Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.

Alternative splicing plays a major role in the adaptation of cardiac function exemplified by the isoform switch of titin, which adjusts ventricular filling. We previously identified a rat strain deficient in titin splicing. Using genetic mapping, we found a loss-of-function mutation in RBM20 as the underlying cause for the pathological titin isoform expression. Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. We showed that the phenotype of Rbm20 deficient rats resembles the human pathology. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20 dependent regulation of alternative splicing. Additionally to titin we identified a set of 30 genes with conserved regulation between human and rat. This network is enriched for genes previously linked to cardiomyopathy, ion-homeostasis, and sarcomere biology. Our studies emphasize the importance of posttranscriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

sorLA (sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid ␤-peptide (A␤), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to A␤, whereas ablation of sorLA expression in knockout mice results in increased levels of A␤ in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into A␤ and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase A␤ production and plaque formation and promote spontaneous AD.endocytic receptors ͉ knockout mouse ͉ neurodegeneration ͉ Vps10p-domain receptors S orting protein-related receptor (sorLA), also known as LR11, is a 250-kDa type-1 membrane protein of unknown function that is expressed in neurons of the central and peripheral nervous system (1-4). The protein is a member of a family of neuronal receptors that share structural similarity with the vacuolar protein sorting 10 protein (Vps10p), a sorting protein in yeast that transports carboxypeptidase Y from the Golgi to the vacuole (5). Other family members include the proneurotrophin receptor sortilin (6) and the head activator-binding protein in hydra (7). Because sorLA interacts with the family of GGA (Golgi-localizing, ␥-adaptin ear homology domain, ARFinteracting) adaptors that shuttle between the Golgi and endosomes͞lysosomes, the receptor was proposed to act in intracellular protein trafficking (8). The relevance of such sorLAmediated protein transport in neurons is unclear at present. However, expression profiling has demonstrated reduction of sorLA expression in the brain of patients suffering from Alzheimer's disease (AD), suggesting a causal role for the receptor in the pathogenesis of this disease (9).Central to the pathogenesis of AD is the proteolytic processing of a neuronal membrane protein called the amyloid precursor protein (APP). APP follows a complex intracellular trafficking pathway that influences processing to either a soluble fragment sAPP␣ (nonamyloidogenic) or to sAPP␤ and the insoluble amyloid ␤-peptide (A␤), the principal component of senile plaques (10). The rate of A␤ production is considered the major risk factor for onset of AD (10). En route through the secretory pathway to the cell surface, most newly synthesized APP molecules are cleaved into sAPP␣ by ␣-secretase;...

Abstract-Titin is a giant vertebrate striated muscle protein with critical importance for myofibril elasticity and structural integrity. We show here that the complete sequence of the human titin gene contains 363 exons, which together code for 38 138 residues (4200 kDa). In its central I-band region, 47 novel PEVK exons were found, which contribute to titin's extensible spring properties. Additionally, 3 unique I-band titin exons were identified (named novex-1 to -3). Novex-3 functions as an alternative titin C-terminus. The novex-3 titin isoform is Ϸ700 kDa in size and spans from Z1-Z2 (titin's N-terminus) to novex-3 (C-terminal exon). Novex-3 titin specifically interacts with obscurin, a 721-kDa myofibrillar protein composed of 57 Ig/FN3 domains, followed by one IQ, SH3, DH, and a PH domain at its C-terminus. The obscurin domains Ig48/Ig49 bind to novex-3 titin and target to the Z-line region when expressed as a GFP fusion protein in live cardiac myocytes. Immunoelectron microscopy detected the C-terminal Ig48/Ig49 obscurin epitope near the Z-line edge. The distance from the Z-line varied with sarcomere length, suggesting that the novex-3 titin/obscurin complex forms an elastic Z-disc to I-band linking system. This system could link together calcium-dependent, SH3-, and GTPase-regulated signaling pathways in close proximity to the Z-disc, a structure increasingly implicated in the restructuring of sarcomeres during cardiomyopathies.

