Absence of dystrophin results in Duchenne muscular dystrophy (DMD), a lethal neuromuscular d isorder that afflicts 1 in 3500 live male births. In the sarcolemma, dystrophin is associated with a complex of proteins and glycoproteins, known as the dystrophin glycoprotein complex. The DGC constituents are dystrophin, a-dys troglycan, b-dystroglycan, syntrophin, a-sarcoglycan, b-sarcoglycan, g-sarcoglycan, d-sarcoglycan, and sarcospan. Not al l of these are single protein species. The syntrophins consists of a group of three homologous proteins composed of acidic (a) and basic (b) components. Syntrophins are known to self-associate to form oligomers.
In this dissertation, syntrophin's oligomerization and its interactions with the cell signaling components in vitro in skeletal muscle were investigated.
Mouse a1-syntrophin sequences, produced as chimeric fusion proteins in bacteria, als o oligomerize and in a micromolar Ca2+-dependent manner. Oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to se lf-associate and calmodulin or Ca2+ chelating agents such as EGTA could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca2+. Ca2+-binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.
Syntrophins have been proposed to serve as adapter proteins. Blot overlay experiment s demonstrate that a-, b-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse a1-syntrophin chimeric fusion proteins bind Grb2 in a Ca2+-independent manner. This binding was localized to two proline rich sequences near the N terminal PH1 domain. This domain is interrupted by a PDZ domain inserted nearly in middle of the PH1 domain dividing it into two parts: the N ter minal PH1 and C terminal PH1b subdomains. One proline rich sequence is Cterminal of PH1b while the other is adjacent to and overlapping with the N terminal of PH1b. Grb2 contains two SH3 domains and both contribute to binding. Intact, bacterial expressed Grb2 bound syntrophin with an apparent KD of 563 ± 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity does not bind these s yntrophin sequences.
Dystrophin glycoprotein complex has been proposed to be involved in signal transduction. We have shown that laminin binding to a–dystroglycan causes syntrophin to recruit Rac1. Laminin-Sepha rose precipitates Rac1, and to a lesser extent Rho-A and Ras, from the rabbit skeletal muscle membranes in a pull-down assay. The presence of heparin, which inhibits the interaction between laminin and a–dystroglycan preven ts recruitment of Rac1. A syntrophin antibody blocks recruitment of Rac1 suggesting that the signaling pathway requires syntrophin. Sos1 is also present in the recruited complex. Jun N terminal kinase 2 is phosphorylated and activated only when laminin is attached to the dystrophin glycoprotein complex. Thus, dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2-Sos complex leading to activation of Jun N terminal kinase 2 only when it is attached to laminin. We postulate this signals the muscle cell to grow.
","abstract_html":"<p>Absence of dystrophin results in Duchenne muscular dystrophy (DMD), a lethal neuromuscular d isorder that afflicts 1 in 3500 live male births. In the sarcolemma, dystrophin is associated with a complex of proteins and glycoproteins, known as the dystrophin glycoprotein complex. The DGC constituents are dystrophin, a-dys troglycan, b-dystroglycan, syntrophin, a-sarcoglycan, b-sarcoglycan, g-sarcoglycan, d-sarcoglycan, and sarcospan. Not al l of these are single protein species. The syntrophins consists of a group of three homologous proteins composed of acidic (a) and basic (b) components. Syntrophins are known to self-associate to form oligomers.</p> <p>In this dissertation, syntrophin's oligomerization and its interactions with the cell signaling components <em>in vitro</em> in skeletal muscle were investigated.</p> <p>Mouse a1-syntrophin sequences, produced as chimeric fusion proteins in bacteria, als o oligomerize and in a micromolar Ca<sup>2+</sup>-dependent manner. Oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to se lf-associate and calmodulin or Ca<sup>2+</sup> chelating agents such as EGTA could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca<sup>2+</sup>. Ca<sup>2+</sup>-binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.