{"created":"2023-06-20T13:21:12.316512+00:00","id":1322,"links":{},"metadata":{"_buckets":{"deposit":"85634de2-7311-4a30-a7d8-450695afebc2"},"_deposit":{"created_by":1,"id":"1322","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1322"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001322","sets":["2:430:27"]},"author_link":["9600","9601","9602"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"新谷, 隆史"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"シンタニ, タカフミ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"1997-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"Protein tyrosine phosphorylation plays a crucial role in regulation of cell proliferation, differentiation and transformation.  The level of tyrosine phosphorylation is determined by the balance between the activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).  Molecular cloning of PTPs have revealed that PTPs comprise a diverse family of cytoplasmic and transmembrane enzymes.  The receptor-like PTPs (RPTPs) are structurally composed of intracellular one or two phosphatase domains, a single transmembrane domain and extracellular segments.  Extracellular segments of several RPTPs display structural motifs that are suggestive of a role in cell-cell or cell-extracellular matrix interactions, such as immunoglobulin (Ig)-like domains, fibronectin type III (FN-III)-like domains and carbonic anhydrase (CAH)-like domains.<br />  RPTPy belongs to RPTPs and shows a high degree of structural similarity to PTPζ/RPTPβ, a nervous system-specific RPTP.  The extracellular regions of these molecules contain a CAH-like domain at the N-terminal, followed by a FN-III-like domain.  In the central nervous system, RPTPγ is up-regulated during cortical development and maintained in the regions containing postmitotic neurons, suggesting that it plays a role in neuronal differentiation.  Despite these observations, RPTPγ gene products have not been fully characterized.<br />  In this study, I identified four isoforms of RPTPγ from rat brain by cDNA cloning.  We designated these molecules as RPTPγ-A, -B, -C and -S.  RPTPγ-A was the longest form, contained an open reading frame of 4,326 bp encoding a protein of 1,442 amino acids, and had the same structure as human and mouse RPTPγ reported.  RPTPγ-B lacked intracellular juxtamembrane 29 amino acids of RPTPγ-A.  RPTPγ-C is a protein of 1,176 amino acids.  The sequence of this isoform differed from that of RPTPy-A from nucleotide residue 3,503, and terminated translation after adding 9 novel amino acids resulting in loss of the carboxy-terminal phosphatase domain.  RPTPγ-S was a protein of 717 amino acids.  The sequence of this isoform from nucleotide residue 2,147 was distinct from that of RPTPY-A, where the translation terminated after adding 2 amino acids causing loss of the transmembrane segment and the intracellular region.  Thus, RPTPγ-S is an extracellular variant of RPTPγ.  mRNAs of the four isoforms were expressed in the brain, kidney, lung and heart.  By Northern blot analysis, four RPTPγ transcripts of about 9.0, 7.0, 4.5 and 3.5 kb were detected in the rat brain.  The former two transcripts were expressed at high levels and the latter two transcripts were expressed at low levels.  The 9.0- and 7.0-kb transcripts likely encode RPTPγ-A and -B, and the 4.5-kb transcript likely encodes RPTPγ-S.<br />  Transfection of RPTPγ-A and -S expression plasmids into COS7 cells resulted in the expression of membrane-bound 190-kDa and secreted 120-kDa proteins, respectively.  These proteins were highly glycosylated by sialic acid.  However, RPTPγ isoforms were not expressed as proteoglycans like PTPζ/RPTPβ.  In RPTPγ-A-expressing PC12D cell lines, a doublet RPTPγ-A protein bands with apparent molecular mass of 184 and 187 kDa were detected, suggesting that RPTPγ-A is proteolytically cleaved in PC12D cells.<br />  To elucidate the functions of RPTPγ in neuronal cells, I first established PC12D cell clones expressing rat RPTPγ-A.  PC12D cells, a subline of PC12 cells, extends neurites immediately in response to NGF, FGF or cAMP.  In PC12 and PC12D cells, the 3.5-kb transcript was the sole transcript of RPTPγ, and enzymatically active RPTPγ isoforms were not expressed.  RPTPγ-A-expressing PC12D cells showed remarkably reduced neurite outgrowth in response to NGF.  Surprisingly, there were no obvious differences in tyrosine-phosphorylation of major cellular proteins between control and RPTPγ-A-expressing transfectants.  Activation of MAP kinases was equally detected in the RPTPγ-A-expressing cells as well as in control cells.  