CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.

Highlights d The SLC1A5 variant is a mitochondrial glutamine transporter d The SLC1A5 variant has a mitochondrial targeting sequence d Hypoxia controls SLC1A5 variant expression through HIF-2a d The SLC1A5 variant mediates mitochondrial glutamine metabolism in cancer

Metabotropic glutamate receptors (mGluRs) 1-8 are G proteincoupled receptors (GPCRs) that modulate excitatory neurotransmission, neurotransmitter release, and synaptic plasticity. PKC regulates many aspects of mGluR function, including protein-protein interactions, Ca 2؉ signaling, and receptor desensitization. However, the mechanisms by which PKC regulates mGluR function are poorly understood. We have now identified calmodulin (CaM) as a dynamic regulator of mGluR5 trafficking. We show that the major PKC phosphorylation site on the intracellular C terminus of mGluR5 is serine 901 (S901), and phosphorylation of this residue is up-regulated in response to both receptor and PKC activation. In addition, S901 phosphorylation inhibits mGluR5 binding to CaM, decreasing mGluR5 surface expression. Furthermore, blocking PKC phosphorylation of mGluR5 on S901 dramatically affects mGluR5 signaling by prolonging Ca 2؉ oscillations. Thus, our data demonstrate that mGluR5 activation triggers phosphorylation of S901, thereby directly linking PKC phosphorylation, CaM binding, receptor trafficking, and downstream signaling.phosphorylation ͉ protein kinase C ͉ receptor trafficking T he group I metabotropic glutamate receptor mGluR5 is highly expressed in the forebrain, where it regulates synaptic plasticity (1, 2). In addition, mGluR5 plays a role in pain (3) and addiction (4) and in neurological disorders such as fragile X syndrome (5, 6). Group I mGluRs are G protein-coupled receptors (GPCRs), which are coupled to phospholipase C, and receptor activation triggers phosphoinositide turnover, release of intracellular Ca 2ϩ , and activation of Protein Kinase C (PKC) (7). Although PKC activity regulates mGluR5-mediated Ca 2ϩ signaling and receptor function (8-11), there are no studies linking PKC phosphorylation of mGluR5 to receptor surface expression, endocytosis, or intracellular trafficking.Like other GPCRs, mGluR5 interacts with many proteins in addition to the guanine nucleotide-binding proteins (G proteins). Most of the binding sites for these protein-protein interactions reside within the long intracellular C-terminal domain of mGluR5, suggesting that this region is critical in the functional regulation of mGluR5. For example, the Homer proteins bind to the PPxxFR motif within the distal C terminus, the Tamalin protein associates with the distal C terminus, and calmodulin (CaM) and the E3 ligase Siah-1A bind to the first one-third of the mGluR5 C terminus (12-15). However, the dynamic regulation of these protein-protein interactions has not been described. CaM is a particularly intriguing candidate as an mGluR5 regulator because of its Ca 2ϩ dependence and its key role in synaptic plasticity (16,17). Furthermore, CaM binding to other GPCRs, including dopamine, opioid, and serotonin receptors, has been documented, consistent with a conserved regulatory role for CaM in regulating . We now show that PKC phosphorylation of serine 901 (S901) on mGluR5 inhibits CaM binding and decreases mGluR5 surface expression. Furthermore, preve...

Metabotropic glutamate receptors (mGluRs) play important roles in neurotransmission, neuronal development, synaptic plasticity, and neurological disorders. Recent studies have revealed a sophisticated interplay between mGluRs and protein kinases: activation of mGluRs regulates the activity of a number of kinases, and direct phosphorylation of mGluRs affects receptor signaling, trafficking, and desensitization. Here we review the emerging literature on mGluR phosphorylation, signaling, and synaptic function.

Na؉ /H ؉ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na ؉ and HCO 3 ؊ absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/ NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.Maintenance of intracellular and systemic pH, Na ϩ concentration, and fluid volume is essential for maintaining the physiological status in cells and whole organisms (1, 2). First demonstrated almost 30 years ago (3), members of the mammalian Na ϩ /H ϩ exchanger (NHE) 2 family participate in the regulation of these parameters at both cellular and systemic levels. To date, nine NHE family members have been identified in mammalian cells with unique tissue distribution and functional properties (2). As a better characterized isoform, NHE3 is primarily found in the apical membrane of epithelial cells of the renal and gastrointestinal tracts, where it mediates transepithelial absorption of Na ϩ and HCO 3 Ϫ (2, 4). Lack of NHE3 activity impairs acid-base balance and extracellular fluid volume homeostasis (5).NHE3 is known to be regulated by a large variety of hormones, such as ␣-and ␤-adrenergic agonists, dopamine, parathyroid hormone, and angiotensin II via multiple signaling systems (6, 7), but the exact underlying mechanisms are still only partially understood. Nevertheless, it has been known for many years that acute regulation of NHE3 activity is linked to protein phosphorylation, as in the case of inhibition by cAMPdependent protein kinase A (PKA) (7,8). Subsequently, it has been demonstrated that adaptor proteins with PDZ (PSD-95/discs large/ ZO-1) domains play an important role in the cAMP-dependent inhibition of NHE3 in a number of systems (7, 9, 10). For example, EBP50 (also known as NHERF1) and E3KARP (also known as NHERF2, SIP-1, or TKA-1) were found to be necessary modular proteins that participated in the cAMP-dependent PKA phosphorylation of NHE3 by forming a multiprotein signaling complex (11,12).In recent years, there has been a growing interest in PDZ domains and modular proteins having PDZ domains. Best studied in the post-synaptic density (PSD) region of neurons, PDZ domain proteins have em...