Protein-Protein Interactions and Muscle cell Signaling Via Syntrophin

<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 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.</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 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.</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>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Year dc:date.available

2002

Author dc:creator

• Oak, Shilpa A.

Contributors dc:contributor

• Harry W. Jarrett, Ph.D.

• muscular dystrophy
• muscle cell signaling
• syntrophin
• Amino Acids, Peptides, and Proteins
• Chemicals and Drugs
• Medicine and Health Sciences

Repository record dc:identifier

https://dc.uthsc.edu/dissertations/193

OAI identifier oai:identifier

oai:dc.uthsc.edu:dissertations-1192