(1/2696) Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding.
Internalization of agonist-activated G protein-coupled receptors is mediated by non-visual arrestins, which also bind to clathrin and are therefore thought to act as adaptors in the endocytosis process. Phosphoinositides have been implicated in the regulation of intracellular receptor trafficking, and are known to bind to other coat components including AP-2, AP180 and COPI coatomer. Given these observations, we explored the possibility that phosphoinositides play a role in arrestin's function as an adaptor. High-affinity binding sites for phosphoinositides in beta-arrestin (arrestin2) and arrestin3 (beta-arrestin2) were identified, and dissimilar effects of phosphoinositide and inositol phosphate on arrestin interactions with clathrin and receptor were characterized. Alteration of three basic residues in arrestin3 abolished phosphoinositide binding with complete retention of clathrin and receptor binding. Unlike native protein, upon agonist activation, this mutant arrestin3 expressed in COS1 cells neither supported beta2-adrenergic receptor internalization nor did it concentrate in coated pits, although it was recruited to the plasma membrane. These findings indicate that phosphoinositide binding plays a critical regulatory role in delivery of the receptor-arrestin complex to coated pits, perhaps by providing, with activated receptor, a multi-point attachment of arrestin to the plasma membrane. (+info)
(2/2696) Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells.
We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTP gamma S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug. (+info)
(3/2696) TNF-alpha upregulates Gialpha and Gqalpha protein expression and function in human airway smooth muscle cells.
Chronic inflammation is a characteristic feature of asthma. Multiple inflammatory mediators are released within the asthmatic lung, some of which may have detrimental effects on signal transduction pathways in airway smooth muscle. The effects of tumor necrosis factor (TNF)-alpha on the expression and function of muscarinic receptors and guanine nucleotide-binding protein (G protein) alpha-subunits were examined in human airway smooth muscle cells. Cultured human airway smooth muscle cells were incubated in serum-free culture medium for 72 h in the presence and absence of 10 ng/ml of TNF-alpha, after which the cells were lysed and subjected to electrophoresis and Galphai-2, Gqalpha, and Gsalpha protein subunits were detected by immunoblot analysis with specific antisera. TNF-alpha treatment for 72 h significantly increased the expression of Galphai-2 and Gqalpha proteins and enhanced carbachol (10(-7) M)-mediated inhibition of adenylyl cyclase activity and inositol phosphate synthesis. These data provide new evidence demonstrating that TNF-alpha not only increases expression of Galphai-2 and Gqalpha proteins but also augments the associated signal transduction pathways that would facilitate increased tone of airway smooth muscle. (+info)
(4/2696) Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio).
Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores. (+info)
(5/2696) Identification of multiple phosphoinositide-specific phospholipases D as new regulatory enzymes for phosphatidylinositol 3,4, 5-trisphosphate.
In the course of delineating the regulatory mechanism underlying phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) metabolism, we have discovered three distinct phosphoinositide-specific phospholipase D (PI-PLD) isozymes from rat brain, tentatively designated as PI-PLDa, PI-PLDb, and PI-PLDc. These enzymes convert [3H]PI(3,4,5)P3 to generate a novel inositol phosphate, D-myo-[3H]inositol 3,4,5-trisphosphate ([3H]Ins(3,4,5)P3) and phosphatidic acid. These isozymes are predominantly associated with the cytosol, a notable difference from phosphatidylcholine PLDs. They are partially purified by a three-step procedure consisting of DEAE, heparin, and Sephacryl S-200 chromatography. PI-PLDa and PI-PLDb display a high degree of substrate specificity for PI(3,4, 5)P3, with a relative potency of PI(3,4,5)P3 >> phosphatidylinositol 3-phosphate (PI(3)P) or phosphatidylinositol 4-phosphate (PI(4)P) > phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) > phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). In contrast, PI-PLDc preferentially utilizes PI(3)P as substrate, followed by, in sequence, PI(3,4,5)P3, PI(4)P, PI(3,4)P2, and PI(4,5)P2. Both PI(3, 4)P2 and PI(4,5)P2 are poor substrates for all three isozymes, indicating that the regulatory mechanisms underlying these phosphoinositides are different from that of PI(3,4,5)P3. None of these enzymes reacts with phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine. All three PI-PLDs are Ca2+-dependent. Among them, PI-PLDb and PI-PLDc show maximum activities within a sub-microM range (0.3 and 0.9 microM Ca2+, respectively), whereas PI-PLDa exhibits an optimal [Ca2+] at 20 microM. In contrast to PC-PLD, Mg2+ has no significant effect on the enzyme activity. All three enzymes require sodium deoxycholate for optimal activities; other detergents examined including Triton X-100 and Nonidet P-40 are, however, inhibitory. In addition, PI(4,5)P2 stimulates these isozymes in a dose-dependent manner. Enhancement in the enzyme activity is noted only when the molar ratio of PI(4,5)P2 to PI(3,4, 5)P3 is between 1:1 and 2:1. (+info)
(6/2696) Specific galpha11beta3gamma5 protein involvement in endothelin receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in rat portal vein myocytes.
