Five members of a novel Ca2؉ -binding protein subfamily (CaBP), with 46 -58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca 2؉ -binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca 2؉ coordination and the fact that their central ␣-helixes are extended by one ␣-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca 2؉ -mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.Among organisms as diverse as yeast and human, changes in the intracellular Ca 2ϩ ion concentration initiate an array of signaling pathways. Ca 2ϩ ions function as a diffusible signal that exerts its effect directly or through Ca 2ϩ -binding proteins on plasma membrane and intracellular channels, intracellular proteins involved in membrane trafficking, and a broad range of enzymes, including kinases, phosphatases, and adenylyl cyclases. Ca 2ϩ -binding proteins sense changes in [Ca 2ϩ ] through either 130-amino acid (aa) 1 structural elements called C2 domains, 29-aa EF-hand motifs, or through acidic regions of proteins or protein-lipid interfaces. In a growing number of eukaryotic signaling proteins, C2 and EF-hand motifs are present as either a single copy or clustered in multiple copies (1).The largest group of Ca 2ϩ -binding proteins belongs to the calmodulin (CaM) superfamily. They are structurally related and comprise four EF-hand motifs, some of which (one or two) may be nonfunctional in Ca 2ϩ coordination (2). Neuronal Ca 2ϩ -binding proteins (NCBP) are a subset of the EF-hand-containing proteins, whose function is largely unknown. The sequence similarity among members of the NCBP family varies from ϳ25% between CaM and visinin to ϳ60% between GCAP1 and GCAP3 (3). NCBPs are acidic and similar in length. CaM and CaM-like proteins are the shortest (149 -150 aa; molecular mass, 16,837 Da); other members of this family are ϳ200 aa long (molecular mass, ϳ23,000 Da) (2).NCBPs also display a variety of interesting structural features. Multifunctional CaM contains a pair of N-terminal (EFhand 1 and EF-hand 2) and C-terminal EF-hand (EF-hand 3 and EF-ha...

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (␣, ␤, ␥, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. ␣-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the ␣ and ␤ isoforms, whereas GRK5 prefers ␣-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and G␤␥ subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.G protein-coupled receptor kinases (GRKs) 1 are involved in the regulation of G protein-coupled receptor (GPCR) signaling (1, 2). GRKs specifically recognize and phosphorylate agonistoccupied GPCRs. Receptor phosphorylation and subsequent binding of another protein, arrestin, uncouples activated receptor from G protein. These events can also promote receptor endocytosis. Internalized receptors are then either dephosphorylated and recycled back to the cell surface or targeted to lysosomes for degradation. The seven mammalian GRKs that have been identified can be divided into three subfamilies based on their overall structural organization and homology: GRK1 (rhodopsin kinase) and GRK7; GRK2 (␤ARK1) and GRK3 (␤ARK2); and GRK4, GRK5, and GRK6. Common features shared by the GRKs include a centrally localized catalytic domain of ϳ270 amino acids, an N-terminal domain of ϳ190 amino acids that has been implicated in receptor interaction and GRK regulation, and a variable length C-terminal domain of 105-233 amino acids that is involved in phospholipid association.Until recently, GPCRs were considered to be the only natural substrates for GRKs. Common protein kinase substrates, such as casein, pho...

Umami is one of the 5 basic taste qualities. The umami taste of L-glutamate can be drastically enhanced by 5 ribonucleotides and the synergy is a hallmark of this taste quality. The umami taste receptor is a heteromeric complex of 2 class C G-protein-coupled receptors, T1R1 and T1R3. Here we elucidate the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling. We propose a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region, and 5 ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This unique mechanism may apply to other class C G-protein-coupled receptors.glutamate ͉ G protein-coupled receptors ͉ IMP ͉ T1R H umans can detect at least 5 basic taste qualities, including sweet, umami, bitter, salty, and sour. Umami, the savory taste of L-glutamate, was first discovered in 1908 by K. Ikeda, but only recently accepted as a basic taste quality by the general public. The most unique characteristic of umami taste is synergism. Purinic ribonucleotides, such as IMP and GMP, can strongly potentiate the umami taste intensity (1). In human taste tests, 200 M of IMP, which does not elicit any umami taste by itself, can increase one's umami taste sensitivity to glutamate by 15-fold (2).Among the 5 basic taste qualities, sweet, umami, and bitter taste are mediated by G protein-coupled receptors (GPCRs) (3). Receptors for umami taste and sweet taste are closely related to each other. The 3 subunits of the T1R family form 2 heteromeric receptors: umami (T1R1/T1R3) (2, 4) and sweet (T1R2/T1R3) (2, 5). T1R receptors belong to the class C GPCRs, along with metabotropic glutamate receptors (mGluRs), ␥-aminobutyric acid receptor B (GABA B R), calcium sensing receptors (CaSR), and so forth. The defining motif in these receptors is an outer membrane N-terminal Venus flytrap (VFT) domain that consists of 2 globular subdomains, the N-terminal upper lobe and the lower lobe, that are connected by a 3-stranded flexible hinge. The VFT domain of C-GPCRs contains the ligand-binding site (6), as demonstrated by studies on mGluRs, GABA B R, and the sweet taste receptor (7). The crystal structures of mGluR VFT domains revealed that the bi-lobed architecture can form an open or closed conformation (8, 9). Glutamate binding stabilizes both the active dimer and the closed conformation. This scheme in the initial receptor activation has been applied generally to other C-GPCRs.Studies on sweet taste-receptor functional domains revealed multiple binding sites for its structurally diverse ligands. Using human-rat chimeric receptors, we demonstrated the T1R2 VFT domain of the human sweet receptor interacts with 2 structurally related synthetic sweeteners aspartame and neotame, while the transmembrane domain (TMD) of human T1R3 interacts with another sweetener cyclamate and a human sweet-taste inhibitor lactisole (7). Works from several other laborator...

