant receptors are also within the scope of this invention . assays which make up further aspects of this invention include binding assays ( competition for 125 i - galanin binding ), coupling assays ( including galanin - mediated inhibition of forskolin - stimulated adenylate cyclase in cells expressing galanin receptors ), measurement of galanin - stimulated calcium release in cells expressing galanin receptors ( such as aequorin assays ), stimulation of inward rectifying potassium channels ( girk channels , measured by voltage changes ) in cells expressing galanin receptors , and measurement of ph changes upon galanin stimulation of cells expressing galanin receptors as measured with a microphysiometer . host cells may be cultured under suitable conditions to produce galr3 . an expression vector containing dna encoding the receptor may be used for expression of receptor in a recombinant host cell . recombinant host cells may be prokaryotic or eukaryotic , including but not limited to bacteria such as e . coli , fungal cells such as yeast , mammalian cells including but not limited to cell lines of human , bovine , porcine , monkey and rodent origin , and insect cells including but not limited to drosophila , spodoptera , and silkworm derived cell lines . cell lines derived from mammalian species which are suitable and which are commercially available include , but are not limited to , l cells l - m ( tk − ) ( atcc ccl 1 . 3 ), l cells l - m ( atcc ccl 1 . 2 ), 293 ( atcc crl 1573 ), raji ( atcc ccl 86 ), cv - 1 ( atcc ccl 70 ), cos - 1 ( atcc crl 1650 ), cos - 7 ( atcc crl 1651 ), cho - k1 ( atcc ccl 61 ), 3t3 ( atcc ccl 92 ), nih / 3t3 ( atcc crl 1658 ), hela ( atcc ccl 2 ), c127i ( atcc crl 1616 ), bs - c - 1 ( atcc ccl 26 ) and mrc - 5 ( atcc ccl 171 ). the specificity of binding of compounds showing affinity for the receptor is shown by measuring the affinity of the compounds for cells transfected with the cloned receptor or for membranes from these cells . expression of the cloned receptor and screening for compounds that inhibit the binding of radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor . these compounds identified by the above assays may be agonists or antagonists of the receptor and may be peptides , proteins , or non - proteinaceous organic molecules . alternatively , functional assays of the receptor may be used to screen for compounds which affect the activity of the receptor . such functional assays range from ex vivo muscle contraction assays to assays which determine second messenger levels in cells expressing the receptor . the second messenger assays include , but are not limited to , assays to measure cyclic amp or calcium levels or assays to measure adenyl cyclase activity . these compounds identified by the above assays may be agonists , antagonists , suppressors , or inducers of the receptor . the functional activity of these compounds is best assessed by using the receptor either natively expressed in tissues or cloned and exogenously expressed . using the assays of this invention , galanin agonists and antagonists may be identified . a galanin agonist is a compound which binds to the galr3 , such as a galanin mimetic , and produces a cellular response which is at least about equivalent to that of galanin , and which may be greater than that of galanin . such compounds would be useful in situations where galanin insufficiency causes anorexia , or for treatment of pain . also using this embodiment of the assay , galanin antagonists may be identified . a galanin antagonist is a compound which can bind to the galr3 , but produces a lesser response than that of native galanin . such compounds would be useful in the treatment of obesity . one assay of this invention is a method of identifying a compound which modulates galr3 receptor comprising : a ) culturing cells expressing the galr3 receptor in the presence of the compound and b ) measuring galr3 receptor activity or second messenger activity . if desired , the determined activity can be compared to a standard , such as that measured using galanin as the compound . in preferred embodiments , the cells are transformed and express the galr3 receptor . the consultant cdna clone ( or shorter portions of , for instance , only 15 nucleotides long ) may be used to probe libraries under hybridization conditions to find other receptors which are similar enough so that the nucleic acids can hybridize , and is particularly useful for screening libraries from other species . in this step , one of ordinary skill in the art will appreciate that the hybridization conditions can vary from very stringent to relaxed . proper temperature , salt concentrations , and buffers are well known . the following non - limiting examples are presented to better illustrate the invention . automated searching of sequence data from genbank ( national center for biotechnology information , bethesda , md .) were queried using sequences from known receptor clones . using a list of 50 - 60 rhodopsin family amino acid sequences , the new division of genbank was searched . the query algorithm is tfastx and the output is placed in a file where alignments are sorted by query sequence and scored ( cut - off based on the expectation value , set for example , at 0 . 