Patent Application: US-72157191-A

Abstract:
novel polypeptides having a β - adrenergic receptor activity containing the sequence of 402 amino acids , or a fragment of this sequence , said fragment being such that , in particular , either it nonetheless includes the sites contained in said sequence and whose presence is necessary so that , when the fragment is exposed to the surface of a cell , it is capable of participating in the activation of the cyclase adenylate in the presence of an agonist , or it is likely to be recognized by antibodies which also recognize the above succession of 402 amino acids , but fail to recognize the β1 adrenergic receptor and the β2 adrenergic receptor . these polypeptides are useful for screening drugs which act on said polypeptides and for treating obesity , fat diabetes and hyperlipidemias .

Description:
other characteristics and advantages of the invention will become apparent in the remainder of the description and the examples , in particular in relation to the drawings and figures in which : fig1 represents the amino acid sequences of the human β1 adrenergic receptor ( second line ), the human β2 adrenergic receptor ( third line ), aligned with those of the human β3 adrenergic receptor ( first line ). in the sequences of the β1 and β2 adrenergic receptors the positions bearing amino acid residues identical with those of the β3 adrenergic receptor are indicated by dashes . in order to demonstrate the homologies , deletions have been made in the three sequences and these deletions are represented by the spaces between square brackets . the seven hydrophobic regions of 21 to 27 residues which are considered to form the δ - helical transmembrane domains are indicated by thick lines and delimit extra - cellular and intra - cellular loops , the extra - cellular loops being represented by 01 - 04 in the figure and the intra - cellular loops by i1 to i4 . in the n - terminal extra - cellular regions of the three receptors , the consensus sequences for the glycosylation sites linked to asparagine ( nxs / t ) are underlined , it being recalled that in the representation of the amino acids according to dayhoff , n represents asparagine , s represents serine , t represents threonine , x represents any amino acid . in the third intra - cellular loop and in the c - terminal region , triangles show the ser and thr residues which are probably implicated in the desensitization of the receptor . fig2 a and 2b represents the accumulation of cyclic amp in cho - β3 cells cells after exposure to ( fig2 a ), ( 1 )- isoproterenol (), ( 1 )- noradrenalin (∘), ( 1 )- adrenalin () or ( d )- isoproterenol (∇) and ( fig2 b ); brl 37344 (), oxprenolol (□), pindolol (∘) or salbutamol (). the k act ( nm ) values are : 3 . 86 ± 0 . 43 for ( 1 )- isoproterenol , 5 . 65 ± 1 . 32 for brl 37344 , 6 . 28 ± 0 . 80 for noradrenalin , 47 . 4 ± 6 . 9 for adrenalin , 76 . 3 ± 1 . 57 for oxprenolol , 108 ± 3 for ( d )- isoproterenol , 167 ± 10 for pindolol and 273 ± 25 for salbutamol . in these experiments , the accumulation of cyclic amp varies from 300 % to 400 % compared with base levels and the results are expressed as % of the maximal response ( mean ± standard deviation / n = 4 to n = 6 /) induced by each of the ligands . in the cho cells , neither noradrenalin , nor adrenalin , nor isoproterenol activates the adenylate cyclase . the agonist procaterol , specific for β2 , stimulates the adnylate cyclase at concentrations higher than 10 - 5 m ( not shown ). fig2 c , 2d , and 2e shown stimulation of cyclase by isoproterenol , brl 37344 , brl 28140 and salbutamol in cho cells expressing β1 , β2 or β3 genes . for each of the panels , the results are the means of two or three experiments done twice . half of the standard deviation is shown for each by vertical bars . three concentrations of ligands were used : 5 × - 9 ( 1 ), 5 × 10 - 7 ( 2 ) and 10 - 4 ( 3 ). the results represent the % mean ± standard deviation ( n = 4 to 6 )/ of the response induced by 10 - 4 m isoproterenol . the method used is the following : the coding region of the gene for the β3 adrenergic receptor was inserted under the control of a sv40 promoter in an expression vector ( 20 ) which also contains the gene for murine dihydrofolate reductase ( dhfr ) used for the selection of the transfected cells . this construction was introduced into cho cells ( 21 ) deficient in dhfr , giving rise to resistant colonies on selective media from which stable sub - clones are obtained . the expression of the β3 adrenergic receptor gene is demonstrated by hybridization of the rna with a specific probe for the β3 adrenergic receptor ( fig4 ). for the cyclic amp assay , cells are cultivated and harvested after treatment with ethylenediamme tetracatic acid ( eurobio , paris ), washed and resuspended in a hank medium containing 20 mm of hepes buffered at ph 7 . 4 , 1 mm ascorbic acid and 0 . 