A single phenylalanine residue in β-arrestin2 critically regulates its binding to G protein–coupled receptors

Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of β-arrestin2 (β-arr2) and blocks its association with activated G protein–coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of β-arr2–GPCR complexes. β-arr2 Phe116Ala mutant has negligible effect on blunting β₂-adrenergic receptor–induced cAMP generation unlike β-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6–activated forms of bovine β-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of β-arr2. Surprisingly, Phe116 is dispensable for the association of β-arr2 with its non-GPCR partners. β-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with β-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent β-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that β-arr2 Phe116Ala interaction with activated β₂-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of β-arr2, regulates the formation of β-arr2–GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent β-arr2 localization and trafficking.     

Structural Basis of Multifunctional Bovine Mitochondrial Cytochrome bc 1 Complex

The mitochondrial cytochrome bc ₁ complex is a multifunctional membrane protein complex. Itcatalyzes electron transfer, proton translocation, peptide processing, and superoxide generation.Crystal structure data at 2.9 Å resolution not only establishes the location of the redox centersand inhibitor binding sites, but also suggests a movement of the head domain of the iron–sulfurprotein (ISP) during bc ₁ catalysis and inhibition of peptide-processing activity during complexmaturation. The functional importance of the movement of extramembrane (head) domain ofISP in the bc ₁ complex is confirmed by analysis of the Rhodobacter sphaeroides bc ₁ complexmutants with increased rigidity in the ISP neck and by the determination of rate constants foracid/base-induced intramolecular electron transfer between [2Fe–2S] and heme c ₁ in nativeand inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovineheart mitochondrial bc ₁ complex by nonionic detergent at concentrations that inactivate electrontransfer activity. This peptide-processing activity is shown to be associated with subunits Iand II by cloning, overexpression and in vitro reconstitution. The superoxide-generation siteof the cytochrome bc ₁ complex is located at reduced b _L and Q^–. The reaction is membranepotential-, and cytochrome c-dependent.     

Conformation of the cecropin-melittin hybrid, cecropin A(1-8)-melittin (1-18) antibacterial Peptide

Cecropin A (1-8)-Melittin (1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. The primary sequence of the peptide is as follows: KWKLPKKIGIGAVLKVLTTGLPALIS-NH2. 1H and 13C 2D NMR techniques were used to deduce the conformational parameters of chemical shift, 3JNHalpha coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter-residue nOe's. NMR studies were carried out in water (pH 6.0) and hexafluoroacetone (HFA). The peptide was found in a beta-pleated structure in water, and in HFA it adopts a right-handed alpha-helix conformation. Solution structures generated using restrained molecular dynamics simulations were refined by Mardigras to R factors ranging from 0.5 to 0.6.     

Conformation of the peptide antibiotic — histatin 8 in aqueous and non aqueous media

Histatin 8 (Lys¹-Phe-His-Glu-Lys⁵-His-His-Ser-His-Arg¹⁰-Gly-Tyr¹²)belongs to a group of related neutral and basic histidine richpeptides present in human salivary secretions that possess fungicidal and bactericidal activities. The conformation of thispeptide has been examined by ¹H and ¹³C 2D-NMR in DMSO-d₆, water (pH 4.0) and 40% HFA solutions. MD simulations incorporating NMR data was used to generate the solution conformations. The structures were refined by MARDIGRAS employing the RANDMARDI approach. In both DMSO-d₆ and water, the peptide is seen to adopt a -pleated sheet, while HFA induces an -helix structure. The role of these structures in its mechanism of action has been explained.     

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.     

Differential spectrum of mutations that activate the Escherichia coli bgl operon in an rpoS genetic background

The bgl promoter is silent in wild-type Escherichia coli under standard laboratory conditions, and as a result, cells exhibit a beta-glucoside-negative (Bgl-) phenotype. Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS. Mutations that confer a Bgl+ phenotype arise spontaneously at a detectable frequency. Transposition of DNA insertion elements within the regulatory locus, bglR, constitutes the major class of activating mutations identified in laboratory cultures. The rpoS-encoded sigmaS, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions. In an attempt to see if the rpoS status of the cell influences the nature of the mutations that activate the bgl promoter, we analyzed spontaneously arising Bgl+ mutants in rpoS+ and rpoS genetic backgrounds. We show that the spectrum of activating mutations in rpoS cells is different from that in rpoS+ cells. Unlike rpoS+ cells, where insertions in bglR are the predominant activating mutations, mutations in hns make up the majority in rpoS cells. The physiological significance of these differences is discussed in the context of survival of natural populations of E. coli.     

Influence of putrescine on anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> mediated through calcium ATPase

The influence of Putrescine (Put) on the growth and elicitation of anthocyanin in callus cultures of Daucus carota var. Nantes scarlet was investigated through the use of α-DL-difluoromethylarginine (DFMA), the polyamine (PA) biosynthetic inhibitor. It was observed that the addition of Put (0.05 mM) resulted in enhancement of growth and anthocyanin content. The anthocyanin content was found to be enhanced by 1.68 fold on the 21^st day as compared to the untreated controls. The PA inhibitor was found to result in lowering of the growth and the anthocyanin accumulation, which could be partially restored by the addition of Put in combination with this inhibitor. The levels of Ca²⁺ ATPase were also found to be elevated in treatment with Put suggesting the involvement of calcium in the elicitation of anthocyanin. The endogenous titres of PAs and the ethylene production under these treatments were also studied. The treatment with DFMA resulted in lower levels of endogenous PAs and higher levels of ethylene. Lowering of ethylene by putrescine treatment shows that PA treatment also inhibited ethylene formation, which would also imply that endogenous ethylene does not influence anthocyanin production in carrot callus cultures.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.     

Kinetics and mechanism of protection of adenine from sulphate radical anion by caffeic acid under anoxic conditions

The rates of photo-oxidation of adenine in the presence of peroxydisulphate (PDS) have been determined by measuring the absorbance of adenine at 260.5 nm spectrophotometrically. The rates and the quantum yields (phi) of oxidation of adenine by sulphate radical anion (SO4(-)) have been determined in the presence of different concentrations of caffeic acid. Increase in the concentration of caffeic acid is found to decrease the rate of oxidation of adenine suggesting that caffeic acid acts as an efficient scavenger of SO4(-) and protects adenine from it; SO4(-) competes for adenine as well as for caffeic acid. From competition kinetics, the rate constant of SO4(-) with caffeic acid has been calculated to be 1.24 +/- 0.2 x 10(10) mol(-1)dm(3)s(-1). The quantum yields of photo-oxidation of adenine have been calculated from the rates of oxidation of adenine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to SO4' -. The results of experimentally determined quantum yields (phi exptl) and the quantum yields calculated (phi cl) by assuming that caffeic acid acts only as a scavenger of SO4(-) radicals show that phi exptl values are lower than phi cl values. The phi prime values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for SO4(-) scavenging by caffeic acid, are also found to be greater than phi exptI values. These observations suggest that the adenine radicals are repaired by caffeic acid, in addition to scavenging of sulphate radical anions.