DYNLT1 gene expression is downregulated in whole blood of patients at different Huntington's disease stages.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG nucleotide expansion, which encodes the amino acid glutamine, in the huntingtin gene. HD is characterized by motor, cognitive, and psychiatric dysfunctions. In a previous study, we showed by qPCR that some genes altered in an HD mouse model were also altered in blood of HD patients. These alterations were mainly with respect to the dynein family. Therefore, this study aimed to investigate whether dynein light chain Tctex type 1 (DYNLT1) is altered in HD patients and if there is a correlation between DYNLT1 gene expression changes and disease progression. We assessed the DYNLT1 gene expression in the blood of 19 HD patients and 20 healthy age-matched controls. Also, in 6 of these patients, we analyzed the DYNLT1 expression at two time points, 3 years apart. The DYNLT1 gene expression in the whole blood of HD patients was significantly downregulated and this difference was widened in later stages. These data suggest that DYNLT1 could emerge as a peripheral prognostic indicator in HD and, also, might be a target for potential intervention in the future.

Biased agonism of the angiotensin II type 1 receptor.

G protein-coupled receptors (GPCRs) can be activated by multiple ligands and exhibit the capacity to couple to numerous intracellular signal transduction pathways. This property allows GPCRs to be modulated by biased agonists that selectively activate specific subsets of GPCR-regulated cellular signaling proteins. The angiotensin II type 1 receptor (AT1R) is a GPCR that endogenously binds to the peptide ligand angiotensin II. More recently it has been demonstrated that a modified peptide, [Sar1I-le4-Ile8]-angiotensin II (SII) acts as a biased agonist towards the AT1R. SII binds to the AT1R without promoting heterotrimeric G protein-coupling, but serves to link the receptor to the beta-arrestin-dependent activation of the mitogen activated protein kinase pathway. The present mini-review summarizes current knowledge regarding the role of biased agonists in stimulating biased AT1R signaling.

Rapid, transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter.

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.

Induction of hepatic cytochrome P450 enzymes: methods, mechanisms, recommendations, and in vitro-in vivo correlations.

Induction of drug-clearance pathways (Phase 1 and 2 enzymes and transporters) can have important clinical consequences. Inducers can (1) increase the clearance of other drugs, resulting in a decreased therapeutic effect, (2) increase the activation of pro-drugs, causing an alteration in their efficacy and pharmacokinetics, and (3) increase the bioactivation of drugs that contribute to hepatotoxicity via reactive intermediates. Nuclear receptors are key mediators of drug-induced changes in the expression of drug-clearance pathways. However, species differences in nuclear receptor activation make the prediction of cytochrome P450 (CYP) induction in humans from data derived from animal models problematic. Thus, in vitro human-relevant model systems are increasingly used to evaluate enzyme induction. In this review, the authors' current understanding of the mechanisms of enzyme induction and the in vitro methods for assessing the induction potential of new drugs will be discussed. Relevant issues and considerations surrounding proper study design and the interpretation of in vitro results will be discussed in light of the current US Food and Drug Administration (FDA) recommendations.

Analysis of a missense variant of the human N-formyl peptide receptor that is associated with agonist-independent beta-arrestin association and indices of inflammation.

Formyl-Met-Leu-Phe (fMLP) is a potent chemoattractant molecule released from both bacteria and damaged mitochondria that activates fMLP receptors (FPR) leading to neutrophil chemotaxis, degranulation and superoxide production. A common missense single nucleotide polymorphism in the human FPR1 gene at nucleotide c.32C>T results in the amino-acid substitution, p.I11T, in the FPR1 extracellular amino-terminus. The minor (c.32T) allele frequencies were 0.25, 0.27, 0.25, 0.15 and 0.14 in healthy Caucasian, African, East Indian, Chinese and Native Canadian individuals, respectively. In subjects homozygous for the p.T11 allele, we find elevated serum concentrations of C-reactive protein, increased absolute counts of blood leukocytes and neutrophils, and erythrocyte sedimentation rates. When expressed in HEK 293 and RBL-2H3 cells a substantial proportion of FPR1 p.I11T variant is retained intracellularly and agonist-independent internalization of the FPR1 p.I11T variant, but not the wild-type FPR1, is constitutively associated with beta-arrestin2-GFP in vesicles. Moreover, basal N-acetyl-D-glucosaminidase release is increased in primary neutrophils isolated from subjects either heterozygous or homozygous for the FPR1 p.T11 allele. Taken together, the data suggest an increased receptor activity and phenotypic expression of increased inflammatory indices in subjects with the p.T11 allele.

