No abstract

The electroretinograms (ERGs) of patients with Duchenne muscular dystrophy and an allelic variant of the mdx mouse (mdxCv3) have been shown to be abnormal. Analysis of five allelic variants of the mdx mouse with mutations in the dystrophin gene has shown that there is a correlation between the position of the mutation and the severity of the ERG abnormality. Three isoforms are expressed in the retina: Dp427, Dp260 and Dp71. Using indirect immunofluorescence and isoform-specific antibodies on retinal sections from three allelic mdx mouse strains, we have examined the localization of each of the isoforms. We show that Dp71 expression does not overlap with Dp427 and Dp260 expression at the outer plexiform layer (OPL). Instead, Dp71 is localized to the inner limiting membrane (ILM) and to retinal blood vessels. Moreover, we show that Dp260 and Dp71 differ structurally at their respective C-termini. In addition, we find that the proper localization of the beta-dystroglycan is dependent upon both Dp260 at the OPL and Dp71 expression at the ILM. Thus, Dp260 and Dp71 are non-redundant isoforms that are located at different sites within the retina yet have a common interaction with beta-dystroglycan. Our data suggest that both Dp71 and Dp260 contribute distinct but essential roles to retinal electrophysiology.

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Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259 -27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71⌬ 110 ) in the membrane cytoskeleton of human platelets. Both Dp71⌬ 110 and utrophin sediment from lysed platelets along with the high speed detergentinsoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71⌬ 110 and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombinstimulated platelets. Immunoelectron microscopy provided further evidence that Dp71⌬ 110 was localized to the submembranous cytoskeleton. In addition to Dp71⌬ 110 , platelets contained several components of the dystrophin-associated protein complex, including ␤-dystroglycan and syntrophin. To better understand the potential function of Dp71⌬ 110 , collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx 3cv ) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx 3cv mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71⌬ 110 is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.

The dystrophin gene defective in Duchenne muscular dystrophy (DMD) is extreme in size and complexity with several promoters which direct expression of different isoforms in different tissues. In contrast with adult skeletal muscle which expresses 427 kDa dystrophin, fetal muscle tissue expresses the 71 kDa ubiquitous isoform Dp71 as well as 427 kDa muscle dystrophin. To examine Dp71 expression in fetal muscle further, we have monitored its expression pattern in differentiating myogenic cultures of human fetal muscle origin. The presence of transcripts initiated from the Dp71 promoter was demonstrated by quantitative RT-PCR. The level of transcript expressed from the Dp71 promoter did not change significantly during myogenic differentiation, consistent with the housekeeping nature of the promoter. Measurements to determine the stability of the Dp71 mRNA indicated that it has a half-life of -20 h and, therefore, is somewhat more stable than the larger 14 kb muscle dystrophin mRNA (t1/2 = 16 h). In contrast with the constant level of Dp71 transcript during myogenic differentiation, the level of Dp71 protein increased significantly, perhaps due to changes in translation efficiency or protein stability. These results demonstrate expression and posttranscriptional upregulation of Dp71 in human fetal myogenic cultures.