</p> <p>Syntrophins have been proposed to serve as adapter proteins. Blot overlay experiment s demonstrate that a-, b-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse a1-syntrophin chimeric fusion proteins bind Grb2 in a Ca<sup>2+</sup>-independent manner. This binding was localized to two proline rich sequences near the N terminal PH1 domain. This domain is interrupted by a PDZ domain inserted nearly in middle of the PH1 domain dividing it into two parts: the N ter minal PH1 and C terminal PH1b subdomains. One proline rich sequence is Cterminal of PH1b while the other is adjacent to and overlapping with the N terminal of PH1b. Grb2 contains two SH3 domains and both contribute to binding. Intact, bacterial expressed Grb2 bound syntrophin with an apparent KD of 563 ± 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity does not bind these s yntrophin sequences.</p> <p>Dystrophin glycoprotein complex has been proposed to be involved in signal transduction. We have shown that laminin binding to a–dystroglycan causes syntrophin to recruit Rac1. Laminin-Sepha rose precipitates Rac1, and to a lesser extent Rho-A and Ras, from the rabbit skeletal muscle membranes in a pull-down assay. The presence of heparin, which inhibits the interaction between laminin and a–dystroglycan preven ts recruitment of Rac1. A syntrophin antibody blocks recruitment of Rac1 suggesting that the signaling pathway requires syntrophin. Sos1 is also present in the recruited complex. Jun N terminal kinase 2 is phosphorylated and activated only when laminin is attached to the dystrophin glycoprotein complex. Thus, dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2-Sos complex leading to activation of Jun N terminal kinase 2 only when it is attached to laminin. We postulate this signals the muscle cell to grow.</p>","abstract_has_math":false,"creators":["Oak, Shilpa A."],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Harry W. Jarrett, Ph.D."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2002,"date_issued":"2002-06-01T07:00:00Z","date_published":"2002-06-01T07:00:00Z","updated_at":"2026-07-24T05:00:11Z","subjects":["muscular dystrophy","muscle cell signaling","syntrophin","Amino Acids, Peptides, and Proteins","Chemicals and Drugs","Medicine and Health Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://dc.uthsc.edu/dissertations/193","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Harry W. Jarrett, Ph.D."]},{"key":"dc:creator","label":"Author","values":["Oak, Shilpa A."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2016-06-09T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["muscular dystrophy","muscle cell signaling","syntrophin","Amino Acids, Peptides, and Proteins","Chemicals and Drugs","Medicine and Health Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://dc.uthsc.edu/dissertations/193"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Absence of dystrophin results in Duchenne muscular dystrophy (DMD), a lethal neuromuscular d isorder that afflicts 1 in 3500 live male births. In the sarcolemma, dystrophin is associated with a complex of proteins and glycoproteins, known as the dystrophin glycoprotein complex. The DGC constituents are dystrophin, a-dys troglycan, b-dystroglycan, syntrophin, a-sarcoglycan, b-sarcoglycan, g-sarcoglycan, d-sarcoglycan, and sarcospan. Not al l of these are single protein species. The syntrophins consists of a group of three homologous proteins composed of acidic (a) and basic (b) components. Syntrophins are known to self-associate to form oligomers.
In this dissertation, syntrophin's oligomerization and its interactions with the cell signaling components in vitro in skeletal muscle were investigated.
Mouse a1-syntrophin sequences, produced as chimeric fusion proteins in bacteria, als o oligomerize and in a micromolar Ca2+-dependent manner. Oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to se lf-associate and calmodulin or Ca2+ chelating agents such as EGTA could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca2+. Ca2+-binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.
Syntrophins have been proposed to serve as adapter proteins. Blot overlay experiment s demonstrate that a-, b-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse a1-syntrophin chimeric fusion proteins bind Grb2 in a Ca2+-independent manner. This binding was localized to two proline rich sequences near the N terminal PH1 domain. This domain is interrupted by a PDZ domain inserted nearly in middle of the PH1 domain dividing it into two parts: the N ter minal PH1 and C terminal PH1b subdomains. One proline rich sequence is Cterminal of PH1b while the other is adjacent to and overlapping with the N terminal of PH1b. Grb2 contains two SH3 domains and both contribute to binding. Intact, bacterial expressed Grb2 bound syntrophin with an apparent KD of 563 ± 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity does not bind these s yntrophin sequences.