Small GTP-binding protein-coupled receptors.

Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins, GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways. The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42, Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range 20-30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition, RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs, and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling and intracellular trafficking.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

Agonist-stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells: agonist-stimulated endocytosis is beta-arrestin1 isoform-specific.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that contribute to the regulation of integrative brain functions such as cognition, motor control, and neural development. Metabotropic glutamate receptors are members of a unique class of GPCRs (class III) that include the calcium sensing and gamma-aminobutyric acid type B receptors. Although mGluRs bear little sequence homology to well-characterized members of the GPCR superfamily, both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) contribute to mGluR desensitization. Therefore, in the present study, we examined whether beta-arrestins, regulators of GPCR desensitization and endocytosis, are required for mGluR1a desensitization and internalization in human embryonic kidney (HEK) 293 cells. Unlike what has been reported for other GPCRs, we find that in response to agonist stimulation, mGluR1a internalization is selectively mediated by beta-arrestin1 in HEK 293 cells. However, even though beta-arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither beta-arrestin1 nor beta-arrestin2 seems to contribute to mGluR1a desensitization in HEK 293 cells. We also observed extensive tonic mGluR1a internalization via clathrin-coated vesicles in the absence of agonist. The tonic internalization of mGluR1a is insensitive to antagonist treatment, dominant-negative mutants of GRK2, beta-arrestin1, and dynamin as well as treatments that disrupt caveolae, but is blocked by hypertonic sucrose and concanavalin A treatment. Internalized mGluR1a is colocalized with clathrin, transferrin receptor, beta2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with beta-arrestin in response to agonist, the agonist-independent internalization of mGluR1a involves the beta-arrestin-independent targeting of mGluR1a to clathrin-coated vesicles.

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line.

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations: protein kinase C-dependent receptor phosphorylation is not required.

The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via G(q) to the hydrolysis of phosphoinositides, the release of Ca(2+) from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca(2+) concentrations. The mGluR1/5-stimulated Ca(2+) oscillations are translated into the synchronized repetitive redistribution of PKCbetaII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca(2+), and PKCbetaII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCbetaII oscillations. Furthermore, oscillations in Ca(2+) continued in the presence of PKC inhibitors, which blocked PKCbetaII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase.

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.

Acute hypoglycemia impairs the functioning of the central but not peripheral nervous system.

Acute hypoglycemia impairs functions of the central nervous system, but few controlled studies have assessed the impact of hypoglycemia on the function of the peripheral nervous system. Sixteen non-diabetic humans underwent two separate hyperinsulinemic glucose clamp procedures on different study days, in a counter-balanced fashion. On one occasion, euglycemia was maintained (blood glucose, 5.0 mmol l(-1)), and on the other occasion, hypoglycemia (blood glucose, 2.6 mmol l(-1)) was induced. During each condition, subjects performed a combined psychometric, cognitive-experimental and psychophysical test battery, and measures were made (in the dominant median and common peroneal nerves) of the motor nerve conduction velocities and the amplitudes of the motor action potentials. Hypoglycemia caused impaired performance of general cognitive and information processing tasks (P<.05), but nerve conduction velocities and the amplitudes of motor action potentials were unaffected. Conduction velocities of the common peroneal nerve decreased from baseline within each experimental condition, perhaps due to hyperinsulinemia. Overall, these results demonstrate that multiple levels of information processing in the brain may alter while peripheral nerve function remains intact, and imply that peripheral neurons do not have the same obligate requirement for glucose as a metabolic fuel as neurons of the central nervous system.

Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling.

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.

Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors.

Beta-arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. Beta-arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT1AR), but not beta2-adrenergic receptor (beta2AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a beta2AR bearing the AT1AR tail acquired the capacity to stimulate beta-arrestin redistribution to endosomes, whereas this property was lost for an AT1AR bearing the beta2AR tail. Using beta2AR/AT1AR chimeras, we tested whether the beta2AR and AT1AR carboxyl-terminal tails, in part via their association with beta-arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that beta-arrestin formed a stable complex with the AT1AR tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the beta2AR chimera bearing the AT1AR tail was observed in the absence of agonist and was inhibited by a dominant-negative beta-arrestin1 mutant. Agonist-independent AT1AR internalization was also observed after beta-arrestin2 overexpression. After internalization, the beta2AR, but not the AT1AR, was dephosphorylated and recycled back to the cell surface. However, the AT1AR tail prevented beta2AR dephosphorylation and recycling. In contrast, although the beta2AR-tail promoted AT1AR recycling, the chimeric receptor remained both phosphorylated and desensitized, suggesting that receptor dephosphorylation is not a property common to all receptors. In summary, we show that the carboxyl-terminal tails of GPCRs not only contribute to regulating the patterns of receptor desensitization, but also modulate receptor intracellular trafficking and resensitization patterns.

The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression.

The chemokine stromal derived factor-1alpha (SDF-1alpha) has been implicated recently in the chemotaxis of primitive human hematopoietic cells, suggesting that pluripotent human stem cells express the SDF-1alpha receptor, CXCR4. By using flow cytometry and confocal microscopy, we have identified and isolated primitive subsets of human CXCR4(+) and CXCR4(-) cells. Distinctions in the progenitor content and response to SDF-1alpha in vitro indicate that CXCR4(+) and CXCR4(-) cells represent discrete populations of primitive blood cells. The i.v. transplantation of these subfractions into immune-deficient mice established that both possess comparable engraftment capacity in vivo. Human myeloid, lymphoid, and primitive CD34(+) CXCR4(+) cells were present in chimeric animals transplanted with either subset, indicating that CXCR4(+) and CXCR4(-) stem cells have equivalent proliferative and differentiative abilities. Our study indicates that the human stem cell compartment is heterogeneous for CXCR4 expression, suggesting that the relationship between CXCR4 expression and stem cell repopulating function is not obligatory.

G protein-coupled receptor kinase-mediated desensitization of metabotropic glutamate receptor 1A protects against cell death.

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.

beta 2-adrenergic receptor internalization, endosomal sorting, and plasma membrane recycling are regulated by rab GTPases.

Rab GTPases are recognized as critical regulatory factors involved in vesicular membrane transport and endosomal fusion. For example, Rab5 directs the transport and fusion of endocytic vesicles to and with early endosomes, whereas Rab4 is thought to control protein trafficking from early endosomes back to the plasma membrane. In the present study, we investigated the role of Rab5 and Rab4 GTPases in regulating the endocytosis, intracellular sorting, and the plasma membrane recycling of the beta(2)AR. In cells expressing the dominant-negative Rab5-S34N mutant, beta(2)AR internalization was impaired, and beta(2)AR-bearing endocytic vesicles remained in either close juxtaposition or physically attached to the plasma membrane. In contrast, a constitutively active Rab5-Q79L mutant redirected internalized beta(2)AR to enlarged endosomes but did not prevent beta(2)AR dephosphorylation and recycling. The expression of either wild-type Rab4 or a Rab4-N121I mutant did not prevent beta(2)AR dephosphorylation. However, the dominant-negative Rab4-N121I mutant blocked beta(2)AR resensitization by blocking receptor recycling from endosomes back to the cell surface. Our data indicate that, in addition to regulating the intracellular trafficking and fusion of beta(2)AR-bearing endocytic vesicles, Rab5 also contributes to the formation and/or budding of clathrin-coated vesicles. Furthermore, beta(2)AR dephosphorylation occurs as the receptor transits between Rab5- and Rab4-positive compartments.