؊ . This revealed the specific ability of bovine brain G␣ q/11 to bind to both GRK2 and GRK3 in an AlF 4 ؊ -dependent manner. In contrast, G␣ s , G␣ i , and G␣ 12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain G␣ q/11 could also bind to an N-terminal construct of GRK2, while no binding of G␣ q/11 , G␣ s , G␣ i , or G␣ 12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified G␣ q revealed significant binding of both G␣ q GDP/AlF 4 ؊ and G␣ q (GTP␥S), but not G␣ q (GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active G␣ q (R183C) but not wild type G␣ q . In vitro analysis revealed that GRK2 possesses weak GAP activity toward G␣ q that is dependent on the presence of a G proteincoupled receptor. However, GRK2 effectively inhibited G␣ q -mediated activation of phospholipase C-␤ both in vitro and in cells, possibly through sequestration of activated G␣ q . These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

Variation in human taste is a well-known phenomenon. However, little is known about the molecular basis for it. Bitter taste in humans is believed to be mediated by a family of 25 G protein-coupled receptors (hT2Rs, or TAS2Rs). Despite recent progress in the functional expression of hT2Rs in vitro, up until now, hT2R38, a receptor for phenylthiocarbamide (PTC), was the only gene directly linked to variations in human bitter taste. Here we report that polymorphism in two hT2R genes results in different receptor activities and different taste sensitivities to three bitter molecules. The hT2R43 gene allele, which encodes a protein with tryptophan in position 35, makes people very sensitive to the bitterness of the natural plant compounds aloin and aristolochic acid. People who do not possess this allele do not taste these compounds at low concentrations. The same hT2R43 gene allele makes people more sensitive to the bitterness of an artificial sweetener, saccharin. In addition, a closely related gene's (hT2R44's) allele also makes people more sensitive to the bitterness of saccharin. We also demonstrated that some people do not possess certain hT2R genes, contributing to taste variation between individuals. Our findings thus reveal new examples of variations in human taste and provide a molecular basis for them.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC 50 ϳ50 nM) > GRK6 > > GRK2 (IC 50 ϳ2 M) > > GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20 -39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity. G protein-coupled receptor kinases (GRKs)1 form a family of serine/threonine protein kinases with the unique ability to recognize specifically the agonist-activated state of G proteincoupled receptors (1, 2). GRK-mediated phosphorylation promotes the binding of an arrestin protein, thereby uncoupling the receptor from G protein and terminating receptor signaling. Six members of the GRK family have been identified, and based on their sequence homology they have been divided into three subfamilies (2). GRK1 (rhodopsin kinase) forms one group; GRK2 (␤-adrenergic receptor kinase) and GRK3 a second; and GRK4, GRK5, and GRK6 combine into a third subfamily.All GRKs share a similar structural organization with a poorly conserved amino-terminal domain of ϳ185 residues, a conserved protein kinase catalytic domain of ϳ270 residues, and a variable length carboxyl-terminal domain of 105-230 residues (3). However, although all GRKs have a similar overall structure and function, various subfamily members also have certain unique features. For example, various GRKs utilize different mechanisms to promote membrane association, an event critical for receptor interaction. GRK1 is farnesylated (4), GRK2 and 3 interact with phospholipids and G protein ␤␥ subunits via pleckstrin homology domains (5-8), GRK4 (9) and GRK6 (10) are palmitoylated, and GRK5 binds to phospholipids via polybasic regions in the amino-and carboxyl-terminal domains (11,12).Another characteristic that appears specific for the GRK subtype involves regulation of kinase activity. For example, in the visual system, GRK1 ha...

SUMMARY 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 is still unclear if serotonin is relevant for regular islet physiology and hormone secretion. Here we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cAMP levels in neighboring alpha cells via 5-HT1F receptors and inhibits glucagon secretion. Without serotonergic input, alpha cells lose their ability to regulate glucagon secretion in response to changes in glucose concentration, suggesting that diminished serotonergic control of alpha cells can cause glucose blindness and the uncontrolled glucagon secretion associated with diabetes. Supporting this model, pharmacological activation of 5-HT1F receptors reduces glucagon secretion and has hypoglycemic effects in diabetic mice. Thus, modulation of serotonin signaling in the islet represents a drug intervention opportunity.

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