LDL Receptor-related Proteins Research Articles

Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR/HDAC9

BackgroundDiabetic angiogenesis is closely associated with disabilities and death caused by diabetic microvascular complications. Advanced glycation end products (AGEs) are abnormally accumulated in diabetic patients and are a key pathogenic factor for diabetic angiogenesis. The present study focuses on understanding the mechanisms underlying diabetic angiogenesis and identifying therapeutic targets based on these mechanisms.MethodsIn this study, AGE-induced angiogenesis serves as a model to investigate the mechanisms underlying diabetic angiogensis. Mouse aortic rings, matrigel plugs, and HUVECs or 293T cells were employed as research objects to explore this pathological process by using transcriptomics, gene promoter reporter assays, virtual screening and so on.ResultsHere, we found that AGEs activated Wnt/β-catenin signaling pathway and enhanced the β-catenin protein level by affecting the expression of β-catenin degradation-related genes, such as FZDs (Frizzled receptors), LRPs (LDL Receptor Related Proteins), and AXIN1. AGEs could also mediate β-catenin Y142 phosphorylation through VEGFR1 isoform5. These dual effects of AGEs elevated the nuclear translocation of β-catenin and sequentially induced the expression of KDR (Kinase Insert Domain Receptor) and HDAC9 (Histone Deacetylase 9) by POU5F1 and NANOG, respectively, thus mediating angiogenesis. Finally, through virtual screening, Bioymifi, an inhibitor that blocks VEGFR1 isoform5-β-catenin complex interaction and alleviates AGE-induced angiogenesis, was identified.ConclusionCollectively, this study offers insight into the pathophysiological functions of β-catenin in diabetic angiogenesis.

Inhibition of a novel Dickkopf-1-LDL receptor-related proteins 5 and 6 axis prevents diabetic cardiomyopathy in mice.

Anti-hypertensive agents are one of the most frequently used drugs worldwide. However, no blood pressure-lowering strategy is superior to placebo with respect to survival in diabetic hypertensive patients. Previous findings show that Wnt co-receptors LDL receptor-related proteins 5 and 6 (LRP5/6) can directly bind to several G protein-coupled receptors (GPCRs). Because angiotensin II type 1 receptor (AT1R) is the most important GPCR in regulating hypertension, this study examines the possible mechanistic association between LRP5/6 and their binding protein Dickkopf-1 (DKK1) and activation of the AT1R and further hypothesizes that the LRP5/6-GPCR interaction may affect hypertension and potentiate cardiac impairment in the setting of diabetes. The roles of serum DKK1 and DKK1-LRP5/6 signalling in diabetic injuries were investigated in human and diabetic mice. Blood pressure up-regulation positively correlated with serum DKK1 elevations in humans. Notably, LRP5/6 physically and functionally interacted with AT1R. The loss of membrane LRP5/6 caused by injection of a recombinant DKK1 protein or conditional LRP5/6 deletions resulted in AT1R activation and hypertension, as well as β-arrestin1 activation and cardiac impairment, possibly because of multiple GPCR alterations. Importantly, unlike commonly used anti-hypertensive agents, administration of the anti-DKK1 neutralizing antibody effectively prevented diabetic cardiac impairment in mice. These findings establish a novel DKK1-LRP5/6-GPCR pathway in inducing diabetic injuries and may resolve the long-standing conundrum as to why elevated blood DKK1 has deleterious effects. Thus, monitoring and therapeutic elimination of blood DKK1 may be a promising strategy to attenuate diabetic injuries.

Effects of Wnt10a and Wnt10b Double Mutations on Tooth Development.