Vascular smooth muscle cell (SMC) proliferation and migration are important events in the development of atherosclerosis. The low-density lipoprotein receptor-related protein (LRP1) mediates suppression of SMC migration induced by platelet-derived growth factor (PDGF). Here we show that LRP1 forms a complex with the PDGF receptor (PDGFR). Inactivation of LRP1 in vascular SMCs of mice causes PDGFR overexpression and abnormal activation of PDGFR signaling, resulting in disruption of the elastic layer, SMC proliferation, aneurysm formation, and marked susceptibility to cholesterol-induced atherosclerosis. The development of these abnormalities was reduced by treatment with Gleevec, an inhibitor of PDGF signaling. Thus, LRP1 has a pivotal role in protecting vascular wall integrity and preventing atherosclerosis by controlling PDGFR activation.

The recent discovery of heterozygous human mutations that truncate full-length titin (TTN, an abundant structural, sensory, and signaling filament in muscle) as a common cause of end-stage dilated cardiomyopathy (DCM) provides new prospects for improving heart failure management. However, realization of this opportunity has been hindered by the burden of TTN truncating variants (TTNtv) in the general population and uncertainty about their consequences in health or disease. To elucidate the effects of TTNtv, we coupled TTN gene sequencing with cardiac phenotyping in 5,267 individuals across the spectrum of cardiac physiology, and integrated these data with RNA and protein analyses of human heart tissues. We report diversity of TTN isoform expression in the heart, define the relative inclusion of TTN exons in different isoforms, and demonstrate that these data, coupled with TTNtv position, provide a robust strategy to discriminate pathogenic from benign TTNtv. We show that TTNtv is the most common genetic cause for DCM in ambulant patients in the community, identify clinically important manifestations of TTNtv-positive DCM, and define the penetrance and outcomes of TTNtv in the general population. By integrating genetic, transcriptome, and protein analyses we provide evidence for a length-dependent, dominant negative mechanism of disease. These data inform diagnostic criteria and management strategies for TTNtv-positive DCM patients and for TTNtv that are identified as incidental findings.

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface. The low density lipoprotein (LDL)1 receptor gene family has traditionally been regarded as a class of constitutively recycling cell surface receptors that merely mediate the endocytosis and lysosomal delivery of various ligands (such as lipoproteins, proteases, and protease inhibitors) that bind to their extracellular domains (1). Recently, we have reported that the cytoplasmic adaptor or scaffold proteins Disabled-1 (DAB1) and FE65 interact with the cytoplasmic tails of certain LDL receptor family members (2). DAB1 and FE65 have no known role in endocytosis but rather function in cellular signal transduction pathways that involve tyrosine kinases and remodeling of the cytoskeleton. LDL receptor family members do not merely bind these proteins in a fortuitous manner, they rather act in concert with these adaptors and play pivotal roles in cellular signal transduction cascades. This was revealed by the analysis of knockout animals lacking the very low density lipoprotein (VLDL) receptor and the apolipoprotein E (apoE) receptor-2 (3). Mice lacking both receptors exhibit a phenotype that is indistinguishable from that of animals deficient for the extracellular signaling molecule Reelin (4, 5) or DAB1 (6 -8), suggesting that these genes function in a linear signaling pathway. Reelin does indeed bind to both the VLDL receptor and the apoER2 but does so only weakly to the structurally closely related LDL receptor (9, 10). Both the VLDL receptor ...

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) has been postulated to participate in a number of diverse physiological and pathological processes ranging from the homeostasis of plasma lipoproteins, atherosclerosis, and fibrinolysis to neuronal regeneration and survival. It has not been possible to demonstrate in vivo the physiological significance of LRP for each of these complex processes by a conventional gene knockout approach because LRP is essential for embryonic development. Here we have used the Cre/loxP recombination system to achieve inducible, tissue-specific and quantitative disruption of the LRP gene in adult mice. Inactivation of LRP in the livers of LDL receptor-deficient mice resulted in the accumulation of cholesterol-rich remnant lipoproteins in the circulation. In normal animals, this caused a compensatory upregulation of the LDL receptor in the liver. Conditional gene targeting has thus allowed us to isolate a specific physiological function of LRP for in vivo analysis and has provided unequivocal evidence for another LDL receptor-independent cholesterol clearance pathway in liver.