Dystrophin glycoprotein complex has been proposed to be involved in signal transduction. We have shown that laminin binding to a–dystroglycan causes syntrophin to recruit Rac1. Laminin-Sepha rose precipitates Rac1, and to a lesser extent Rho-A and Ras, from the rabbit skeletal muscle membranes in a pull-down assay. The presence of heparin, which inhibits the interaction between laminin and a–dystroglycan preven ts recruitment of Rac1. A syntrophin antibody blocks recruitment of Rac1 suggesting that the signaling pathway requires syntrophin. Sos1 is also present in the recruited complex. Jun N terminal kinase 2 is phosphorylated and activated only when laminin is attached to the dystrophin glycoprotein complex. Thus, dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2-Sos complex leading to activation of Jun N terminal kinase 2 only when it is attached to laminin. We postulate this signals the muscle cell to grow.
"]},{"key":"dc:title","label":"Title","values":["Protein-Protein Interactions and Muscle cell Signaling Via Syntrophin"]}]}],"canonical_facts":{"dc:contributor":["Harry W. Jarrett, Ph.D."],"dc:creator":["Oak, Shilpa A."],"dc:date.available":["2016-06-09T07:00:00Z"],"dc:description.abstract":["Absence of dystrophin results in Duchenne muscular dystrophy (DMD), a lethal neuromuscular d isorder that afflicts 1 in 3500 live male births. In the sarcolemma, dystrophin is associated with a complex of proteins and glycoproteins, known as the dystrophin glycoprotein complex. The DGC constituents are dystrophin, a-dys troglycan, b-dystroglycan, syntrophin, a-sarcoglycan, b-sarcoglycan, g-sarcoglycan, d-sarcoglycan, and sarcospan. Not al l of these are single protein species. The syntrophins consists of a group of three homologous proteins composed of acidic (a) and basic (b) components. Syntrophins are known to self-associate to form oligomers.
In this dissertation, syntrophin's oligomerization and its interactions with the cell signaling components in vitro in skeletal muscle were investigated.
Mouse a1-syntrophin sequences, produced as chimeric fusion proteins in bacteria, als o oligomerize and in a micromolar Ca2+-dependent manner. Oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to se lf-associate and calmodulin or Ca2+ chelating agents such as EGTA could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca2+. Ca2+-binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.
Syntrophins have been proposed to serve as adapter proteins. Blot overlay experiment s demonstrate that a-, b-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse a1-syntrophin chimeric fusion proteins bind Grb2 in a Ca2+-independent manner. This binding was localized to two proline rich sequences near the N terminal PH1 domain. This domain is interrupted by a PDZ domain inserted nearly in middle of the PH1 domain dividing it into two parts: the N ter minal PH1 and C terminal PH1b subdomains. One proline rich sequence is Cterminal of PH1b while the other is adjacent to and overlapping with the N terminal of PH1b. Grb2 contains two SH3 domains and both contribute to binding. Intact, bacterial expressed Grb2 bound syntrophin with an apparent KD of 563 ± 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity does not bind these s yntrophin sequences.
Dystrophin glycoprotein complex has been proposed to be involved in signal transduction. We have shown that laminin binding to a–dystroglycan causes syntrophin to recruit Rac1. Laminin-Sepha rose precipitates Rac1, and to a lesser extent Rho-A and Ras, from the rabbit skeletal muscle membranes in a pull-down assay. The presence of heparin, which inhibits the interaction between laminin and a–dystroglycan preven ts recruitment of Rac1. A syntrophin antibody blocks recruitment of Rac1 suggesting that the signaling pathway requires syntrophin. Sos1 is also present in the recruited complex. Jun N terminal kinase 2 is phosphorylated and activated only when laminin is attached to the dystrophin glycoprotein complex. Thus, dystrophin glycoprotein complex recruits Rac1 via syntrophin through a Grb2-Sos complex leading to activation of Jun N terminal kinase 2 only when it is attached to laminin. We postulate this signals the muscle cell to grow.
"],"dc:identifier":["https://dc.uthsc.edu/dissertations/193"],"dc:subject":["muscular dystrophy","muscle cell signaling","syntrophin","Amino Acids, Peptides, and Proteins","Chemicals and Drugs","Medicine and Health Sciences"],"dc:title":["Protein-Protein Interactions and Muscle cell Signaling Via Syntrophin"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T05:00:11Z"}