WNT molecules are the regulators of various biological functions, including body axis formation, organ development, and cell proliferation and differentiation. WNTs have been extensively studied as causative genes for an array of diseases. WNT10A and WNT10B, which are considered to be genes of the same origin, have been identified as causative genes for tooth deficiency in humans. However, the disrupted mutant of each gene does not show a decrease in teeth number. A negative feedback loop, interacting with several ligands based on a reaction-diffusion mechanism, was proposed to be important for the spatial patterning of tooth formation, and WNT ligands have been considered to play a pivotal role in controlling tooth patterning from mutant phenotypes of LDL receptor-related proteins (LRPs) and WNT co-receptors. The Wnt10a and Wnt10b double-mutants demonstrated severe root or enamel hypoplasia. In Wnt10a-/- and Wnt10a+/-;Wnt10b-/- mice, changes in the feedback loop may collapse the modulation of fusion or split a sequence of tooth formation. However, in the double-knockout mutant, a decrease in the number of teeth was observed, including the upper incisor or third molar in both jaws. These findings suggest that there may be a functional redundancy between Wnt10a and Wnt10b and that the interaction between the two genes functions in conjunction with other ligands to control the spatial patterning and development of teeth.

The WNT/β-catenin signalling pathway induces chondrocyte apoptosis in the cartilage injury caused by T-2 toxin in rats

We investigated whether the WNT/β-catenin signalling pathway is involved in paediatric Kashin–Beck disease (KBD) and T-2-toxin-induced cartilage injury in rats to better understand the mechanism of KBD. One hundred twenty-two children were selected and assigned to the case (31), internal control (41), and external control (50) groups. The serum β-catenin and bone morphogenetic protein 2（BMP2）levels in each group were measured and compared. Thirty-six rats were randomly assigned to three groups, which received no intervention, T-2 toxin, or solvent. After 18 weeks, the expression of LDL receptor related proteins 5 (LRP5), β-catenin, BMP2, BAX, BCL2, APAF1, and caspase 9 was measured and compared. The serum BMP2 levels were significantly elevated in the children with KBD and in the rats treated with T-2 toxin. In the T-2 toxin group, LRP5 and β-catenin expression was reduced, whereas BAX, APAF1, and caspase 9 expression was increased. In conclusion, the WNT/β-catenin signalling pathway is suppressed in KBD, which induces chondrocyte apoptosis, leading to cartilage injury. Therefore, BMP2 may play a role in the pathogenesis of KBD.

LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells.

LDL receptor-related proteins 6 (LRP6) is a type I transmembrane receptor (C-terminus in cytosol), which appears to be essential in numerous biological processes, since it is an essential co-receptor of Wnt ligands for canonical β-catenin dependent signal transduction. It was shown that tissue plasminogen activator (tPA), physically interacting with LRP6, induces protein phosphorylation, which may have large implication in the regulation of neural processes. In this investigation we analyzed whether LRP6 is associated with lipid rafts following tPA triggering in neuroblastoma cells and the role of raft integrity in LRP6 cell signaling. Sucrose gradient separation revealed that phosphorylated LRP6 was mainly, but not exclusively present in lipid rafts; this enrichment became more evident after triggering with tPA. In these microdomains LRP6 is strictly associated with ganglioside GM1, a paradigmatic component of these plasma membrane compartments, as revealed by coimmunoprecipitation experiments. As expected, tPA triggering induced LRP6 phosphorylation, which was independent of LRP1, as revealed by knockdown experiments by siRNA, but strictly dependent on raft integrity. Moreover, tPA induced β-catenin phosphorylation was also significantly prevented by previous pretreatment with methyl-β-cyclodextrin. Our results demonstrate that LRP6 mediated signal transduction pathway triggered by tPA acts through lipid rafts in neuroblastoma cells. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents. Indeed, these data, pointing to the key role of lipid rafts in tPA triggered signaling involving β-catenin, may have pharmacological implications, suggesting that cyclodextrins, and potentially other drugs, such as statins, may represent an useful tool.

Expression of LDL receptor-related proteins (LRPs) in common solid malignancies correlates with patient survival.

