Down regulation of yeast G protein-coupled receptors

In this research, totally 200 raw milk samples in different areas of Ankara were collected from various dairy plant. The isolated psychotrophic bacteria from the raw milk samples are the species of Pseudomonas spp., Acinetobacter spp., Alcaligenes and Aeromonas. Isolation of Pseudomonas and other gram(-) psychrotrophic bacteria types are determined as P. aeruginosa 11 (5.5%), P. putida 11 (5.5%), P. fluorescens biotype I 10 (5.0%), P. fluorescens biotype II 4 (2.0%), P. fluorescens biotype III 6 (3.0%), P. aurefaciens 2 (1.0%), P. pseudomallei 3 (1.5%), P. cepacia 1 (0.5%), A. calcoaceticus lowffii 5 (2.5%), A. calcoaceticus anitratum 4 (2.0%), A. faecalis 3 (1.5%) and A. hydrophilia 1 (0.5%).     

A maize Myb homolog is encoded by a multicopy gene complex

Assumed oxygen consumption (VO2) is increasingly used as a convenient surrogate for measured VO2 for calculation of cardiac output. This substitution is often based on empirical formulae, previously validated only in relatively young patients. To assess the inaccuracy introduced by extrapolating these formulae to older patients, we compared measured VO2 with assumed VO2 in 57 patients. VO2 was measured using an open circuit analyzer. Assumed VO2 was calculated according to the LaFarge or Bergstra formulae. Agreement between both methods was assessed according to the method of Bland and Altman. The mean difference of measured VO2 minus assumed VO2 was 7.9 ml/min/m2 (P < 0.02) using the LaFarge formula, and -15.6 ml/min/m2 (P < 0.0002) using the Bergstra formula across a range of measured VO2 from 70 to 176 ml/min/m2. A systematic error was introduced by assumed VO2 from both formulae of underestimating higher and overestimating lower values of VO2, resulting in poor overall agreement with measured VO2. The same error and poor agreement was found when analyzing subgroups of patients > or =60 or <70 years of age. In summary, use of assumed VO2 introduces large, unpredictable errors in adult patients, suggesting requirement for measurement of VO2 when calculating cardiac output.     

Binding and phosphorylation of tubulin by G protein-coupled receptor kinases

Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During our attempts to identify novel betaARK interacting proteins, we found that the cytoskeletal protein tubulin could specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrated that betaARK and G protein-coupled receptor kinase-5 (GRK5) were able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of betaARK with tubulin from brain tissue; (ii) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (iii) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells. In addition, GRK-phosphorylated tubulin was found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function.     

Molecular mechanisms of G protein-coupled receptor signaling: role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization

Dynamic regulation of G protein-coupled receptor signaling demands a coordinated balance between mechanisms leading to the generation, turning off and re-establishment of agonist-mediated signals. G protein-coupled receptor kinases (GRKs) and arrestin proteins not only mediate agonist-dependent G protein-coupled receptor desensitization, but also initiate the internalization (sequestration) of activated receptors, a process leading to receptor resensitization. Studies on the specificity of beta-arrestin functions reveal a multiplicity of G protein-coupled receptor endocytic pathways and suggest that beta-arrestins might serve as adaptors specifically targeting receptors for dynamin-dependent clathrin-mediated endocytosis. Moreover, inactivation of the GRK2 gene in mice has lead to the discovery of an unexpected role of GRK2 in cardiac development, further emphasizing the pleiotropic function of GRKs and arrestins.     

