Patent Application: US-58961900-A

Abstract:
the present invention features a method for treatment of an organism having a disease or condition characterized by an abnormality in a signal transduction pathway , wherein the signal transduction pathway includes a rdgb protein . the invention also features methods for diagnosing such diseases and for screening for agents that will be useful in treating such diseases . the invention also features purified and / or isolated nucleic acid encoding a rdgb protein .

Description:
the present invention relates to rdgb polypeptides , nucleic acids encoding such polypeptides , cells , tissues and animals containing such nucleic acids , antibodies to such polypeptides , assays utilizing such polypeptides , and methods relating to all of the foregoing . those skilled in the art will recognize that many of the methods described below in relation to rdgb , pyk - 2 , a nbp , or a complex of rdgb with pyk - 2 or a nbp could also be utilized with respect to the other members of this group . we describe the isolation and characterization of a novel non - receptor tyrosine kinase binding protein , termed rdgb . hrdgb1 is expressed in the brain , spleen , and ovary . hrdgb2 is expressed in many human tissues including brain , heart , thymus , and peripheral blood leukocytes . hrdgb3 is highly expressed in the thymus but is also expressed in the brain , heart , ovary , and testis . the examples presented for pyk2 , supra , reveal a novel mechanism for the coupling , between g - protein coupled receptors and the map kinase signaling pathway . these examples also showed that calcium influx induced by membrane depolorization following activation of the nicotinic acetylcholine receptor or other stimuli that cause calcium influx or release from internal stores lead to the activation of pyk2 , tyrosine phosphorylation of shc , recruitment of grb2 / sos and activation of the map kinase signaling pathway . pyk2 can also link extracellular signals with the jnk / sap kinase signaling pathway . rdgb proteins represent a link in the observations discolosed above . rdgb proteins are shown to bind to pyk2 with high affinity both in vitro and in vivo . evidence of this high affinity interaction is visualized in experiments pulling pyk2 out of a cell lysate with glutathione s - transferase fused rdgb proteins . these experiments are described in the examples section below . in addition the drosphila homologs of the rdgb proteins contain a phosphitidylinositol trasferase domain as well as a ca2 + binding domain . although the phosphitidyl inositol transferase domain is missing in an alternatively spliced variant , all forms of rdgb proteins contain a ca2 + binding domain . thus the ca2 + binding domain of rdgb proteins are potentially involved in the ca2 + response observed in pyk2 signaling . the model presented herein may represent the mechanism underlying calcium mediated regulation of gene expression in neuronal cells induced by mmda receptor or voltage sensitive calcium channels . the expression pattern of pyk2 , the external stimuli that activate the kinase together with its role in the control of map kinase and jnk signaling pathways suggests a potential role for pyk2 and rdgb proteins in the control of a broad array of processes in the central nervous system including neuronal plasticity , highly localized control of ion channel function , as well as , localized activation of the map kinase and jnk signaling pathways , cell excitability , and synaptic efficacy . various other features and aspects of the invention are : nucleic acid encoding a rdgb polypeptide ; a nucleic acid probe for the detection of rdgb ; probe based method and kit for detecting rdgb ; dna constructs comprising a rdgb nucleic acid molecule and cells containing these constructs ; purified rdgb polypeptides ; rdgb . antibody and hybridoma ; an antibody based method and kit for detecting rdgb ; isolation of compounds which interact with rdgb ; compositions ; disruption of protein complexes ; antibodies to complexes ; pharmaceutical formulations and modes of administration ; identification of agents ; purification and production of complexes ; derivatives of complexes ; and evaluation of disorders . all of these aspects and features are explained in detail with respect to pyk - 2 in pct publication wo 96 / 18738 , which is incorporated herein by reference in its entirety , including any drawings . those skilled in the art will readily appreciate that such description can be easily adapted to rgdb as well , and is equally applicable to the present invention . the examples