These results suggest that RPTPγ functions at the downstream of MAP kinases or through independent signal transduction pathways.<br />  Rabin et al. (1993) reported that tyrosine phosphorylation of some proteins purified with p13suc1 beads was augmented by NGF in PC12 cells.  p13suc1 was initially identified as a gene product which rescued p34cdc2 mutations.  Recently, it was also reported that complexes purified from squid axoplasm with p13suc1 beads contained cdc2-like kinases, some other kinases and cytoskeletal proteins (Takahashi et al., 1995).  These results suggested that p13suc1 complexes are involved in phosphorylation/dephosphorylation processes underlying axonal cytoskeletal dynamics.  Therefore, I next examined phosphorylation of the complexes purified with p13suc1-agarose, and found that the pattern of protein phosphorylation was different between control and RPTPγ-A-expressing transfectants, which was not detected in the total cell lysates;  tyrosine-phosphorylation of 140- and 117-kDa proteins were specifically reduced in the p13suc1-complexes from RPTPγ-expressing cells.  These 140- and 117-kDa proteins may be directly dephosphorylated by RPTPγ or indirectly through activation of other protein tyrosine phosphatases.  p13suc1-complexes from PC12D cells also contained cytoskeletal proteins such as actin, tubulin and neurofilaments, and kinases including cdk5 and MAP kinases.  While the association of tubulin and actin with p13suc1 beads-complexes did not change after NGF-treatment, the binding of MAP2 to the complexes and phosphorylation of neurofilaments in the complexes increased rapidly.  MAP2 is a high molecular weight, neuron-specific phosphoprotein copurified with microtubules, and neurofilaments are neuron-specific proteins that constitute the major cytoskeleton in axons.  Since neurite formation and extension are regulated by cytoskeletal proteins and their associated proteins, abnormality of tyrosine phosphorylation in this complex caused by RPTPγ expression may explain the inhibition of neurite extension in RPTPγ-A-expressing cells.<br />  Neurite extension and axon guidance are regulated by neurite outgrowth-promoting and -inhibiting molecules distributed in the local environment.  Recently, it was suggested that activities of RPTPs might  be down-regulated by the ligand-mediated dimerization (Bilwes et al., 1996).  Ligand molecules of RPTPγ may control neurite extension or axon guidance by regulating RPTPγ activity:  in the absence of ligands, active RPTPy on neuron may dephosphorylate the proteins such as 140- and 117-kDa proteins and inhibit the neurite outgrowth, while ligand binding to RPTPγ may lead to dimerization and inactivation of this receptor resulting in promotion of neurite outgrowth.<br />  The RPTPγ-expressing PC12D transfectants suggested that RPTPγ plays an important role in neuronal differentiation.  The findings obtained in this study will contribute to elucidating the regulation mechanism of neurite extension in neuronal cells.  From this standpoint of views, it is important to characterize the tyrosine phosphorylated 140- and 117-kDa proteins.  Moreover identification of ligand molecules which regulate RPTPγ activity is a prerequisite for revealing the functional roles of RPTPγ in the brain development.  The RPTPγ-expressing PC12D transfectants which I prepared here will be usefulfor these investigations.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第277号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"X2 分子生物機構論専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"1996"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"SHINTANI, Takafumi","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲277_要旨.pdf","filesize":[{"value":"313.8 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/1322/files/甲277_要旨.pdf"},"version_id":"b9d2927b-1557-4435-92b7-9cf2fa5eb27b"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Molecular characterization and functionalanalysis of rat receptor-like protein tyrosinephosphatase γ","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Molecular characterization and functionalanalysis of rat receptor-like protein tyrosinephosphatase γ"},{"subitem_title":"Molecular characterization and functionalanalysis of rat receptor-like protein tyrosinephosphatase γ","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["27"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1322","relation_version_is_last":true,"title":["Molecular characterization and functionalanalysis of rat receptor-like protein tyrosinephosphatase γ"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:45:43.073439+00:00"}