In this study, we identified the receptor subtype activated by endothelin-1 (ET-1) and the subunit composition of the G protein coupling this receptor to increase in cytosolic Ca2+ concentration in rat portal vein myocytes. We used intranuclear antisense oligonucleotide injection to selectively inhibit the expression of G protein subunits. We show here that the endothelin receptor subtype A (ETA)-mediated increase in cytosolic Ca2+ concentration was mainly dependent on Ca2+ release from the intracellular store. ETA receptor-mediated Ca2+ release was selectively inhibited by antisense oligonucleotides that inhibited the expression of alpha11, beta3, and gamma5 subunits, as checked by immunocytochemistry. Intracellular dialysis of a carboxyl terminal anti-betacom antibody and a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase-1 had no effect on the ETA receptor-mediated Ca2+ release. In contrast, a synthetic peptide corresponding to the carboxyl terminus of the alphaq/alpha11 subunit, heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors), and U73122 (an inhibitor of phosphatidylinositol-phospholipase C) inhibited, in a concentration-dependent manner, the ETA receptor-mediated Ca2+ responses. Accumulation of [3H]inositol trisphosphate evoked by norepinephrine peaked at approximately 15 s, whereas that evoked by ET-1 progressively increased within 2 min. In myocytes injected with anti-alphaq antisense oligonucleotides, both amplitude and time course of the norepinephrine-induced Ca2+ release became similar to those of the ET-1-induced Ca2+ response. We conclude that the ETA receptor-mediated Ca2+ release is selectively transduced by the heterotrimeric G11 protein composed of alpha11, beta3, and gamma5 subunits, and that a delayed stimulation of phospholipase C occurs via the alpha11 subunit. (+info)
(7/2696) N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane.
The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn-->Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies. (+info)
(8/2696) Effects of varying the expression level of recombinant human mGlu1alpha receptors on the pharmacological properties of agonists and antagonists.
1. Different expression levels of the human type 1alpha metabotropic glutamate (mGlu1alpha) receptor were obtained in transfected Chinese hamster ovary cells using an isopropyl beta-D-thiogalactopyranoside (IPTG) inducible system. Expression of mGlu1alpha receptors could not be detected using immunoblotting or immunocytochemical approaches in non-induced cells, however, controlled expression could be induced following IPTG addition in a time- and concentration-dependent manner. 2. In induced cells (100 microM IPTG, 20 h) the agonists L-quisqualate or 1-aminocyclopentane-1S,3R-dicarboxylic acid stimulated large increases in [3H]-inositol (poly)phosphate (in the presence of Li+) and inositol, 1,4,5-trisphosphate levels. 3. Induction with 1-100 microM IPTG allowed the receptor density to be increased incrementally and this not only resulted in an increase in the maximum response to L-quisqualate, 1-aminocyclopentane-1S,3R-dicarboxylic acid and (S)-3,5-dihydroxy-phenylglycine, but also in an increase in the respective potencies of each agent to activate phosphoinositide hydrolysis. 4. The intrinsic activity of the partial agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid dramatically increased with increasing receptor expression. 5. The activities of the competitive mGlu1alpha receptor antagonists (S)-alpha-methyl-4-carboxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine for inhibition of the effects of L-quisqualate or (S)-3,5-dihydroxyphenylglycine were found to be independent of the receptor expression level. 6. When the mGlu1alpha receptor was expressed at very high levels, no evidence for receptor constitutive activity could be detected, and none of the antagonists tested revealed either any intrinsic activity or negative efficacy. 7. These data demonstrate that both the potency and efficacy of mGlu1alpha receptor agonists are influenced by expression level, whilst mGlu1alpha receptor antagonist activities are independent of expression level. (+info)