A rat G protein-coupled receptor selectively expressed in myelin-forming cells

We have examined the role of the p75 neurotrophin receptor in survival-promoting effects of nerve growth factor (NGF) and neurotrophin-3 (NT-3) on cultured Purkinje cells. Previously, we showed that NGF promotes Purkinje cell survival in conjunction with (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), an agonist of metabotropic excitatory amino acid receptors, whereas NT-3 by itself increases cell number. We now present evidence that p75 plays different roles in Purkinje cell responses to the two neurotrophins. A metabotropic receptor of the mGluR1 subtype may interact with p75 function, so as to regulate Purkinje cell responsiveness to neurotrophins. When cerebellar cultures were grown for 6 days in the presence of ACPD and a mutant form of NGF that does not bind to p75, no increase in Purkinje cell number was observed. Moreover, the survival-promoting effect of wild-type NGF and ACPD could be inhibited by a neutralizing antiserum to p75 or by a pyrazoloquinazolinone inhibitor of neurotrophin binding to p75. In contrast, the response to NT-3 was potentiated by anti-p75 treatment and by the quinazolinone. These data indicate the mediation of p75 in the trophic response to NGF-ACPD and a negative modulatory role of p75 in the action of NT-3. To probe the role of ACPD in the p75-dependent response to NGF, metabotropic receptor subtype-specific ligands were tested. The pattern of agonist specificity implicated the mGluR1 subtype, a receptor that is expressed at high levels by Purkinje cells and linked to activation of protein kinase C (PKC). Down-regulation or blockade of PKC abolished the response to NGF-ACPD. Consistent with the opposite roles of p75 in effects of the two neurotrophins, blockade of mGluR1 or PKC potentiated the survival response elicited by NT-3. In sum, our data suggest that afferent excitatory transmitters activate specific metabotropic receptors to elicit a p75-mediated action of NGF. NT-3 acts on Purkinje cells by a different mechanism that is not absolutely p75-dependent and that is reduced by neurotrophin access to p75 and metabotropic receptor activity.     

Inhibition of G protein-coupled receptor kinase subtypes by Ca2+/calmodulin

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous betaARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.     

Molecular mechanisms of G protein-coupled receptor desensitization and resensitization

Beta-arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, beta-arrestins were also shown to be critical determinants for beta2-adrenergic receptor (beta2AR) resensitization. This was demonstrated by overexpressing wild-type beta-arrestins to rescue the resensitization-defect of a beta2AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative beta-arrestin inhibitor of beta2AR sequestration to impair beta2AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the beta2AR to resensitize in different cell types was shown to be dependent upon beta-arrestin expression levels. To further study the mechanisms underlying beta-arrestin function, green fluorescent protein was coupled to beta-arrestin2 (beta arr2GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of beta-arrestin in living cells. Beta arr2GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. Beta arr2GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because beta-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, beta arr2GFP provides a valuable new tool to dissect the biological function and regulation of beta-arrestin proteins.     

Factors to consider in the naming of a G protein-coupled receptor subtype

Receptors that mediate their effects through G proteins are predicted to have a seven transmembrane domain architecture. The last few years have seen a remarkable increase in the cloning of members of this superfamily leading to the identification of many more receptors than previously thought to exist on the basis of differences in agonist and antagonist specificities. This has important implications for nomenclature and classification, especially in view of the difficulty in relating receptors identified through cloning techniques to endogenously expressed receptors. Receptor cloning has also identified important differences in receptors between species raising the question as to whether these should be considered as species homologues or distinct subtypes. It is also becoming increasingly apparent that the pharmacology of this superfamily of receptors is influenced by the nature of the G protein present in the host cell and by alternative splicing of the receptor. The rapid pace of developments in this area necessitate the need for a regular publication summarizing recent developments. In the future, the cloning of G protein-coupled receptors will enable rationalization of the naming of individual receptor subtypes and identification of their interrelationships.     

Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking

A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.     

Development of pig oocytes activated by stimulation of an exogenous G protein-coupled receptor

