The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The beta(2) adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Ca(v)1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate-dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein-coupled receptor.

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartatetype glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.The prevailing excitatory neurotransmitter in the mammalian brain is glutamate (1, 2). Upon its release from presynaptic sites, this neurotransmitter binds to ionotropic glutamate receptors that mediate rapid excitatory synaptic transmission in the mammalian brain (1, 2). Several immuno-electron microscopic studies have demonstrated that ionotropic glutamate receptors are clustered at postsynaptic sites of excitatory synapses (3-5). Two major glutamate receptor families exist, namely N-methyl-D-aspartate (NMDA) 1 receptors, which mediate Ca 2ϩ influx, and non-NMDA receptors, which are usually not Ca 2ϩ -permeable (1, 2, 6). Non-NMDA receptors are further divided into ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and kainate receptors. At low frequency, synaptic transmission normally depends nearly exclusively upon AMPA receptors. On the other hand, kainate and NMDA receptors require higher frequencies for activation. NMDA receptor-mediated Ca 2ϩ influx is necessary for different forms of synaptic plasticity, such as long term potentiation (1,7,8). At different synapses in the hippocampus and in other brain areas, a few bursts of high frequency electric stimulation that activate NMDA receptors induce a long lasting increase in synaptic transmission, the hallmark of long term potentiation.Glutamate receptors are thought to be heterotetramers consisting of homologous subunits (39). AMPA recepto...

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca 2؉ inf lux through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca 2؉ ͞calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKII␣ by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca 2؉ inf lux but also close to ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP. Caϩ ͞calmodulin-dependent protein kinase II (CaMKII) mediates a variety of different cellular responses to Ca 2ϩ influx (1, 2). An important source of Ca 2ϩ influx into neurons is the N-methyl-D-aspartate (NMDA)-type glutamate receptor, which is activated by the excitatory neurotransmitter glutamate (2). NMDA-and ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors are clustered at postsynaptic sites opposing presynaptic neurotransmitter release sites (3, 4). Brief trains of presynaptic highfrequency stimulation efficiently activate NMDA receptors (5), resulting in postsynaptic Ca 2ϩ influx and long-term potentiation (LTP). LTP is a long-lasting increase in neurotransmission thought to represent the physiological correlate of learning and memory (5, 6). The induction of NMDA receptor-dependent LTP requires activation of CaMKII in the postsynaptic neuron (6, 7). CaMKII is enriched at postsynaptic densities (8, 9), where it is well placed for activation by Ca 2ϩ influx through NMDA receptors and subsequent phosphorylation of neighboring AMPA receptors, an event contributing to LTP (7, 10). A kinase anchoring proteins, usually called AKAPs, place cyclic AMP-dependent protein kinase (PKA) next to selected substrates such as AMPA receptors (11), and receptors for activated C kinase (RACKs) are important for subcellular localization of different protein kinase C (PKC) isozymes (12). Crucial information about the subcellular targeting of CaMKII is lacking. NMDA receptors would be ideal postsynaptic adapter sites for CaMKII, where it would have full access to Ca 2ϩ influx through these receptors. Cortical NMDA receptors consist of one or two NR1 and two or three NR2A and 2B subunits whose C termini are intracellular (13-16). We show that CaMKII is directly associated with NR1 a...

Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl, A. (1997) J. Biol. Chem. 272, 12650 -12661). We tested the hypothesis that cyclin G2 may be a negative regulator of cell cycle progression and found that ectopic expression of cyclin G2 induces the formation of aberrant nuclei and cell cycle arrest in HEK293 and Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a detergentresistant compartment, suggesting an association with cytoskeletal elements. We determined that cyclin G2 and its homolog cyclin G1 directly interact with the catalytic subunit of protein phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nM) phosphatase activity coprecipitates with endogenous and ectopic cyclin G2. We found that cyclin G2 also associates with various PP2A B regulatory subunits, as previously shown for cyclin G1. The PP2A/A subunit is not detectable in cyclin G2-PP2A-B-C complexes. Notably, cyclin G2 colocalizes with both PP2A/C and B subunits in detergent-resistant cellular compartments, suggesting that these complexes form in living cells. The ability of cyclin G2 to inhibit cell cycle progression correlates with its ability to bind PP2A/B and C subunits. Together, our findings suggest that cyclin G2-PP2A complexes inhibit cell cycle progression.

