Gaucher's disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinson's disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.

Angiotensin II is the effector molecule of the reninangiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT 1 . These receptors contain the structural features of the seven-transmembrane, G-proteincoupled receptor superfamily. Angiotensin II, acting through the AT 1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT 1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT 1A receptor motif YIPP (amino acids 319 -322). The wild-type AT 1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT 1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT 1A receptor. The binding of the AT 1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.The analysis of cytokines and their receptors has implicated the intracellular Jak family of kinases as critically important for the intracellular signaling initiated in response to ligand (1-3). Cytokines induce receptor dimerization and the activation, via tyrosine phosphorylation, of the associated Jak kinases. The Jak kinases phosphorylate the cytokine receptors, leading to the binding and eventual activation of intermediate signaling molecules referred to as STAT (signal transducers and activators of transcription). The STAT proteins are a family of transcription factors that migrate to the nucleus and induce gene transcription (4). The Jak/STAT pathway was first elucidated through the study of interferon signaling, but it is now known that this pathway participates in the signaling initiated by a wide variety of cytokines and growth factors. Recently, the vasoactive peptide angiotensin II was also found to activate the Jak/STAT pathway (5).Angiotensin II is the effector molecule of the renin-angiotensin system. It is an 8-amino acid peptide that induces several physiologic responses that act to raise blood pressure. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT 1 (6). Whereas humans have a single AT 1 receptor gene, rodents possess two genes encoding highly homologous receptor isoforms termed AT 1A and AT 1B . These proteins are 95% identical and appear to bind ligand and to signal in an identical fashion (7,8). All AT 1 receptors...

The angiotensin II type 1 (AT 1 ) receptor signals via heterotrimeric G-proteins and intracellular tyrosine kinases. Here, we investigate a modified AT 1 receptor, termed M5, where the last five tyrosines (residues 292, 302, 312, 319, and 339) within the intracellular carboxyl tail have been mutated to phenylalanine. This receptor did not elevate cytosolic free calcium or inositol phosphate production in response to angiotensin II, suggesting an uncoupling of the receptor from G-protein activation. Despite this, the M5 receptor still activated tyrosine kinases, induced STAT1 tyrosine phosphorylation, and stimulated cell proliferation. We also studied another AT 1 mutant receptor, D74E, stably expressed in Chinese hamster ovarian cells and a fibroblast cell line from mice with a genetic inactivation of G␣ q/11 . Both cell lines have a deficit in calcium signaling and in G-protein activation, and yet in both cell lines, angiotensin II induced the time-dependent tyrosine phosphorylation of STAT1. These studies are the first to show the ability of a seven-transmembrane receptor to activate intracellular tyrosine kinase pathways in the absence of a G-protein-coupled rise in intracellular calcium.The AT 1 1 receptor is a seven-transmembrane receptor that signals via heterotrimeric G-proteins (1, 2). Ligand binding activates G q , leading to the generation of inositol 1,4,5-trisphosphate (IP 3 ) and a rise in cytosolic free calcium. This, in turn, affects cell contractility, secretion, gene transcription, and cell proliferation.More recently, the AT 1 receptor has also been shown to signal by activating nonmembrane tyrosine kinases including Src, Fyn, and Pyk2 (3). In 1995, our group reported that the binding of angiotensin II to the AT 1 receptor activated the intracellular tyrosine kinase Jak2 and that this led to the tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 (4). Although the activation of tyrosine kinases by G-protein-coupled receptor has now been established in several systems including vascular smooth muscle cells (5, 6), mesangial cells (7), zona glomerulosa cells (8), and cardiac cells (9, 10), the interplay between G-protein activation and receptor-mediated kinase activation is less clear. Structure-function studies of the AT 1 receptor have defined amino acids critical for coupling the receptor to G-proteins. For example, mutation of either Asp 74 or Tyr 292 results in the loss of G-protein coupling and blocks the ligand-dependent production of IP 3 (11, 12). However, the effect of these mutations on AT 1 receptor-dependent tyrosine kinase signaling is not known. In fact, dissecting the importance of angiotensin II-dependent tyrosine phosphorylation signaling from G-protein activation is difficult because of multiple conversion points. For example, both the G-protein and tyrosine kinase pathways activate mitogen-activated protein kinase (9, 10), and both also give rise to an increase in cytosolic free calcium (5, 6, 7).To separate the effects of AT 1 receptor activat...

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8. Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

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