Occupancy of the Dictyostelium cAMP receptor, cAR1, induces a reduction in affinity which depends upon COOH-terminal serine residues

Many G-protein-coupled receptors display a rapid decrease in ligand binding following pretreatment with agonist. cAR1, a cAMP receptor expressed early in the developmental program of Dictyostelium, mediates chemotaxis, activation of adenylyl cyclase, and gene expression changes that bring about the aggregation of 10(5) amoebae to form a multicellular structure. Occupancy of cAR1 by cAMP initiates multiple desensitization processes, one of which is an apparent reduction in binding sites. In transformed cells expressing cAR1 constitutively, Scatchard analyses revealed that this apparent loss of ligand binding is largely due to a significant reduction in the affinity of cAR1 for cAMP. A parallel increase in the dose dependence of cAR1-mediated cAMP uptake was observed. Consistent with these findings, proteolysis of intact cells and immunofluorescence suggested that cAR1 remains on the cell-surface following cAMP treatment. Finally, agonist-induced loss of ligand binding is impaired in cAR1 mutants lacking a cluster of cytoplasmic serine residues, which are targets of cAMP-induced phosphorylation.     

A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgammaS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgammaS stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac- piaA- double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes.     

Two transmembrane signaling mechanisms control expression of the cAMP receptor gene CAR1 during Dictyostelium development

Dictyostelium discoideum is among the best characterized organisms for the study of receptor/guanine nucleotide binding protein-mediated control of differentiation. Dictyostelium grow unicellularly but form fully differentiated multicellular organisms through a developmental program regulated by secreted cAMP activating specific cell-surface receptors. Dictyostelium respond differentially to cAMP at different developmental stages. During early development, expression of certain genes is induced by low-level oscillations of extracellular cAMP. Later, continuous, high cAMP concentrations will promote expression of specific genes in multicellular structures. Here, we show that the cAMP receptor gene CAR1, which is essential for development, utilizes two promoters that are activated at distinct stages of development and respond to different extracellular cAMP conditions. One promoter is active with low-level oscillations of cAMP; exposure to high cAMP concentrations will repress this promoter and induce a second promoter. The CAR1 mRNAs are alternatively spliced but encode identical proteins. Thus, through differential sensitivity to its own ligand, cAMP, two promoters and alternative splicing regulate CAR1 expression during Dictyostelium development.     

Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.     

Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier

We previously found in human blood a fraction of low-density lipoprotein (LDL) that is characterized by a reduced content of sialic acid. Desialylated LDL also has a low neutral carbohydrate level, decreased content of major lipids, small size, high density, increased electronegative charge and altered tertiary apolipoprotein B structure. Unlike native LDL, this fraction of desialylated (multiple-modified) LDL induces the accumulation of lipids in smooth muscle cells cultured from unaffected human aortic intima, i.e. it exhibits atherogenic properties. In this study, we attempted to elucidate the mechanism of desialylation and other changes in the multiple-modified LDL by investigating the possibility of LDL modification by different cells and the blood plasma. A 24-h incubation at 37 degrees C of lipoprotein with intact endotheliocytes, hepatocytes, macrophages and smooth muscle cells or cell homogenates did not cause alterations either in the physical properties or in the chemical composition of native LDL. On the other hand, a significant fall in the lipoprotein sialic acid level was observed already after a 1-h incubation of native LDL with an autologous plasma-derived serum. While LDL sialic acid level continuously decreased, LDL became capable of inducing the accumulation of total cholesterol in the smooth muscle cells cultured from unaffected human aortic intima after 3 h of incubation. Starting from the sixth hour of LDL incubation with serum, a steady decrease in the lipoprotein lipid content was observed as well as the related reduction of LDL size. Following 36 h of incubation, an increase in the negative charge of lipoprotein particles was also seen. Prolonged incubation of LDL with plasma-derived serum (48 and 72 h) leads to the loss of alpha-tocopherol by the LDL as well as to an increase in LDL susceptibility to copper oxidation and to accumulation of cholesterol covalently bound to apolipoprotein B, a marker of lipoperoxidation. Degradation of apolipoprotein B starts within the same period of time. Hence, desialylation of LDL particles represents one of the first or the primary act of modification which is, apparently, a sufficient prerequisite for the development of atherogenic properties. Subsequent modifications just enhance the atherogenic potential of LDL. The loss of sialic acid by LDL occurred at neutral pH and was not inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The [3H]sialic acid removed from LDL was not found in free form, but in the plasma fraction precipitated by trichloroacetic acid. These data along with the fact that cytidine-5'-triphosphate inhibited LDL desialylation suggest that enzymes close to sialyltransferases play a role in this process. Thus, this study demonstrated that the LDL modification processes imparting atherogenic properties to this lipoprotein can take place in human blood plasma. Multiple modification of LDL is a cascade of successive changes in the lipoprotein particle: desialylation, loss of lipids, reduction in particle size, increase of its electronegative charge and peroxidation of lipids.     

Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAA receptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.     

Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non-targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the 'post-P loop' (PP) regions. These PP mutations specifically caused marked defects in the 'C1' states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non-targeted mutagenesis and microbial selection can be a valuable tool for probing structure-function relationships of ion channels.     

