{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:62336"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:62336","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Funktionelle und strukturelle Analysen von Mutationen im Caveolin-3-Gen","abstract":"The protein caveolin-3 is the most important scaffolding-protein of the caveolar membrane domains in the smooth-, the skeletal- and the heart muscles. Mutations in the caveolin-3 gene are proposed to be responsible for a variety of hereditary myopathies which range from mild forms such as HyperCKemia to severe pheno-types like Limb-girdle muscular dystrophy or the Rippling Muscle Disease. In the context of the present study four different point mutations in the caveolin-3 gene had to be characterized in detail to gain insights into the pathomechanism of the specific myopathies. Therefore, caveolin-3 constructs carrying the mutations R26Q, P28L, A45T and G55S were created. After the verification of protein expression, the localization of the mutated proteins was analyzed. It became apparent that the mutations lead to a mislocalization of the proteins in the Golgi apparatus, but only the mutations P28L and A45T act in a dominant-negative way with respect to the WT-caveolin-3 protein, which was also located in the Golgi apparatus after cotransfection. Since signaling molecules are concentrated in caveolae which therefore are named “signaling platform” (Michel and Bakovic, 2007) it was analyzed if the point mutations R26Q and P28L can influence the signal transduction of three different receptor molecules. With the help of western blot-analysis it became obvious that not only the signaling but also the trafficking of the epidermal growth factor receptor EGFR and the receptor tyrosine kinase TrkA is disturbed by the point mutations. Interestingly, the receptors acted controversially after transfection with the constructs carrying the mutations. The analysis of the phosphorylation of the receptors revealed that TrkA was less phosphorylated after transfection with the construct carrying the mutation P28L whereas EGFR was phosphorylated stronger. The trafficking analysis showed that the mutation R26Q led to increased accumulation of the TrkA receptors on the cell surface. After the transfection of the construct with the P28L mutation a higher amount of EGFR could be detected at the cell surface. The results of the analysis demonstrate that caveolin-3 possibly has direct effects on the receptor signaling pathways. Additionally the results could explain the aetiology of the different phenotypes of the disease caused by two mutations which are located very close to each other. The mutation R26Q leads to Limb-Girdle Muscular Dystrophy or Rippling Muscle Disease whereas the mutation P28L causes HyperCKaemia. The signaling of the IL-6 receptor which was analyzed utilizing a reporter gene assay seems to be caveolin-3 independent. Cotransfection with the mutated caveolin-3 constructs did not cause any changes. In the last part of the study an in vivo model was established to verify the cell culture data. Therefore the tibialis anterior muscle of Lewis rats was electroporated three times either with WT-caveolin-3 expression constructs or with constructs carrying the mutations R26Q. Initial experiments confirmed the in vitro data, showing that WT-caveolin-3 protein is located at the sarcolemma of the muscle fiber whereas the mutated protein is spread over the whole muscle fiber.","abstract_html":"The protein caveolin-3 is the most important scaffolding-protein of the caveolar membrane domains in the smooth-, the skeletal- and the heart muscles. Mutations in the caveolin-3 gene are proposed to be responsible for a variety of hereditary myopathies which range from mild forms such as HyperCKemia to severe pheno-types like Limb-girdle muscular dystrophy or the Rippling Muscle Disease. In the context of the present study four different point mutations in the caveolin-3 gene had to be characterized in detail to gain insights into the pathomechanism of the specific myopathies. Therefore, caveolin-3 constructs carrying the mutations R26Q, P28L, A45T and G55S were created. After the verification of protein expression, the localization of the mutated proteins was analyzed. It became apparent that the mutations lead to a mislocalization of the proteins in the Golgi apparatus, but only the mutations P28L and A45T act in a dominant-negative way with respect to the WT-caveolin-3 protein, which was also located in the Golgi apparatus after cotransfection. Since signaling molecules are concentrated in caveolae which therefore are named “signaling platform” (Michel and Bakovic, 2007) it was analyzed if the point mutations R26Q and P28L can influence the signal transduction of three different receptor molecules. With the help of western blot-analysis it became obvious that not only the signaling but also the trafficking of the epidermal growth factor receptor EGFR and the receptor tyrosine kinase TrkA is