PC12 cells express different Dp71 isoforms originated from alternative splicing; one of them, Dp71ab lacks exons 71 and 78. To gain insight into the function of Dp71 isoforms we identified dystrophin associated proteins (DAPs) that associate in vivo with Dp71ab during nerve growth factor (NGF) induced differentiation of PC12 cells. DAPs expression was analyzed by RT-PCR, Western blot and indirect immunofluorescence, showing the presence of each mRNA and protein corresponding to alpha-, beta-, gamma-, delta-, and epsilon-sarcoglycans as well as zeta-sarcoglycan mRNA. Western blot analysis also revealed the expression of beta-dystroglycan, alpha1-syntrophin, alpha1-, and beta-dystrobrevins. We have established that Dp71ab forms a complex with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and alpha-, beta- and gamma-sarcoglycans in undifferentiated PC12 cells. In differentiated PC12 cells, the complex composition changes since Dp71ab associates only with beta-dystroglycan, alpha1-syntrophin, beta-dystrobrevin, and delta-sarcoglycan. Interestingly, neuronal nitric oxide synthase associates with the Dp71ab/DAPs complex during NGF treatment, raising the possibility that Dp71ab may be involved in signal transduction events during neuronal differentiation.
Function of dystrophin Dp71 isoforms is unknown but seems related to neurite outgrowth and synapse formation. To evaluate Dp71 role in myoneural synapses, we established a coculture model using PC12 cells and L6 myotubes and analyzed expression and localization of Dp71 and related proteins, utrophin and beta-dystroglycan, in PC12 cells. Confocal microscopy showed Dp71d isoform in PC12 nuclei, golgi-complex-like and endoplasmic reticulum-like structures, whereas Dp71ab concentrates at neurite tips and cytoplasm, colocalizing with beta-dystroglycan, utrophin, synaptophysin and acetylcholine receptors. Evidences suggest that Dp71ab isoform, unlike Dp71d, may take part in neurite-related processes. This is the first work on Dp and members of Dp-associated protein complex roles in a cell-line based coculturing system, which may be useful in determining Dp71 isoforms associations.
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