G-Protein-coupled receptor kinase activity in hypertension : increased vascular and lymphocyte G-protein receptor kinase-2 protein expression.

Impaired receptor-stimulated adenylyl cyclase activation has been observed in lymphocytes from hypertensive subjects and has been linked to an increase in lymphocyte G-protein receptor kinase-2 (GRK-2) protein expression. However, whether the increase in lymphocyte GRK-2 reflected an increase in vascular GRK-2 was unknown. Therefore, we compared GRK-2 protein expression in lymphocytes and aortas obtained from normotensive Wistar rats, Wistar-Kyoto rats (WKY), and spontaneously hypertensive rats (SHR) and from aortas of Dahl rats. Impaired beta-adrenergic responsiveness was observed in lymphocytes and vascular tissues obtained from hypertensive SHR (10 and 15 weeks old) but not in those obtained from prehypertensive SHR (5 weeks old). Immunodetectable lymphocyte GRK-2 protein expression was increased in 10-week-old SHR (143+/-10% of the expression in 10-week-old Wistar rats and 131+/-11% of the expression in 10-week-old WKY, n=5 in each group). Immunodetectable vascular smooth muscle cell GRK-2 was comparably increased (169+/-14% of the expression in Wistar rats and 138+/-7% of the expression in WKY, n=5 in each group). Also, in hypertensive Dahl salt-sensitive rats, vascular GRK-2 protein expression was increased (185+/-14% of the expression in Dahl salt-resistant rats, n=5 in each group) compared with Dahl salt-resistant controls. These studies support a generalized defect in vascular GRK-2 protein expression in hypertension, which could be an important factor in the impairment of beta-adrenergic-mediated vasodilation, characteristic of the hypertensive state.

Regulation of tyrosine kinase activation and granule release through beta-arrestin by CXCRI.

Chemoattractant-stimulated granule release from neutrophils, basophils and eosinophils is critical for the innate immune response against infectious bacteria. Interleukin 8 (IL-8) activation of the chemokine receptor CXCRI was found to stimulate rapid formation of beta-arrestin complexes with Hck or c-Fgr. Formation of beta-arrestin-Hck complexes led to Hck activation and trafficking of the complexes to granule-rich regions. Granulocytes expressing a dominant-negative beta-arrestin-mutant did not release granules or activate tyrosine kinases after IL-8 stimulation. Thus, beta-arrestins regulate chemokine-induced granule exocytosis, indicating a broader role for beta-arrestins in the regulation of cellular functions than was previously suspected.

Nuclear localization of the 82-kDa form of human choline acetyltransferase.

Choline acetyltransferase is the enzyme catalyzing synthesis of the neurotransmitter acetylcholine in cholinergic neurons. In human, transcripts encoding two forms of the enzyme with apparent molecular masses of 69 and 82 kDa are found in brain and spinal cord; the 82-kDa form differs from the 69-kDa enzyme only in terms of a 118-amino acid extension on its amino terminus. Using green fluorescent protein-tagged choline acetyltransferase, we show that the 82-kDa enzyme is targeted to nuclei of cells, whereas the 69-kDa protein is found in cytoplasm. Expression of site-directed and deletion mutants of the 82-kDa isoform reveals that the extended amino terminus contains a nuclear localization signal in the first nine amino acids which targets the protein to nucleus. This represents the first report of a neurotransmitter-synthesizing enzyme that is localized to the cell nucleus.