1 mm isobutyl - methyl - xanthine . aliquots of 10 6 cells are incubated for 30 min . in a total volume of 1 ml with the indicated concentrations of agonists and the camp levels are measured according to the specifications of the amersham assay kit . fig3 represents the inhibition by the β - adrenergic antagonists of the accumulation of cyclic amp induced by isoproterenol in β1 - cho , β2 - cho and β3 - cho cells . the results are expressed in %/ mean ± sd ( n = 4 to 6 ) of the accumulation of cyclic amp induced by isoproterenol alone . the compounds ici , 118551 ( manufactured by the imperial chemical industry company ) and cgp 20712a ( manufactured by the ciba - geigy company ) block the accumulation of cyclic amp with calculated ki of 0 . 77 ± 0 . 08 μm and 6 . 70 ± 0 . 87 μm , respectively . metropol has no effect at concentrations lower than 10 - 5 m . the method used is the following : the cells , as in fig2 are incubated for 30 min . with the inhibitors at 10 - 4 m before the addition of 5 . 10 - 9 m of isoproterenol . the incubation is continued for a further 30 min . and the levels of cyclic amp are measured . fig4 represents the northern blot analyses of rna from tissues and line cells . the rna to be analysed is obtained from rat tissues ( 2 μg of rna polya + from : total brain , cortex , hippocampus , hypothalamus , hypophysis , olfactory bulb , striatum , cerebellum , ileum , liver , heart , lung , kidney , skin and muscle ) and human cell lines ( 5 μg of rna polya + of : neuroblastomas , lung and fibroblasts of lung and epidermis , b and t lymphocytes , erythroblasts and myeloblasts ). none of these rnas give any detectable signal ( the results are shown for the ileum ). a specific messenger is detected in the rna ( 15 μg of total rna ) from adipose cell lines 3t3 - f442a or from transfected β3 - cho cells . the s values for the molecular weight markers of rna are shown in the lefthand margin . the method used is the following : the rna is subjected to electrophoresis on a 0 . 7 % agarose gel , transferred to nylon membranes and hybridized with probes specific for the β3 adrenergic receptors radioactively labelled by the random initiation method . this probe is derived from the third intracytoplasmic loop of the β3 adrenergic receptor , the sequence of which is weakly conserved between the three receptors ( positions 1313 to 1513 of fig1 b ). following hybridization , the filters are washed in 0 . 1 × ssc ( i . e . 0 . 015m nacl and 0 . 0015m sodium citrate , ph 7 . 0 )+ 0 . 05 % sds at 55 ° c . under these conditions , no signal is obtained with the rna prepared from cho cells expressing the β1 or β2 adrenergic receptor . determination and expression of the human β3 adrenergic receptor in cho cells a human genomic library was investigated with the entire coding regions of the gene for the β1 adrenergic receptor of the turkey ( 14 ) and of the human gene for the β2 adrenergic receptor ( 8 ). the genes coding respectively for the human β1 and β2 adrenergic receptors were identified among the positive clones . the homology between their coding regions is 48 . 9 %. other clones were obtained containing a gene without an intron , the coding region of which shows 50 . 7 % and 45 . 5 % of homology with the coding regions of β1 and β2 , respectively ( fig1 a ). this gene was designated as the gene for the β3 adrenergic receptor . more precisely , in fig1 a , each group of three lines corresponds to β3 in the case of the first line , to β1 in the case of the second line and to β2 in the case of the third line . the gene for the β3 adrenergic receptor codes for a polypeptide of 402 amino acid residues ( molecular weight 42881d ) which possesses the main characteristics common to the other membrane receptors of the r 7 g family ( fig1 a ). it possesses seven groups of 21 to 27 essentially hydrophobic amino acids , likely to constitute α - helical transmembrane domains . these transmembrane domains play an important role in the formation of the recognition sites of the catecholamines by the β - adrenergic receptors ( 15 - 18 ) and are certainly the most homologous regions between the three proteins β1 , β2 and β3 ( fig1 a ). in particular , the asp residues at positions 79 and 113 of the β2 adrenergic receptor , which may act as counter - ions to the positively charged amine of the adrenergic ligands ( 18 ), are conserved at similar positions of the β3 adrenergic receptor . other functionally important residues such as the cys residues at positions 106 and 184 , asn at position 318 and pro at position 323 of the sequence of the β2 adrenergic receptor are present at the corresponding positions in the β3 adrenergic receptor . like the other r 7 g proteins , the β3 adrenergic receptor contains in its amino terminal region consensus sequences for the glycosylation sites associated with asn . in its third intra - cytoplasmic loop and in its c - terminal region , the β3 adrenergic receptor possesses several ser and thr residues surrounded by basic residues ( arg and lys ) and residues which break up the α - helical structure ( pro and gly ), which may serve as substrates for the kinases possibly involved in the desensitization of the receptor ( 19 ). in order to better characterize the β3 adrenergic receptor , its gene was transfected into cho cells and several clones producing the corresponding rna were stabilized . these cells are designated as &# 34 ; β3 - cho &# 34 ; cells . cho cells expressing either the gene for the β1 adrenergic receptor or the gene for the β2 adrenergic receptor were also prepared in order to compare the pharmacological properties of the three receptors in an identical environment . these cells are designated as β1 - cho and β2 - cho cells . the β3 - cho cells synthesize a protein of an apparent molecular weight of about 65000d , which may be visualized by affinity labelling with 125 i - iodocyanopindololdiazirine . the post - transcriptional addition of sugar residues to one or two glycosylation sites at the amino - terminus of the β3 adrenergic receptor is certainly responsible for this molecular weight being higher than that deduced from the amino acid sequence . the exposure of the β3 - cho cells to β - adrenergic agonists ( fig2 ) such as adrenalin , noradrenalin , isoproterenol , salbutamol , brl 28410 or brl 37344 increases the intracellular concentration of cyclic amp between 300 % and 400 % compared with basal levels , whereas there is no effect on non - transfected cho cells . this affect is stereospecific , in view of the fact that ( 1 )- isoproterenol is almost 30 times more potent than ( d ) isoproterenol in the activation of the cyclase . other β - agonists such as procaterol and cgp 361a lead to a stimulation of the cyclase only when they are used at concentrations higher than 10 - 5 m . the order in which the following compounds : ( 1 )- isoproterenol , brl 37344 , brl 28410 and salbutamol activate the adenylate cyclase in the β3 - cho cells is clearly different from that obtained for the responses mediated by the β1 or β2 adrenergic receptors ( fig2 c ). in the case of the β3 - cho cells , this order is similar to that determined for the stimulation of lipolysis in rat adipose cells . the only difference is the relative order of isoproterenol compared with brl 37344 . however , in the reference cited , it was racemic ( d1 )- isoproterenol which was used whereas in the invention , the levo - rotatory ( 1 )- isoproterenol was used . the analysis of the capacity of several classical β - antagonists to block the activation of the adenylate cylase stimulated by isoproterenol was carried out in β3 - cho cells ( fig3 ). apart from ici 118 , 551 , cgp 20 , 712a and metoprolol , none of these compounds used at 10 - 4 m is capable of inhibiting this effect . at concentrations lower than 10 - 5 m , metoprolol no longer has an effect and ici 118 , 551 and cgp 20 , 712a block the accumulation of cyclic amp with ki of 0 . 77 ± 0 . 08 μm and 6 . 70 ± 0 . 87 μm , respectively . in agreement with these results , neither the intact β3 - cho cells nor their membrane fractions display specific and saturable binding of alprenolol or cgp 12 , 177 labelled with 3 h . furthermore , the k d of 125 i - iodocyanopindolol for the β3 - adrenergic receptor ( 488 ± 90 pm ) is about 10 times higher than that for the β1 or β2 adrenergic receptors . according to in vitro studies , pindolol and oxprenolol are considered to be β - adrenergic antagonists . however , they are also considered to be partial agonists because in vivo they may exhibit a slight sympathomimetic activity ( 22 ). in the cho cells transfected with a single type of receptor , these compounds are complete agonists towards the β3 - adrenergic receptor ( fig2 ), whereas they totally block the accumulation of cyclic amp mediated by the β1 and β2 adrenergic receptors ( fig3 ). this new subtype of β - adrenergic receptor might be able to modulate various functions such as lipolysis , the secretion of insulin or intestinal relaxation . the contractions of the guinea pig ileum induced by the cholinergic pathway are modulated by adrenergic agonists . however , in spite of the total blockage of the α and β - adrenergic receptors with phentolamine and propanolol , isoproterenol can always inhibit the occurrence of contractions . the β3 receptor exhibits a low affinity for propanolol and other classic β blockers , but shows a marked response toward the agonist brl 37344 , which is a powerful stimulant of lipolysis in adipose tissue ( 1 ). the hypothesis , according to which the β3 receptor is present in adipose tissues was confirmed by the analysis of the capacity of the rna derived from tissues of various origins to hybridize with probes specific for the β3 - adrenergic receptor ( fig4 ): a hybridization signal with the probes used was observed only with the rna of the adipocyte line 3t3 - f442a ( 23 ), this β3 - adrenergic receptor might also be implicated in the regulation by the catecholamines of the action of insulin on the metabolism of glucose and fatty acids . 1 . arch , j . r . s ., ainsworth , a . t ., cawthorne , m . a ., piercy , v ., sennitt , m . v ., thody , v . e ., wilson , c . and wilson , s . 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