below are non - limiting and are merely representative of various aspects and features of the procedures used to identify the full - length nucleic and amino acid sequences of a series of rdgb proteins . experiments demonstrating rdgb expression , interaction and signalling activities are also provided . the yeast strain l40 containing the reporter genes his3 and β - gal under control of upstream lexa - binding site , was used as a host for the two - hybrid screening . pyk2 - n terminal domain ( aa 2 - 245 ), pykn - δi ( aa 2 - 237 ), pyk - nn ( aa2 - 285 ) and fak ( aa 2 - 412 ) n - terminal domain ( aa 2 - 412 ) were fused in frame to lexa dna binding domain . yeast strain that express the lexa - pykn fusion protein was transfected with human brain cdna library ( clontech # hl404ab ) fused to gal4 transcriptional activation domain . transformants were plated on agar selection medium lacking uracil ( ura -), tryptophane ( trp -), leucine ( leu -) and histidine ( his -). resulting colonies were isolated and retested for growth on - ura - trp - leu - his plates and for β - galactosidase activity . plasmid dna was purified from colonies that were his +, β - gal + and used for retransformation of yeast strains expressing heterologous baits to determine the specificity of the interaction . hrdgb1 : human brain , substania nigra cdna library ( λgt10 , clontoch hl1179a .) was screened with 32 - p - labelled probe derived from the yeast prey plasmid encoding gal10 - rdgb1 . four independent clones were isolated , subcloned and analyzed by sequence . sequence analysis indicated that the 5 ′ end of the gene is missing from our clones . therefore human fetal brain cdna library ( λgt11 , clontech hl3003b ) was screened with probe derived from the most 5 ′ region of our new cdna contig . sequence analysis of six independent clones that were isolated indicated that all of them are belong to the same gene , hrdgb1 , but they are missing the 5 ′ end of the gene . a specific - primed cdna library was constructed in λzapii utilizing human fetal brain poly ( a )+ rna as templet for our cdna synthcasis ( stratagene kit ). 15 independent clones were isolated and allowed subsequently isolation of the full length cdna of hrdgb1 . hrdgb2 and hrdgb3 : a dna fragment derived from an est fragment ( t12574 ) was amplified by pcr from human fetal brain cdna . the pcr product was subcloned , sequenced and used as a probe for screening a human fetal brain cdna library ( λgt11 , clontech h15015b ). one positive clone was obtained from this screen . sequence analysis indicated that it is a partial cdna clone of a novel gene belongs to the human rdgb family . the cdna insert of this clone ( 1 . 8 kb ) was used as a probe for rescreening the same cdna library . seven independent clones were obtained , subcloned and sequenced . sequence analysis indicated that all of them belong to the same gene ; hrdgb2 , but they are different from the original clone that was isolated from the same library . the 3 ′ end of our first clone ( 1 . 8 kb ), was used as a probe to screen a human heart cdna library ( clontech 7759 - 1 , 7760 - 1 ) and allowed subsequent isolation two alternative spliced isoforms of hrdgb3 . human multiple tissues northern blots ( clontech hl11296 ) were hybridized under high - stringency conditions using 32p - labelled cdna fragment of hrdgb1 ( ecori - eco47iii nuc # 245 - 511 , hrdgb2 ( saci - eco47iii nuc # 1540 - 2661 ) and hrdgb3 bst - x1 nuc # 912 - 1472 as probe according to the instructions of the manufacture . fusion with lexa dna - binding domain : pcr was used to amplified different regions of pyk2 and fak cdnas as indicated , the amplified dna fragments were subcloned into pbtm116 in frame to generate a fusion protein with lexa dna - binding domain . fusion with gal4 activation domain : pcr was used to amplified different regions of hrdgb1 , hrdgb2 or hrdgb3 cdnas as indicated , the amplified dna fragments were subcloned into pgad10 ( clontech ) in frame to generate a fusion protein with gal4 activation domain . the full length cdnas of hrdgb1 , hrdgb2 and hrdgb3 were subcloned into pcmp1 downstream to cmv promoter . an ha - epitope tag ( ypydvpdyas ) seq id no : 10 was fused in frame to their carboxy terminal ends . the pyk2 binding domain of hrdgb2 ( residues 911 - 1243 ) was subcloned into pcmv - neo which encode an initiator methionine codon followed by a myc epitope tag ( eqkliseedl ) seq id no : 1 immediately upstream to the cloning site . antibodies against rdgb1 were raised in rabbit immunized either with a synthetic peptide corresponding to amino - acids 