Dp71ab/DAPs complex composition changes during the differentiation process in PC12 cells

Abstract

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.

Dystrophin Dp71 is Critical for Stability of the DAPs in the Nucleus of PC12 Cells

“…Previously, we demonstrated that the Dp71f variant modulates the adhesion of PC12 cells by interacting with the b1-integrin adhesion protein complex and by this way conferring stability to this complex. Furthermore, it has been reported that Dp71f associates with b-dystroglycan, a1-syntrophin and b-dystrobrevin in the cytoplasm of the PC12 cells to constitute a dystrophin-associated protein complex (DAPC) [36]. These authors propose that the cytoplasmic DAPC is involved in the cytoskeleton network modeling that occurs during the NGF-induced neuronal differentiation of PC12 cells.…”

“…We [17,19], and others [18,35,36], have adopted the PC12 cell line as in vitro neuronal model for studying the function of Dp71. These cells express two Dp71 isoforms called Dp71d and Dp71f, which display different subcellular localization each other [18].…”

Expression of Sarcoglycans in the Human Cerebral Cortex: An Immunohistochemical and Molecular Study

“…Romo-Yáñez et al [2007] detected gene expression of all known SGs ( ␣ -, ␤ -, ␥ -, ␦ -, -and -SG) and confirmed the expression of the proteins by indirect immunofluorescence. Our results in the normal human cerebral cortex evidenced the existence of all proteins that characterized the complex ( ␣ , ␤ , ␦ , ␥ , This study documented the presence of all SGs tested, even in astrocytes of the premotor area.…”

Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with β-Dystroglycan and α1-Syntrophin

“…These results suggest that exons 71-74 play an important role in the cytoplasmic localization of Dp71a and Dp71e isoforms because Dp71c and Dp71ec, which lack these exons, do not show cytoplasmic localization. Most likely, each Dp71 isoform forms a specific complex with the membrane, cytoplasm, or nuclear proteins because Dp71-DAP complexes have been described in the end-feet and cell membranes of retina Müller glial cells (Fort et al 2008) and in the cytoplasm and nuclei of several cell line models (Fuentes-Mera et al 2006;Romo-Yáñez et al 2007;González-Ramírez et al 2008;Villarreal-Silva et al 2010).…”

“…Previously, we reported that β-dystroglycan and α1-syntrophin colocalize and interact with Dp71ab in PC12 cells (Romo-Yáñez et al 2007). To determine if β-dystroglycan formed complexes with the recombinant Dp71 isoforms, colocalization assays were performed.…”