Dual regulation of the glycogen phosphorylase 2 gene Dictyostelium discoideum: the effects of DIF-1, cAMP, NH3 and adenosine

Cell differentiation in Dictyostelium results in the formation of two cell types, stalk and spore cells. The stalk cells undergo programmed cell death, whereas spore cells retain viability. The current evidence suggests that stalk cell differentiation is induced by Differentiation Inducing Factor (DIF), while spore cell differentiation occurs in response to cAMP. We have discovered the first developmentally regulated Dictyostelium gene, the glycogen phosphorylase gene 2 (gp2) gene, that can be induced by both DIF-1 and cAMP, suggesting the possibility of a new group of developmentally regulated genes that have DIF-1 and cAMP dual responsiveness. The gp2 gene was found to be expressed in both prestalk/stalk cells and prespore/spore cells. The DIF-1 competence of the gp2 gene required uninterrupted development, whereas the cAMP-competence for the gene required only starvation. Both DIF-1 and cAMP induction of the gene could be inhibited by NH3, a factor that is thought to act as a developmental signal in Dictyostelium. Another developmental signal, adenosine, was found to repress the DIF-1 induction of the gp2 gene. Two introns in the gp2 gene were examined for their involvement in the regulation of the gene, but no regulatory function was detected. A model for the regulation of the gp2 gene during the development is proposed.     

An inverted cAMP response element mediates the cAMP induction of the ovine beta 1-adrenergic receptor gene

We identified an inverted, functional cAMP response element (CRE) located at--1599 bp relative to the translation start site within the ovine beta 1-adrenergic receptor (beta 1 AR) gene promoter. In transfection studies with SK-N-MC cells, a 40-bp oligonucleotide containing the potential CRE, beta 1 AR-CRE, conferred a 3- to 4-fold increase in luciferase activity mediated by cAMP. The induction was mimicked by co-transfecting the cells with a vector overexpressing the alpha-catalytic subunit of the cAMP-dependent protein kinase (PKA) without treatment, and was blocked by overexpressing a PKA inhibitor (PKI). In electrophoretic mobility shift assays, a discrete binding pattern was shown in cell nuclear extract probed with the 40 bp beta 1 AR-CRE. The binding was shown to be specific and supershifted by addition of a CRE binding protein (CREB-1) antibody. These data demonstrate that cAMP mediates the induction of beta 1 AR gene expression by interacting with an inverted CRE within the promoter region. This is the first reported functional CRE among all beta 1 AR genes.     

A review of mutagenesis studies of angiotensin II type 1 receptor, the three-dimensional receptor model in search of the agonist and antagonist binding site and the hypothesis of a receptor activation mechanism

OBJECTIVE: To seek the mechanism whereby agonists, competitive antagonists and insurmountable antagonists affect the receptor function differently, by reviewing recent mutagenesis studies of angiotensin II type 1 receptor (AT1) in which the binding of the agonist and antagonists and receptor signaling were affected. AT1 RECEPTOR STRUCTURE AND LIGAND BINDING SITES: We built a model of seven transmembrane spanning domains of the AT1 receptors using bacteriorhodopsin as a template. The carboxy terminal of angiotensin II binds to Lys199 in transmembrane domain 5, whereas the guanidinium group of Arg2 binds to Asp281 in transmembrane domain 7. Results of studies using mutagenesis supporting proposed ligand-docking models are discussed. HYPOTHESIS FOR THE LIGAND-INDUCED RECEPTOR SIGNALING MECHANISM: We submit a set of hypotheses for a mechanism whereby the ligand binding induces changes in the receptor conformation by the rotation of transmembrane helices as the initial event for the subsequent activation of a G protein. In this mechanism antagonists are not capable of rotating the helices but agonists are able to do so, which results in the formation of a hydrogen bond between Asp74 in transmembrane domain 2 and Tyr292 in transmembrane domain 7. This mechanism also provides plausible explanation for the activation of monoamine receptors. COMPETITIVE AND INSURMOUNTABLE ANTAGONISTS: Competitive antagonists share the same binding sites with agonists, but insurmountable antagonists do not, and binding of the latter does not preclude agonist binding, for example, to Asp281. CONCLUSION: This hypothesis of the intrareceptor signaling mechanism and the receptor model indicate that some amino acid residues essential for the signaling play their roles in the intrareceptor activation mechanism, whereas others participate directly in ligand binding.     

Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.     

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated alpha-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Summation of activation: Evidence from multiple primes that converge and diverge within semantic memory.

Six experiments addressed the combinatorial influence of multiple related primes in naming, lexical decision, and relatedness judgment performance. Primes either converged on a single semantic representation (e.g., LION-STRIPES-TIGER) or diverged onto distinct semantic representations (e.g., KIDNEY-PIANO-ORGAN). The facilitatory influence of 2 related primes was well predicted by the sum of the influences from the single-related-prime conditions (1) for both convergent and divergent primes, (2) in lexical-decision and naming, (3) across varying prime-target stimulus onset asynchronies, and (4) under target-degradation conditions that increased the priming effects. The relatedness-judgment task yielded an additive pattern of priming for convergent prime conditions; however, an underadditive pattern of priming was found for divergent prime conditions. Discussion focuses on the role of attentional systems that modulate the type of information used to perform a given task. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Selective reaching: Evidence for multiple frames of reference.

Students participated in 3 experiments investigating the use of environment- and action-centered reference frames in selective reaching. They pointed to a green target appearing either with or without a red distractor. Target-distractor distance was manipulated, and distractor interference (difference between distractor trials and no-distractor trials) was measured in reaction time, movement time, and movement endpoint. Target-distractor distance determined the dominant frame of reference. Small distances evoked an environment-centered framework that encoded targets within an external context. Large distances evoked an action-centered framework that encoded targets relative to the start position of the hand. Results support the hypothesis that the brain represents spatial information in multiple frames of reference, with the dominant frame of reference being dependent on the task demands. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence that the cannabinoid CB1 receptor is a 2-arachidonoylglycerol receptor. Structure-activity relationship of 2-arachidonoylglycerol, ether-linked analogues, and related compounds

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.