965 - 974 of hrdgb1 ( c - ter ab ), or with a gst - fusion protein containing residues 231 - 374 ( n - ter ab ) antibodies against hrdgb2 were raised in rabbit immunized with a synthetic peptide corresponding to amino acids 152 - 163 of hrdgb2 . antibodies against hrdgb3 were raised in rabbit against mbp - fusion protein containing residues 7 - 116 of hrdgb3 . the yeast two - hybrid system was used to identify proteins that interact with the amino - terminal domain of pyk2 . the n - terminal domain of pyk2 was fused to the lexa dna binding domain and screened a human brain cdna library . using a his synthetase gene ( his3 ) under the control of lexa operators as a reporter , 124 his + colonies were identified from an initial screen of a million transformants . of these , 24 were also b - galactosidase positives ( gal +). retransformation of these clones into a yeast strain expressing the lexa - pyk2 - n fusion protein indicated that only one interacts with the pyk2 n - terminal domain ( pyk2 - n ). the specificity of the interaction was further determined by transformation of this clone into a yeast strain expressing heterologous baits . an interaction was detected in yeast strain expressing either the pyk - n terminal domain , or a shorter version of pyk - n that was missing 48 amino acids from its c - terminal end . no interaction , however , was detected in strains expressing either the pyk - nn ( amino acids 2 - 285 ), or the n - terminal domain of fak , suggesting that this interaction is very specific . the clone that scored for specific interaction with pyk2 - n contained a partial cdna which allowed subsequent isolation of a 3 . 1 kb cdna with an open reading fram of 975 amino acids . the coding region was flanked by 5 ′ and 3 ′ untranslated regions of 93 and 149 bp respectively . the 5 ′ untranslated region contains triplet repeats ( cgg ), a motif that was identified in many neuropsychiatric disorders . this region showed homology to the untranslated region of the human fragile x mental retardation fmr - 1 gene ( 66 . 3 % match ) using the smith - waterman algorithm . a blast search with the full length cdna sequence revealed that this protein is related to the drosophila retinal degeneration b protein ( rdgb ) and therefore it was named hrdgb1 . the drosophila rdgb protein has an important role in phototransduction pathway . the rdgb mutant was initially identified by defects in the compound eye , in that rdgb mutant flies undergo light - enhanced photoreceptor cell degeneration . the drosophila rdgb protein contains a . phosphatidylinositol transfer domain ( pi - tp ) in its n - terminal portion , and a calcium binding site downstream . the protein contains six hydrophobic regions that were identified as transmembrane domains . the same hydrophobic regions are conserved in the hrdgb1 protein , however , analysis of rdgb1 sequence , as well as the drosophila homolog , using different algorithms ( prosite ) indicated that they are not classical transmembrane domains . an ests data base search with drosophila rdgb sequence allowed the identification of two additional , human genes that belong to the same gene family . a pcr fragment derived from an est fragment ( t12574 ) was used as probe to screen a human brain cdna library and subsequent isolation the hrdgb2 gene . the full length cdna of hrdgb2 ( 4186 bp ) contained an open reading of 1244 amino acids which was flanked by a 5 ′ untranslated region of 174 bp and a 3 ′ untranslated region of 280bp . the 257 amino - acids in the n - terminal end of the hrdgb2 protein have 41 % similarity to the entire human ptdinstp ( m73704 ). the full length cdna of hrdgb3 was obtained by screening human brain and heart cdna libraries . an initial clone of 1 . 8 kb was isolated from a human brain library using the pcr product derived from est fragment ( t12574 ) as a probe . a cdna fragment derived from our 1 . 8 kb clone was used as a probe to screen a human heart cdna library and allowed subsequent isolation of hrdgb3 gene . two isoforms arising from alternative splicing have been identified by cdna cloning , the longest which encodes a protein of 1349 amino - acids with a predicted molecular weight of 150 kda , and a shorter one which lacks amino - acids 50 - 378 , with a predicted molecular weight of 120 kda . the coding sequence is flanked by a 79 bp 5 ′ untranslated region and a 945 bp 3 ′ untranslated region . the n - terminal region of hrdgb3 contains a pi - tp domain that is missing from the alternative spliced isoform . a strecht of glycines and serines was identified within amino acids 612 - 634 ( 78 % glycine , 22 % serine ). multiple alignment analysis of the novel hrdgb1 , hrdgb2 and hrdgb3 revealed high similarity in their primary structure : a pi - tp domain in the amino - terminal region , six conserved hydrophobic regions and very conserved c - terminal region . unlike the other rdgb family members , hrdgb1 does not contain ptdinstp domain , this may suggest that our clone represent an alternative spliced isoform . the levels of hrdgb1 , hrdgb2 and hrdgb3 mrna expression were determined by northern analysis of various human tissues . hrdgb1 has a very restricted expression pattern . it is expressed in the brain , spleen and ovary as a message of approximately 7 . 