Date of Award

1999

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Donald A. Primerano

Second Advisor

Elsa Mangiarua

Third Advisor

Vernon E. Reichenbecher

Fourth Advisor

William D. McCumbee

Fifth Advisor

Todd L. Green

Sixth Advisor

Leonard J. Deutsch

Abstract

Laminin β2 is localized to the synaptic basal lamina (BL) in muscle and glomerular BL in kidney. To find interacting proteins, a mouse kidney cDNA library was screened with domain I of rat laminin β2 using the yeast two-hybrid system. Fifteen positive clones were found. The DNA sequence of three each of these clones corresponded to the phosphoprotein Eps8 (epidermal growth factor receptor pathway substrate clone 8, which is tyrosine phosphorylated after EGF stimulation) and the intermediate filament (IF) protein keratin. To determine if this interaction occurred in muscle cells, C2 cells transfected with the rat laminin β2 cDNA (S-C2 cells) were immunoprecipitated with antibodies to either laminin β2, Eps8, or desmin (the IF protein found in skeletal muscle). When the precipitates were immunoblotted with the same antibodies, both Eps8 and desmin were found to be complexed with laminin β2. Based on migration on SDS-PAGE and detection of laminin β2 in an anti-phosphotyrosine precipitate, the hyperphosphorylated form of Eps8 interacts with laminin β2 and desmin. These results suggest that a complex is formed in muscle cells between laminin β2, hyperphosphorylated Eps8, and desmin. Confocal microscopy of Eps8-EGFP (green fluorescen protein ) transfected C2 cells and immunocytochemistry of S-C2 cells demonstrated not only that a portion of laminin β2 and Eps8 co-localized underneath the muscle cell membrane, but also that a part of laminin β2 and Eps8 was translocated into nuclei of the cells. The nuclear translocation of laminin β2 and Eps8 was carefully verified with three different biochemical methods. The nuclear translocation of Eps8 and laminin β2 is related to the differentiation of muscle cells since more of these proteins were found in differentiated nuclei. ErbB2 and erbB3 receptors, which are related to the epidermal growth factor receptor (EGFR), mediate the neuregulin (NRG)/acetylcholine receptor inducing activity (ARIA) signaling-pathway at the neuromuscular junction. Western analyses of laminin β2-transfected C2 cell lysates using antibodies against erbB receptors revealed that overexpression of laminin β2 in C2 muscle cells results in dramatic down-regulation of erbB3 receptors from these cells. When desmin was expressed in RMo rat muscle cells, it resulted in an up-regulation of erbB3 receptors. The erbB3 mRNA levels of transfectd C2 and RMo cells were not affected, suggesting that laminin β2, Eps8, desmin complex may be involved in the regulation of expression of erbB3 receptors on the post-translational level. The data support the hypothesis that laminin β2, Eps8, and desmin play a role in NRG/ARIA-erbB signaling pathway at the neuromuscular junctions and are involved in the gene regulation of muscle cell differentiation.

Subject(s)

Muscle cells – Genetics.

Myoneural junction.

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