Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide–binding protein G q and is terminated by receptor desensitization and degradation of the second messengers. We show that β-arrestins coordinate both processes for the G q -coupled M1 muscarinic receptor. β-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, β-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the β-arrestin–DGK complex to activated 7TMRs. The dual function of β-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3′,5′-monophosphate phosphodiesterases to G s -coupled β 2 -adrenergic receptors. Thus, β-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol to form phosphatidic acid. In most cases, members of this large family of enzymes appear to bind and regulate proteins activated by either diacylglycerol or phosphatidic acid. Proteins that appear to be regulated, in part, by DGKs include protein kinase Cs, RasGRPs, and phosphatidylinositol kinases. By modulating the activity of these proteins, DGKs potentially affect a number of biological events including-but likely not limited to-cell growth, neuronal transmission, and cytoskeleton remodeling.

To study the physiological function of diacylglycerol (DAG) kinase (DGK ), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGK . DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGK inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGK bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGK by crossing wild-type or DGK -deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGK -deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGK regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGK , which is structurally similar to DGK , inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK. D iacylglycerol (DAG) is a potent activator of several signaling proteins, many of which, when abnormally active, can contribute to the initiation or promotion of cancer (1, 2). Several enzymes can metabolize signaling DAG, but the major route is thought to be by its phosphorylation, a reaction that produces phosphatidic acid and is catalyzed by the DAG kinases (DGKs) (reviewed in ref. 2). DAG exerts its effects by activating classical and novel protein kinase C isoforms, as well as other proteins including the Ras guanyl nucleotide-releasing proteins (RasGRPs) (reviewed in refs. 3-5). Although the transforming effects of DAG have been attributed to activation of PKCs, identification of the RasGRP family suggested that DAG could also transform cells by directly activating RasGRPs, which in turn could lead to excess Ras signaling. The four known RasGRP proteins are RasGRP1 [calcium DAG guanine exchange factor II (CalDAG-GEFII)], RasGRP2 (CalDAG-GEFI), RasGRP3 (CalDAG-GEFIII), and RasGRP4 (reviewed in ref. 5). Each RasGRP activates either Ras or Rap1, except RasGRP3, which is unique because it facilitates exchange for both .Activating mutations in Ras are found in a number of tumors (reviewed in ref. 11). Using a mouse model, Chin et al. (12) showed that Ras expression was an absolute requirement for melanoma tumor maintenance, and other studies have demonstrated a role for Ras in metastasis (reviewed in ref. 13). Together, these data clearly show that abnormally active Ras contributes to the maintenance and progression of many types of cancer.Rap1-dependent signaling is not a...

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Using a phosphorylation-dependent cell-free system to study NADPH . In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22 phox . These findings suggest that phosphorylation of p22 phox by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22 phox , the small subunit of the membranebound oxidase component flavocytochrome b 558 , is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms ( In intact neutrophils, several signaling mechanisms, initiated by receptor-ligand interactions, are involved in the regulation of NADPH oxidase (reviewed in Refs. 1 and 15). These events include activation of phospholipases, production of lipid second messengers, activation of protein kinases, and phosphorylation of neutrophil proteins and NADPH oxidase components. Activation of phospholipase A 2 and production of arachidonic acid is required for NADPH oxidase activity (16,17), although the role of arachidonic acid has not been identified. Phospholipase C also may be involved in NADPH oxidase activation (18 -20). Production of diacylglycerol by phospholipase C can induce activation of protein kinase C (PKC), which may mediate the phosphorylation of several NADPH oxidase components (see below) and is clearly an important regulator of NADPH oxidase activation (21, 22). Activation of phospholipase D (PLD), which cleaves phospholipids to form phosphatidic acid (PA), correlates with NADPH oxidase activation (23-30). The mechanism by which PA regulates NADPH oxidase has not been determined; however, a PA-activated protein kinase has been partially purified from neutrophil cytosol (31). phox , and p67 phox , 22-, 47, and 67-kDa phox component, respectively; dCB, dihydrocytochalasin B; fMLP, N-formyl-methionyl-leucyl-phenylalanine; OPZ, opsonized zymosan; PA, phosphatidic acid; PKC, protein kinase C; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate; PVDF, polyvinylidene difluoride membrane; PAGE, polyacrylamide gel electrophoresis; GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); mAb, monoclonal antibody; Pipes, 1,4-piperazinediethanesulfonic acid.

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

The national importance of telemedicine for safe and effective patient care has been highlighted by the current COVID‐19 pandemic. Prior to the 2020 pandemic the Division of Genetics and Metabolism piloted a telemedicine program focused on initial and follow‐up visits in the patients' home. The goals were to increase access to care, decrease missed work, improve scheduling, and avoid the transport and exposure of medically fragile patients. Visits were conducted by physician medical geneticists, genetic counselors, and biochemical dietitians, together and separately. This allowed the program to develop detailed standard operating procedures. At the onset of the COVID‐19 pandemic, this pilot‐program was deployed by the full team of 22 providers in one business day. Two physicians remained on‐site for patients requiring in‐person evaluations. This model optimized patient safety and workforce preservation while providing full access to patients during a pandemic. We provide initial data on visit numbers, types of diagnoses, and no‐show rates. Experience in this implementation before and during the pandemic has confirmed the effectiveness and value of telemedicine for a highly complex medical population. This program is a model that can and will be continued well‐beyond the current crisis.