disturbed by the point mutations. Interestingly, the receptors acted controversially after transfection with the constructs carrying the mutations. The analysis of the phosphorylation of the receptors revealed that TrkA was less phosphorylated after transfection with the construct carrying the mutation P28L whereas EGFR was phosphorylated stronger. The trafficking analysis showed that the mutation R26Q led to increased accumulation of the TrkA receptors on the cell surface. After the transfection of the construct with the P28L mutation a higher amount of EGFR could be detected at the cell surface. The results of the analysis demonstrate that caveolin-3 possibly has direct effects on the receptor signaling pathways. Additionally the results could explain the aetiology of the different phenotypes of the disease caused by two mutations which are located very close to each other. The mutation R26Q leads to Limb-Girdle Muscular Dystrophy or Rippling Muscle Disease whereas the mutation P28L causes HyperCKaemia. The signaling of the IL-6 receptor which was analyzed utilizing a reporter gene assay seems to be caveolin-3 independent. Cotransfection with the mutated caveolin-3 constructs did not cause any changes. In the last part of the study an in vivo model was established to verify the cell culture data. Therefore the tibialis anterior muscle of Lewis rats was electroporated three times either with WT-caveolin-3 expression constructs or with constructs carrying the mutations R26Q. Initial experiments confirmed the in vitro data, showing that WT-caveolin-3 protein is located at the sarcolemma of the muscle fiber whereas the mutated protein is spread over the whole muscle fiber.","abstract_has_math":false,"creators":["Brauers, Eva"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Weis, Joachim"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2010,"date_issued":"2010","date_published":"2010","updated_at":"2026-07-30T19:43:28Z","subjects":["info:eu-repo/classification/ddc/570","Caveolin-3","Biowissenschaften, Biologie","LGMD1C"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123908%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123908%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123908%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/62336","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Weis, Joachim"]},{"key":"dc:creator","label":"Author","values":["Brauers, Eva"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2010"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-33321"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","Caveolin-3","Biowissenschaften, Biologie","LGMD1C"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/62336","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123908%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The protein caveolin-3 is the most important scaffolding-protein of the caveolar membrane domains in the smooth-, the skeletal- and the heart muscles. Mutations in the caveolin-3 gene are proposed to be responsible for a variety of hereditary myopathies which range from mild forms such as HyperCKemia to severe pheno-types like Limb-girdle muscular dystrophy or the Rippling Muscle Disease. In the context of the present study four different point mutations in the caveolin-3 gene had to be characterized in detail to gain insights into the pathomechanism of the specific myopathies. Therefore, caveolin-3 constructs carrying the mutations R26Q, P28L, A45T and G55S were created. After the verification of protein expression, the localization of the mutated proteins was analyzed. It became apparent that the mutations lead to a mislocalization of the proteins in the Golgi apparatus, but only the mutations P28L and A45T act in a dominant-negative way with respect to the WT-caveolin-3 protein, which was also located in the Golgi apparatus after cotransfection. Since signaling molecules are concentrated in caveolae which therefore are named “signaling platform” (Michel and Bakovic, 2007) it was analyzed if the point mutations R26Q and P28L can influence the signal transduction of three different receptor molecules. With the help of western blot-analysis it became obvious that not only the signaling but also the trafficking of the epidermal growth factor receptor EGFR and the receptor tyrosine kinase TrkA is disturbed by the point mutations. Interestingly, the receptors acted controversially after transfection with the constructs carrying the mutations. The analysis of the phosphorylation of the receptors revealed that TrkA was less phosphorylated after transfection with the construct carrying the mutation P28L whereas EGFR was phosphorylated stronger. The trafficking analysis showed that the mutation R26Q led to increased accumulation of the TrkA receptors on the cell surface. After the transfection of the construct with the P28L mutation a higher amount of EGFR could be detected at the cell surface. The results of the analysis demonstrate that caveolin-3 possibly has