01 ). a dna sequence alignment of 300 bp to a portion of a large bac clone (˜ 100 , 000 bp ) with accession number z97630 was identified from the high through - put genomic sequence ( htgs database , genbank ). the complete open reading frame ( orf ) for the putative gene encoding galr3 was then identified using sequence from bac z97630 and an additional bac clone , with assession number z82241 , from the htgs database . the genbank assession numbers corresponded to the following htgs bac clones ( hs entries ): z97630 , hs466n1 ; z82241 , hs8112 . dna sequences derived from these bacs were used to choose pcr primers . pcr primers begining at the predicted initiating met and ending more 3 ′ than the predicted stop codon were utilized to pcr from human genomic dna a fragment containing the predicted exon i , the intervening intron , and predicted exon ii . this pcr product was subcloned and sequenced , resulting in expression plasmid galr3 — 3 . in a parallel approach , a human genomic dna library ( stratagene , la jolla , calif .) was screened to isolate the putative galr3 gene . primary screening under medium stringency resulted in 6 positive plaques using an exon 2 probe . one hybridizing phage plaque was obtained upon secondary screening . a 13 kb ecor1 / ecorv fragment was identified from the genomic clone by southern blotting , transferred into pbluescript vector ( stratagene , la jolla , calif . ), and confirmed to be galr3 by sequencing . a intronless galr3 expression construct was assembled in a similar manner to that described above using pfu dna polymerase ( stratagene , la jolla , calif .) resulting in expression plasmid galr3 - 2 . the bac clones which were identified by the searches of the htgs dataset have been mapped by the the sanger centre ( cambridge , uk ) genome research laboratory to human chromosome 22 . fish analysis conducted herein has confirmed this assignment and refined it to 22q12 . 2 - 13 . 1 . the human galr3 cdna expression construct was assembled stepwise from pcr products amplified from human genomic dna . each exon was pcr amplified using standard conditions . the primers in for exon i were : forward exon i ( 5 ′- gcg aat tcg gta cca tgg ctg atg ccc aga aca t - 3 ′; seq id no : 13 ) and reverse exon i ( 5 ′- cgc ctg tcg aca gat aca gca gc - 3 ′; seq id no : 14 ). the primers for exon ii were : forward exon ii ( 5 ′- tgt atc tgt cga cag gta acc tgg ccg tgc ggc acc c - 3 ′; seq id no : 15 ) and reverse exon ii ( 5 ′- gcg cgg ccg ctt att ccg gtc ctc ggg c - 3 ′; seq id no : 16 ). pcr products were subcloned into pcrii and sequenced . for expression in mammalian cells , the putative galr3 orf was subcloned into pcdna - 1 / amp ( invitrogen ) resulting in plasmid galr3 — 3 ; and pcdna - 3 ( invitrogen ), resulting in plasmid galr3 - 2 . using rnase protection analysis , the relative levels of human galr3 mrna was assessed . as shown below galr3 is expressed in numerous brain regions and peripheral tissues , as observed for galr1 and galr2 . mammalian cos - 7 cells were transfected by electroporation . cos - 7 cells ( 1 × 10 7 ) were suspended in 0 . 85 ml of ringers &# 39 ; buffer and 15 mg of the galr3 - 2 or galr3 — 3 expression plasmid was added to a 0 . 4 mm electroporation cuvette ( bio - rad , hercules , calif .). current was applied ( 960 mf , 260 v ) using a bio - rad electroporator device and the cells were transferred to a t - 180 flask ( corning ). expression was allowed to proceed for 72 hrs . membranes were prepared from transfected cells following dissociation in enzyme - free dissociation solution ( specialty media , lavallette , n . j .) by disruption in a dounce homogenizer in ice - cold membrane buffer ( 10 mm tris , ph 7 . 4 , 10 mm pmsf , 10 mm phosphoramidon , and 40 mg / ml bacitracin ). after a low speed ( 1100 × g for 10 min . at 4 ° c .) and a high speed centrifugation ( 38 , 700 × g for 15 min . at 4 ° c . ), membranes were resuspended in buffer and protein concentration determined ( bio - rad assay kit ). binding of 125 i - human or porcine galanin ( specific activity of 2200 ci / mmol , dupont nen ) was measured in membranes using a buffer of 25 mm tris ph 7 . 4 , 0 . 5 % bsa , 2 mm mgcl 2 , 40 μg / ml bacitracin , 4 mg / ml phosphoramidon , and 10 μm leupeptin in a total volume of 250 ml . 70 μm 125 i - human or porcine galanin was used . transfected cells expressing plasmid galr3 — 3 were bound with 125 i - human galanin whereas cells expressing plasmid galr3 - 2 were bound with 125 i - porcine galanin . reactions were initiated by the addition of membranes and the incubation was allowed to proceed at room temperature for 1 hour . non - specific binding was defined as the amount of radioactivity remaining bound in the presence of 1 mm respectively unlabeled galanin and was generally not above 200 cpm (& lt ; 10 % of total radioactivity bound ). titration of membrane protein from 1 to 50 μg was conducted . in competition studies various concentrations of unlabeled human or porcine galanin were included along with 125 i - porcine galanin ( 70 pm ) in cells expressing the galr3 - 2 plasmid . incubations were terminated by rapid filtration through gf / c filters which had been presoaked with 0 . 1 % polyethylamine using a tomtec ( orange , conn .) cell harvester . the results were analyzed using the prism software package ( graphpad , san diego , calif .). the table below illustrates that both clones confer specific binding to cos - 7 cells for both human and porcine galanin radioligands as a function of protein concentration . in cos - 7 cells mock transfected with expression vector only ( no galr3 gene ), no specific binding of either radioligand was observed . competition curves for 125 i - porcine galanin against human and porcine galanin were generated to to determine the ic50 for both unlabeled peptides ( clone galr3 - 2 ), as shown in fig7 . the ic50 values for porcine and human galanin were 16 nm and 93 nm , respectively . primers based on the intronless human galr3 sequence from tm4 and tm7 were designed and used to pcr amplify with pfu dna polymerase the rat galr3 ortholog from rat genomic dna . a pcr product of the appropriate size ( approximately 400 bp ) that hybridized with an exon 2 probe from the human galr3 gene was subcloned into pbluescript vector ( stratagene , la jolla , calif .). the dna sequence is shown is fig8 and the deduced amino acid sequence is shown in fig9 . dna sequence analysis revealed significant homology with human galr3 : approximately 95 % protein sequence identity for 129 amino acids spanning tm4 through tm - 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