LDL receptor-related proteins (LRPs) are transmembrane receptors involved in endocytosis, cell-signaling, and trafficking of other cellular proteins. Considerable work has focused on LRPs in the fields of vascular biology and neurobiology. How these receptors affect cancer progression in humans remains largely unknown. Herein, we mined provisional databases in The Cancer Genome Atlas (TCGA) to compare expression of thirteen LRPs in ten common solid malignancies in patients. Our first goal was to determine the abundance of LRP mRNAs in each type of cancer. Our second goal was to determine whether expression of LRPs is associated with improved or worsened patient survival. In total, data from 4,629 patients were mined. In nine of ten cancers studied, the most abundantly expressed LRP was LRP1; however, a correlation between LRP1 mRNA expression and patient survival was observed only in bladder urothelial carcinoma. In this malignancy, high levels of LRP1 mRNA were associated with worsened patient survival. High levels of LDL receptor (LDLR) mRNA were associated with decreased patient survival in pancreatic adenocarcinoma. High levels of LRP10 mRNA were associated with decreased patient survival in hepatocellular carcinoma, lung adenocarcinoma, and pancreatic adenocarcinoma. LRP2 was the only LRP for which high levels of mRNA expression correlated with improved patient survival. This correlation was observed in renal clear cell carcinoma. Insights into LRP gene expression in human cancers and their effects on patient survival should guide future research.

P 44 Low-density-Lipoprotein-related-proteins -1, -2, and -8 in the placenta of IUGR

Introduction Intrauterine growth restriction (IUGR) shows a typical drop of apolipoprotein E (ApoE) concentration levels in fetal blood. ApoE activates LDL receptor related proteins (LRP) thus greatly increases transplacental transport of lipoproteins such as LDL and VLDL. The origin of the fetal ApoE is still unknown but might be maternal. Objectives Our hypothesis is that ApoE increases the effect of transplacental cholesterol transport by induction of ApoE receptors in the placenta which is disturbed in IUGR. Methods Placentas of 40 IUGR and 40 controls (CTRL) arranged in tissue micro arrays (TMA) were analyzed immunohistochemically for LRP1, LRP8, LRP2. The expression was assessed in trophoblasts and endothelium cells semi-quantitatively using Immunoreactivity-Score (IRS). Expression values were related to maternal and fetal lipid profiles. Expression patterns were statistically accomplished via the Mann–Whitney test. Results There was a significant lower expression of LRP1 in trophoblasts and in endothelium cells in IUGR as compared to a healthy control group (p Conclusion: The significant lower expression of the LRP1 receptor in IUGR may contribute to lower ApoE concentrations in fetal blood. Further experimental studies are necessary focusing on functional aspects.

Delivery of circulating lipoproteins to specific neurons in the Drosophila brain regulates systemic insulin signaling.

The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. Here, we demonstrate a function for the Drosophila lipoprotein LTP in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in Blood Brain Barrier glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content.

Structural Basis of Wnt Signaling Inhibition by Dickkopf Binding to LRP5/6

LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

The role of microglia in amyloid clearance from the AD brain

Alzheimer's disease (AD), the most prominent cause of senile dementia, is clinically characterized by the extracellular deposition of beta-amyloid (Abeta) and the intracellular neurofibrillary tangles. It has been well accepted that AD pathogenesis arises from perturbation in the homeostasis of Abeta in the brain. Abeta is normally produced at high levels in the brain and cleared in an equivalent rate. Thus, even a moderate decrease in the clearance leads to the accumulation of Abeta and subsequent amyloid deposition. Microglia are the tissue macrophages in the central nervous system (CNS) and have been shown to play major roles in internalization and degradation of Abeta. Abeta exists in the brain both in soluble and in fibrillar forms. Microglia interact with these two forms of Abeta in different ways. They take up soluble forms of Abeta through macropinocytosis and LDL receptor-related proteins (LRPs) mediated pathway. Fibrillar forms of Abeta interact with the cell surface innate immune receptor complex, initiating intracellular signaling cascades that stimulate phagocytosis. Inflammatory responses influence the activation status of microglia and subsequently regulate their ability to take up and degrade Abeta. ApoE and its receptors have been shown to play critical roles in these processes. In this review, we will explore the mechanisms that microglia utilize to clear Abeta and the effectors that modulate the processes.