direct effects on the receptor signaling pathways. Additionally the results could explain the aetiology of the different phenotypes of the disease caused by two mutations which are located very close to each other. The mutation R26Q leads to Limb-Girdle Muscular Dystrophy or Rippling Muscle Disease whereas the mutation P28L causes HyperCKaemia. The signaling of the IL-6 receptor which was analyzed utilizing a reporter gene assay seems to be caveolin-3 independent. Cotransfection with the mutated caveolin-3 constructs did not cause any changes. In the last part of the study an in vivo model was established to verify the cell culture data. Therefore the tibialis anterior muscle of Lewis rats was electroporated three times either with WT-caveolin-3 expression constructs or with constructs carrying the mutations R26Q. Initial experiments confirmed the in vitro data, showing that WT-caveolin-3 protein is located at the sarcolemma of the muscle fiber whereas the mutated protein is spread over the whole muscle fiber."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University XII, 126 S. : Ill., graph. Darst. (2010). = Aachen, Techn. Hochsch., Diss., 2010"]},{"key":"dc:title","label":"Title","values":["Funktionelle und strukturelle Analysen von Mutationen im Caveolin-3-Gen"]}]}],"canonical_facts":{"dc:contributor":["Weis, Joachim"],"dc:coverage":["DE"],"dc:creator":["Brauers, Eva"],"dc:date":["2010"],"dc:description":["The protein caveolin-3 is the most important scaffolding-protein of the caveolar membrane domains in the smooth-, the skeletal- and the heart muscles. Mutations in the caveolin-3 gene are proposed to be responsible for a variety of hereditary myopathies which range from mild forms such as HyperCKemia to severe pheno-types like Limb-girdle muscular dystrophy or the Rippling Muscle Disease. In the context of the present study four different point mutations in the caveolin-3 gene had to be characterized in detail to gain insights into the pathomechanism of the specific myopathies. Therefore, caveolin-3 constructs carrying the mutations R26Q, P28L, A45T and G55S were created. After the verification of protein expression, the localization of the mutated proteins was analyzed. It became apparent that the mutations lead to a mislocalization of the proteins in the Golgi apparatus, but only the mutations P28L and A45T act in a dominant-negative way with respect to the WT-caveolin-3 protein, which was also located in the Golgi apparatus after cotransfection. Since signaling molecules are concentrated in caveolae which therefore are named “signaling platform” (Michel and Bakovic, 2007) it was analyzed if the point mutations R26Q and P28L can influence the signal transduction of three different receptor molecules. With the help of western blot-analysis it became obvious that not only the signaling but also the trafficking of the epidermal growth factor receptor EGFR and the receptor tyrosine kinase TrkA is disturbed by the point mutations. Interestingly, the receptors acted controversially after transfection with the constructs carrying the mutations. The analysis of the phosphorylation of the receptors revealed that TrkA was less phosphorylated after transfection with the construct carrying the mutation P28L whereas EGFR was phosphorylated stronger. The trafficking analysis showed that the mutation R26Q led to increased accumulation of the TrkA receptors on the cell surface. After the transfection of the construct with the P28L mutation a higher amount of EGFR could be detected at the cell surface. The results of the analysis demonstrate that caveolin-3 possibly has direct effects on the receptor signaling pathways. Additionally the results could explain the aetiology of the different phenotypes of the disease caused by two mutations which are located very close to each other. The mutation R26Q leads to Limb-Girdle Muscular Dystrophy or Rippling Muscle Disease whereas the mutation P28L causes HyperCKaemia. The signaling of the IL-6 receptor which was analyzed utilizing a reporter gene assay seems to be caveolin-3 independent. Cotransfection with the mutated caveolin-3 constructs did not cause any changes. In the last part of the study an in vivo model was established to verify the cell culture data. Therefore the tibialis anterior muscle of Lewis rats was electroporated three times either with WT-caveolin-3 expression constructs or with constructs carrying the mutations R26Q. Initial experiments confirmed the in vitro data, showing that WT-caveolin-3 protein is located at the sarcolemma of the muscle fiber whereas the mutated protein is spread over the whole muscle fiber."],"dc:identifier":["https://publications.rwth-aachen.de/record/62336","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123908%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-33321"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University XII, 126 S. : Ill., graph. Darst. (2010). = Aachen, Techn. 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