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2, NANOG, and OCT-4, the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here, we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency, prevents cell proliferation, and enhances mesoderm and endoderm activities in hESCs. At the molecular level, mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growthinhibitory genes, as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration.differentiation ͉ pluripotency ͉ OCT-4 ͉ long-term undifferentiated growth

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We describe the isolation and characterization of cDNAs encoding full-length human and murine cyclin G1 and a novel human homologue of this cyclin designated cyclin G2. Cyclin G1 is expressed at high levels in skeletal muscle, ovary, and kidney. Following an initial up-regulation from early G 1 to G 1 /S phase, cyclin G1 mRNA is constitutively expressed throughout the cell cycle in T and B cell lines. In contrast, in stimulated peripheral T cells, cyclin G1 mRNA is maximal in early G 1 phase and declines in cell cycle progression. Cyclin G1 levels parallel p53 expression in murine B lymphocytes; however, in several human Burkitt's lymphomas, murine lymphocytes treated with transforming growth factor-␤, early murine embryos, and several tissues of p53 null mice, cyclin G1 levels are either inverse of p53 levels or expressed independent of p53. The cyclin G1 homologue, cyclin G2, exhibits 60% nucleotide sequence identity and 53% amino acid sequence identity with cyclin G1, and like cyclin G1, exhibits closest sequence identity to the cyclin A family. Distinct from cyclin G1, the amino acid sequence for cyclin G2 shows a PESTrich sequence and a potential Shc PTB binding site. Cyclin G2 mRNA is differentially expressed compared to cyclin G1, the highest transcript levels seen in cerebellum, thymus, spleen, prostate, and kidney. In contrast to the constitutive expression of cyclin G1 in lymphocytes, cyclin G2 mRNA appears to oscillate through the cell cycle with peak expression in late S phase.Transitions through the eukaryotic cell division cycle are primarily coordinated by the sequential activation of cyclin-dependent kinases (CDKs) 1 which are, in turn, regulated by subunit associations and phosphorylation (reviewed in Refs. 1-3). The cyclins represent a group of closely related molecules which primarily function at specific stages of the cell cycle as regulators of CDK activity by binding and forming active complexes with specific partner CDKs. This cyclin-CDK association is in part determined by the conserved cyclin region of ϳ110 amino acids referred to as the cyclin box (4 -6). The cyclin box exhibits ϳ30 -50% identity between the different cyclin types, the consensus sequence varying depending on the class and subclass of cyclin (5,7,8). Cyclins have been classified into different groups on the basis of their structural similarity, functional period in the cell division cycle and regulated expression. In addition to providing positive growth control, CDKs and cyclin-CDK pairs may participate in metabolism and signal transduction unrelated to cell cycle as evidenced by pho80-pho85 cyclin-CDK complex participation in yeast phosphate metabolism (9, 10), the expression of CDK5 in nonproliferating brain tissue (11-13), and the SRB10/11 cyclin-CDK regulator of RNA polymerase II (14).To date at least 12 different cyclins in budding yeast, 4 in fission yeast, and 10 in mammalian cells (cyclins A-H with multiple family members for some types) are known, all primarily displaying sequence homology within the cyclin b...

(1996) J. Biol. Chem. 271, 6050 -6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor ␤1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5-14-fold over control cell levels. Proliferation signals promote the coordinated progression of a cell through the cell division cycle. In eukaryotes this process is controlled by the sequential formation, activation, and inhibition of cyclin-cyclin-dependent kinase (CDK) 1 complexes (1). Active cyclin-CDK complexes phosphorylate specific targets such as the tumor suppressor RB, various transcription factors, DNA polymerase ␣, and cytoskeletal proteins (2) and thus trigger progression through the cell cycle. The levels of many cyclins oscillate during the cell cycle and act as rate-limiting positive regulators of CDK activity. Mammalian cyclins are classified into different types based on their structural similarity, functional period in the cell division cycle, and regulated expression (1, 3, 4). 12 different cyclins in mammalian cells (cyclins A-I, some with multiple subtypes) have been identified (1, 5-7) either functionally or through an ϳ110-amino acid homologous region essential for cyclin-CDK complex formation (8 -10) referred to as the cyclin box (3, 11). Cyclin-CDK activity is also subject to regulation by CDK inhibitors (CDKIs) such as p15INK4 and p16 INK4, p21 WAF1/CIP1, and p27 KIP1 which, in response to negative stimuli, bind cyclin-CDK complexes and block cell cycle progression (5, 12). In addition to participation in cellular proliferation, CDKs and cyclin-CDK pairs may participate in processes not directly related to cell cycle regulation as evidenced by Pho80-Pho85 cyclin-CDK participation in yeast phosphate metabolism (13,14), the involvement of p35⅐CDK5 in promoting neurite outgrowth (15-17), the association of the cyclin H/CDK7 pair in the TFIIH transcription factor complex (18,19), and the cyclin C/CDK8 and SRB10/11 cyclin-CDK regulation of RNA polymerase II (20,21).We studied the effects of stimulatory and inhibitory signals on cell cycle components expressed in B lymphocytes representative of two different maturation stages of development. A robust immune system has to deliver specific and effective immune responses to foreign antigens and yet be immunologically tolerant of self-antigens. Such tolerance is achieved because T and B cells pass through stages in their development when ligation of their antigen receptors by self-antigens results in negative regulatory signals that induce either unresponsiveness and functional inactivation (clonal anergy) or their physical elimination (clonal deletion) (22-2...