This study determined whether stimulation of a G protein-coupled receptor could initiate the events that occur at fertilization in pig oocytes and, if so, whether the activated oocytes were competent to form blastocysts. After maturation for 30 h, oocytes received microinjections of mRNA encoding the rat M1 muscarinic receptor, a G protein-coupled acetylcholine (ACh) receptor. Oocytes were then incubated for an additional 15 h to complete maturation of oocytes and translation of microinjected mRNA, and they were subsequently cultured in the presence of ACh. ACh treatment of these oocytes triggered pronuclear formation (50.4%) as well as cortical granule exocytosis. SDS-PAGE showed that mRNA-microinjected oocytes treated with ACh were activated (61.1%), as characterized by the appearance of the 22-kDa polypeptide derived from dephosphorylation of the 25-kDa precursor. Furthermore, after being cultured in a ligated pig oviduct for 6 days, 17.4% of treated oocytes developed to the compact morula or blastocyst stage. Transmission electron microscopy revealed that blastocysts recovered from ligated oviducts contained reticulated nucleoli with fibrillar cores surrounded by fibrillar and granular components. In addition, mitochondria in the blastocysts were dispersed throughout the cytoplasm and contained numerous transverse cristae. These results show that pig oocyte activation mediated by a G protein-coupled signal transduction system can signal a series of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathway showed preimplantation development consistent with normal development.     

Regulation of G protein-coupled receptor kinase 5 (GRK5) by actin

G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806-3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.     

Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin

Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.     

A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lpA1/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [3H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.     

beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.     

Internalization of G protein-coupled receptors in single olfactory receptor neurons

The cardiovascular effects of human albumin (Alb) and three human hemoglobin (Hb) solutions, dextran-benzene-tetracarboxylate Hb, alphaalpha-crosslinked Hb, and o-raffinose-polymerized Hb were compared in anesthetized rabbits undergoing acute isovolemic hemodilution with Hct reduction from 41.4 +/- 2.7 to 28.8 +/- 1.6%. The impact of the vasoconstricting properties of Hb was examined by measuring heart rate (HR), mean arterial pressure (MAP), abdominal aortic, and femoral arterial blood flow, vascular resistance (VR), and aortic distension during the first 3 h after hemodilution. The impact of the hemorheological parameters was assessed by measurements of hemodiluted blood viscosity. In contrast to Alb, the Hb solutions elicited an immediate increase in MAP (20-38%). The effects of Alb and Hb solutions on HR, as well as on aortic and femoral arterial blood flow, were similar. VR decreased with Alb (20-28%) and increased with all three Hb solutions (30-90%), but the MAP and VR rising trends were different with each Hb solution. Aortic distension decreased in Hb groups compared with the Alb group for the first 60 min. The viscosity of hemodiluted blood was similar for all groups at high shear rates but was dependent on the viscosity of the solutions at low shear rates. We conclude that the vasoconstriction elicited by the Hb solutions overrides the vasodilation associated with viscosity changes due to hemodilution and would be the major factor responsible to the cardiovascular changes.     

Transgenic manipulation of myocardial G protein-coupled receptors and receptor kinases

The rare folate-sensitive, fragile sites on chromsomes X, 11, and 16 contain blocks of CCG triplet repeats and large expansions of the CCG block at the FRAXA site produce the fragile X syndrome (FraX). The fragile, poorly staining nature of these sites suggested an altered chromatin structure. Here, repeating CCG DNAs from FraX patients were tested for their ability to assemble into nucleosomes, the basic subunits of chromatin, using in vitro nucleosome reconstitution, electron microscopy and competitive assembly gel retardation assays. CCG blocks of >50 repeats displayed strong nucleosome exclusion, providing a possible explanation for the nature of these sites.     

A DNA damage and stress inducible G protein-coupled receptor blocks cells in G2/M

Cell cycle progression is monitored by highly coordinated checkpoint machinery, which is activated to induce cell cycle arrest until defects like DNA damage are corrected. We have isolated an anti-proliferative cell cycle regulator named G2A (for G2 accumulation), which is predominantly expressed in immature T and B lymphocyte progenitors and is a member of the seven membrane-spanning G protein-coupled receptor family. G2A overexpression attenuates the transformation potential of BCR-ABL and other oncogenes, and leads to accumulation of cells at G2/M independently of p53 and c-Abl. G2A can be induced in lymphocytes and to a lesser extent in nonlymphocyte cell lines or tissues by multiple stimuli including different classes of DNA-damaging agents and serves as a response to damage and cellular stimulation which functions to slow cell cycle progression.     

The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the alpha factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.