Umami is one of the 5 basic taste qualities. The umami L-glutamate can be drastically enhanced by 5' ribonucleotides and synergy a hallmark this quality. receptor heteromeric complex 2 class C G-protein-coupled receptors, T1R1 T1R3. Here we elucidate molecular mechanism using chimeric T1R site-directed mutagenesis, modeling. We propose cooperative ligand-binding model involving Venus flytrap domain T1R1, where binds close to hinge region, bind an adjacent site opening further stabilize...

In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It still unclear whether relevant for regular islet physiology and hormone secretion. Here, we show that human cells produce secrete when stimulated increases in glucose concentration. Serotonin secretion from decreases cyclic AMP (cAMP) levels neighboring alpha via 5-HT1F receptors inhibits glucagon Without serotonergic...

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous receptors (GPCRs), ultimately resulting in desensitization signaling. Until recently, GPCRs were considered to be only natural substrates for GRKs. However, recent discovery that GRKs also tubulin raised possibility additional GRK exist cellular role may much broader than just GPCR regulation. Here we report synucleins are a novel class substrates. Synucleins (α, β, γ,...

G protein-coupled receptor kinases (GRKs) are well characterized regulators of receptors, whereas protein signaling (RGS) proteins directly control the activity α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) <i>Curr. Biol.</i> 6, 211–212) identified region within N terminus GRKs that contained homology to RGS domains. Given domains demonstrate AlF₄⁻-dependent binding subunits, we tested ability from...

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these proteins and CaM include alterations within their second EF-hand loop that render motifs inactive Ca(2+) coordination fact central alpha-helixes are extended by one alpha-helical turn. CaBP1 CaBP2 contain consensus for N-terminal myristoylation, similar recoverin fatty acid acylated vitro. The...

The α1B-adrenergic receptor (α1BAR), its truncated mutant T368, different G protein-coupled kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or various combinations. Coexpression of β-adrenergic kinase (βARK) 1 (GRK2) 2 (GRK3) could increase epinephrine-induced phosphorylation the wild type α1BAR above basal as compared to that alone. On other hand, overexpression dominant negative βARK (K220R) impaired agonist-induced receptor. Overexpression...

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 GRK5 can be phosphorylated either or inhibited, respectively, by protein kinase C. Here we demonstrate calmodulin, another mediator calcium signaling, is a potent inhibitor GRK activity with selectivity for (IC50 ∼50 nm) > GRK6 ≫ ∼2 μm) GRK1. Calmodulin inhibition mediated via...

Earlier, a family of G protein-coupled receptors, termed T2Rs, was identified in the rodent and human genomes through data mining. It suggested that these receptors mediate bitter taste perception. Analysis genome revealed hT2R is composed 25 members. However, ligands have been for only three so far. Here we report identification two novel ligand-receptor pairs. hT2R61 activated by 6-nitrosaccharin, derivative saccharin. hT2R44 denatonium 6-nitrosaccharin. Activation profiles correlate with...

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each these subunits comprises a family proteins. The rules for association between members one with another to form multimer not known; it is clear whether associations specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified contain more subtype beta gamma subunits. Gt alpha subunit associated only 1 1. It known this...

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the hormone-occupied form of numerous receptors, ultimately resulting in termination signaling. While little is presently known about regulation GRK function, recent studies suggest a role for protein kinase C (PKC) phosphorylation beta-adrenergic membrane association activation kinase. To assess potential general PKC regulating we characterized ability to GRK5, recently identified member family. We...

Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation neuroinflammation in the retina. Glial release pro-inflammatory cytokines TNFα IL-1β, complement, other danger factors was shown to facilitate injury loss retinal ganglion cells (RGCs) that send visual information brain. However, cellular events linking neurotoxicity remain poorly characterized. Several signaling pathways, including P2X7 receptors Panx1 channels, are known activate...

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated agonist-dependent phosphorylation GPCR kinases (GRKs), such as GRK2, resulting in protein/receptor uncoupling. GRK2 also regulates signaling binding Gαq/ll inhibiting Gαq stimulation of the effector phospholipase Cβ. The site for resides within...

Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G interaction, the C-terminal tail G gamma subunit, masked in beta complex, exposed establishes high-affinity contact with receptor. This potential conformational switch provides a mechanism to regulate coupling. may also be significant for role complex regulation effector function.

Olfactory receptors (ORs), the largest family of G protein–coupled receptors, are expressed in nasal epithelium where they mediate sense smell. However, ORs also found other non-nasal tissues, but role these ectopic cell signaling, proliferation, and survival is not well understood. Here, using an inducible expression system lymph node carcinoma prostate (LNCaP) line, we investigated two ORs, OR51E1 OR51E2, which have been shown to be upregulated cancer. We that, consistent with previous...

The use of pharmacological inhibitors protein kinases represents a potentially powerful tool in dissecting the regulatory features intracellular signaling pathways. However, although vitro potency, selectivity, and efficacy numerous kinase have been characterized, little is known regarding usefulness these compounds as intact cells. In attempting to characterize role A (PKA) regulating beta-2 adrenergic receptor (AR) human airway cells, we observed seemingly profound capacity isoquinoline...

We studied the function of G protein-coupled receptor kinases (GRKs) in regulation thrombin-activated signaling endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly The these was by expressing wild-type dominant negative (dn) mutants determined responses to thrombin, which activates intracellular cells cleaving NH 2 terminus proteinase-activated receptor-1 (PAR-1). measured changes phosphoinositide hydrolysis Ca 2+ concentration ([Ca ] i ) response thrombin as well...

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied receptors at the inner surface of plasma membrane (PM), leading to desensitization. GRKs utilize a variety mechanisms bind tightly, and sometimes reversibly, cellular membranes. Previous studies demonstrated presence binding domain in C terminus GRK5. Here we define mechanism by which this short C-terminal stretch amino acids GRK5 mediates PM localization. Secondary structure predictions suggest that region...

Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P₁) is increased in response to either SSP phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated stimulated accumulation phosphoserine and phosphothreonine phosphotyrosine. An inhibitor PMA-stimulated EDG1 failed block SSP-stimulated phosphorylation. Additionally, removal 12 amino...

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth classified as either full or partial muscarinic agonist. Here, we report several unexpected results pertaining its interaction with M3 receptor (M3R). We found that pilocarpine was 1000 times less potent in stimulating mouse-eye pupil constriction than agonists oxotremorin-M (Oxo-M) carbachol (CCh), although all three ligands have similar Kd values for M3R. In contrast CCh Oxo-M, does not induce Ca2+ mobilization via...

The functionality and benefits of Klipper, a firmware for 3D printers, are analysed to determine its effectiveness as software solution printing control. Klipper algorithms such ‘look-ahead’, ‘minimum cruise ratio’ ‘input shaping’, which improve print quality by reducing vibration more accurately controlling head movement, analysed. An integrated system is described that includes flexible settings high productivity motion optimisation algorithms.

Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of receptor kinases (GRKs) that specifically target the activated form receptor. Recent studies have revealed GRKs also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited protein kinase C phosphorylation Ca2+-calmodulin binding. binding promotes autophosphorylation, which further contributes inhibition. In this study we identify two important structural domains in...

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism GRK regulation. While GRK1 (rhodopsin kinase) is inhibited the photoreceptor-specific protein recoverin, other GRKs can be Ca2+−calmodulin. To dissect this inhibition at molecular level, we localized domains involved in interaction using series GST−GRK fusion proteins. GRK1, GRK2, and GRK5, which represent three known subclasses, were each found to possess two distinct...