Apolipoprotein E receptors in the nervous system

Over the past few years, apolipoprotein E (ApoE) receptors, also known as LDL receptor-related proteins, have distinguished themselves as functionally diverse signaling receptors with pivotal roles not only in the vascular system but also in the nervous system and during development. The expanding roles of ApoE receptors for cellular signal transduction at the same time transcend and integrate their lipid transport roles into a larger biological and clinical context. ApoE receptors are essential for the development of the nervous system, the regulation of synaptic plasticity, neuroprotection and the innervation of the muscle. They also regulate the metabolism of the amyloid precursor protein on multiple levels, implicating them in the pathogenesis of Alzheimer's disease. ApoE, a common ligand for all members of the evolutionarily ancient LDL receptor gene family, is the major genetic modifier of the age of onset of Alzheimer's disease. The underlying molecular mechanisms remain shrouded in mystery, but the numerous critical functions of ApoE receptors within and outside the nervous system that have recently emerged make it likely that these multifunctional signal modulators participate in Alzheimer's disease pathogenesis. This review attempts to summarize the most recent and relevant findings in this area.

Cleavage of the Wnt Receptor Ryk Regulates Neuronal Differentiation during Cortical Neurogenesis

Ryk is a transmembrane receptor tyrosine kinase (RTK). It functions as a receptor of Wnt proteins required for cell-fate determination, axon guidance, and neurite outgrowth in different organisms; however, the molecular mechanism of Ryk signaling is unknown. Here, we show that Ryk is cleaved, permitting the intracellular C-terminal fragment of Ryk to translocate to the nucleus in response to Wnt3 stimulation. We also show that the cleaved intracellular domain of Ryk is required for Wnt3-induced neuronal differentiation in vitro and in vivo. These results demonstrate an unexpected mechanism of signal transduction for Ryk as a Wnt receptor, in which the intracellular domain itself functions as the transducing molecule to bring extracellular signals from the cell surface into the nucleus, to regulate neural progenitor cell differentiation.

Wnt/β-Catenin Signaling Stimulates Chondrogenic and Inhibits Adipogenic Differentiation of Pericytes

The aberrant differentiation of pericytes along the adipogenic, chondrogenic, and osteogenic lineages may contribute to the development and progression of several vascular diseases, including atherosclerosis and calcific vasculopathies. However, the mechanisms controlling pericyte differentiation and, in particular, adipogenic and chondrogenic differentiation are poorly defined. Wnt/beta-catenin signaling regulates cell differentiation during embryonic and postnatal development, and there is increasing evidence that it is involved in vascular pathology. Therefore, this study tested the hypothesis that Wnt/beta-catenin signaling regulates the chondrogenic and adipogenic differentiation of pericytes. We demonstrate that pericytes express several Wnt receptors, including LDL receptor-related proteins 5 and 6, and Frizzled 1 to 4 and 7, 8, and 10, and that Wnt/beta-catenin signaling is stimulated by both Wnt3a and LiCl. Furthermore, induction of Wnt/beta-catenin signaling by LiCl enhances chondrogenesis in pericyte pellet cultures in the presence of transforming growth factor-beta3, as demonstrated by increased Sox-9 expression and glycosaminoglycan accumulation into the matrix. In contrast, transduction of pericytes with a recombinant adenovirus encoding dominant-negative T-cell factor-4 (RAd/dnTCF), which blocks Wnt/beta-catenin signaling, inhibited chondrogenesis, leading to reduced Sox-9 and type II collagen expression and less glycosaminoglycan accumulation. Together, these data demonstrate that transforming growth factor-beta3 induces the chondrogenic differentiation of pericytes by inducing Wnt/beta-catenin signaling and T-cell factor-induced gene transcription. Induction of Wnt/beta-catenin signaling also attenuates adipogenic differentiation of pericytes in both pellet and monolayer cultures, as demonstrated by decreased staining with oil red O and reduced peroxisome proliferator-activated receptor gamma2 expression. This effect was negated by transduction of pericytes with RAd/dnTCF. Together, these results demonstrate that Wnt/beta-catenin signaling inhibits adipogenic and enhances chondrogenic differentiation of pericytes.