5 kb . by contrast , hrdgb2 is highly expressed in many human tissues as a message of 4 . 5 kb . highest levels of expression were detected in the brain , heart , thymus and peripheral blood leukocytes . hrdgb3 is very highly expressed in the thymus , but it is also expressed in the heart , brain , ovary and testis . two messages were detected for hrdgb3 : 7 . 5 kb and 9 . 5 kb messages that may represent the two alternative spliced isoforms that were isolated . the results discussed above indicate the rdgbs gene family members have very different expression patterns , whereas hrdgb1 is very rare , hrdgb2 is abundant and hrdgb3 has a unique pattern of expression . to map the pyk2 interaction domain within the hrdgb1 protein , a series of hrdgb1 - deletion mutants were constructed and their ability to interact with pyk2 - n was tested utilizing the two hybrid system . our original two hybrid clone containing amino acids 627 - 975 of hrdgb1 was used as a positive control . deletion mutants were constructed , and among all these mutants , only hrdgb1 - δiv , containing amino acids 627 - 936 , interacts with pyk2 - n terminal domain . the interaction of this domain with pyk2 was further confirmed by an in vitro binding experiment , showing binding of pyk2 to immobilized gst - fusion protein containing the same portion of hrdgb1 . no binding was detected , however , to the gst - protein alone or between hrdgb1 - δiv mutant and the focal adhesion kinase . since hrdgb1 shares high homology with hrdgb2 and hrdgb3 in their c - terminal domains , whether the corresponding regions of these two proteins interact with pyk2 was examined . for this purpose amino acids 911 - 1244 and 996 - 1350 of hrdgb2 and hrdgb3 respectively , were fused in frame to the activator domain of gal - 4 , and their ability to interact with pyk2 - n was tested by the two hybrid system . the results indicate that hrdgb2 can strongly bind to pyk2 n - terminal domain , whereas the interaction of rdgb3 with pyk2 is quite weak . to further confirm this interaction in vivo , hrdgb2 - ha or hrdgb3 - ha were coexpressed either with pyk2 or with fak in cos cells . following cell lysis , hrdgb proteins were immunoprecipitated by anti - ha antibodies and the presence of pyk2 or fak in the immunocomplexes was determined by immunoblotting with antibodies against pyk2 or fak respectively . the results indicate that both hrdgb2 and hrsdgb3 interact with pyk2 in vivo . no interaction , however , was detected with the related kinase fak , suggesting that hrdgbs proteins interact strongly and specifically with pyk2 . to explore whether the ‘ pyk2 binding domain ’ of hrdgbs is sufficient to confer association of those two proteins in vivo , a myc - tagged version of the hrdgb2 ‘ pyk2 - binding domain ’ was coexpressed either with pyk2 or with fak in cos cells , and their interaction was analyzed . the results showed that this domain can interact with pyk2 in vivo and therefore represent a separate domain in this family of proteins . to confirm the interaction of hrdgb1 and pyk2 in vivo an hemagglutinin - tagged rdgb1 and pyk2 were coexpressed in 293 cells . the results indicate that hrdgb1 strongly associates with pyk2 . association of hrdgb1 with the related kinase fak could not be detected under the same experimental conditions , suggesting a strong and specific interaction of hrdgb1 and pyk2 . to further characterize the interaction between hrdgb and pyk2 , an adult rat brain was used as a source of these two proteins . when hrdgb1 was immunoprecipitated from a rat brain homogenate , utilizing specific antibodies against hrdgb1 , pyk2 could be detected in the immunocomplex . however , the stochiometry of pyk2 / rdgb1 interaction was not as high as shown in transfected cells . these results indicate that pyk2 and rdgb1 interact in vivo under physiological condition , and this 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