The Switch on the RAPper's Necklace…

The biosynthesis and export of LDL receptor-related proteins rely on specialized chaperones in the endoplasmic reticulum. Two recent papers in Molecular Cell by Fisher et al. (2006) and Lee et al. (2006) reveal a novel mechanism by which one of these chaperones, the receptor-associated protein RAP, accomplishes this task.

A genome scan for loci influencing levels and trends of lipoprotein lipid-related traits since childhood: The Bogalusa Heart Study

Coronary heart disease is the result of life-long processes. Previous genetic linkage analyses of lipid and lipoprotein variables that can be measured throughout life have focused on a single measure at one point in time. Genome-wide linkage analyses were performed in the present study to identify loci influencing the long-term levels and trends of high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC) and triglycerides in a longitudinal cohort. Microsatellite markers ( n = 357) were typed on 779 white and 444 black siblings, ages 14–43 years. Subjects had been examined serially 2–13 times with 6963 serial observations over an average of 22 years from childhood to adulthood. Total and incremental area under the growth curves of lipid traits was calculated and used as measures for long-term levels and trends. After adjusting for age, sex and body mass index, heritability estimates of total area values for all lipid variables were higher than those of a single measurement in either childhood or adulthood. In blacks, significant linkage to LDLC incremental area (peak LOD = 3.6 at 50 cM) was observed on chromosome 1; and suggestive linkage for total area of LDLC (LOD = 2.9 at 21 cM) on chromosome 19. Only one suggestive linkage (LOD = 2.2 at 161 cM) on chromosome 2 was identified in whites for LDLC incremental area. Other suggestive linkage (LOD ≥ 2.0) was noted for LDLC and HDLC in terms of either total or incremental area on chromosomes 2, 5, 7 and 15 for blacks and whites. Several lipid-related candidate genes such as low-density lipoprotein receptor (LDLR), LDL receptor-related proteins 3 and 8, ApoE, ApoAII and ApoCII are located in these regions. Linkage evidence found in this community-based study indicates that regions on these chromosomes harbor genetic loci that affect the propensity to develop dyslipidemia from childhood.

Role of the intracellular domains of LRP5 and LRP6 in activating the Wnt canonical pathway

LDL-receptor related proteins 5 and 6 (LRP5/6) are co-receptors of Frizzled receptors that mediate Wnt-induced activation of the transcription factor family TCF/LEF-1. Even though LRP5 and LRP6 are highly homologous, LRP6, but not LRP5, is expressed primarily in the nervous system and deletion of the LRP6 gene results in significant brain abnormalities, while deletion of LRP5 results in primarily decreased bone density. Additionally, the exact function of LRP5 and LRP6 have not been clearly defined, although it is clear that they both play key roles in the Wnt canonical pathway. In this study the role of the intracellular domains of LRP5/6 in mediating Wnt signaling was examined. In the absence of exogenous Wnt 3a, full-length (FL) LRP6, but not LRP5, increased TCF/LEF-1 transcriptional activity, however both significantly potentiated Wnt 3a-induced TCF/LEF-1 activation. In contrast to the findings with the FL constructs, the intracellular domains (membrane-anchored and cytosolic) of both LRP5 and LRP6 significantly increased TCF/LEF-1 activation in the absence of Wnt 3a, and potentiated the Wnt 3a-induced decrease in beta-catenin phosphorylation, increase in free beta-catenin levels and the increase in TCF/LEF-1 activity. These findings demonstrate that: (1) LRP5 and LRP6 differentially modulate TCF/LEF-1 activation in the absence of Wnt 3a and (2) the intracellular C-terminal domains of LRP5/6 potentiate Wnt 3a-induced TCF/LEF-1 activation whether or not they are membrane-anchored. These findings provide significant new insights into the roles of LRP5/6 in modulating canonical Wnt signaling.

LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way.

Wnt signaling through the canonical beta-catenin pathway plays essential roles in development and disease. Low-density-lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) in vertebrates, and their Drosophila ortholog Arrow, are single-span transmembrane proteins that are indispensable for Wnt/beta-catenin signaling, and are likely to act as Wnt co-receptors. This review highlights recent progress and unresolved issues in understanding the function and regulation of Arrow/Lrp5/Lrp6 in Wnt signaling. We discuss Arrow/Lrp5/Lrp6 interactions with Wnt and the Frizzled family of Wnt receptors, and with the intracellular beta-catenin degradation apparatus. We also discuss the regulation of Lrp5/Lrp6 by other extracellular ligands, and LRP5 mutations associated with familial osteoporosis and other disorders.

A Mechanism for Wnt Coreceptor Activation

LDL receptor related proteins 5 and 6 (LRP5/6) and their Drosophila homolog Arrow are single-span transmembrane proteins essential for Wnt/β-catenin signaling, likely via acting as Wnt coreceptors. How Wnt activates LRP5/6/Arrow to initiate signal transduction is not well defined. Here we show that a PPPSP motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, is necessary and sufficient to trigger Wnt/β-catenin signaling. A single PPPSP motif, upon transfer to the LDL receptor, fully activates the Wnt pathway, inducing complete axis duplication in Xenopus and TCF/β-catenin-responsive transcription in human cells. We further show that Wnt signal-ing stimulates, and requires, phosphorylation of the PPPSP motif, which creates an inducible docking site for Axin, a scaffolding protein controlling β-catenin stability. Our study identifies a critical signaling module and a key phosphorylation-dependent activation step of the Wnt receptor complex and reveals a unifying logic for transmembrane signaling by Wnts, growth factors, and cytokines.

V-Src induces Shc binding to tyrosine 63 in the cytoplasmic domain of the LDL receptor-related protein 1.

We recently observed that the LDL receptor-related protein 1 (LRP-1) is tyrosine phosphorylated in v-Src-transformed cells. Using a GST-fusion protein containing the cytoplasmic domain of LRP-1, we show that LRP-1 is a direct substrate for v-Src in vitro. To study LRP-1 phosphorylation in vivo, we constructed an LRP-1 minireceptor composed of the beta chain linked at the amino-terminus to a Myc epitope (Myc-LRPbeta). When expressed together with v-Src, Myc-LRPbeta becomes phosphorylated on tyrosine. Of the four tyrosine residues present in the cytoplasmic domain of LRP-1, only Tyr 63 is phosphorylated by v-Src in vivo or in vitro. Using fibroblasts deficient in Src, Yes and Fyn, we were able to show that there are multiple kinases present in the cell that can phosphorylate LRP-1. Tyrosine-phosphorylated LRP-1 associates with Shc, a PTB and SH2 domain containing signaling protein that is involved in the activation of Ras. Binding of the purified Shc PTB domain to Tyr 63 containing peptides shows that the interaction between LRP-1 and Shc is direct. We found that DAB, a PTB domain containing signaling protein that is involved in signaling by LDL receptor-related proteins in the nervous system, did not bind to full-length LRP-1. Our observations suggest that LRP-1 may be involved in normal and malignant signal transduction through a direct interaction with Shc adaptor proteins.