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.

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2 O-methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132-p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.cAMP response element-binding (CREB) protein ͉ transcription ͉ CaM kinase ͉ actin cytoskeleton ͉ Rac N euronal activity regulates the development and modification of neuronal circuitry in part by activating genetic programs. Activity-regulated gene expression has been implicated in axon guidance, dendrite elaboration, synapse formation, and long-lasting synaptic plasticity (1, 2). Dendrites are the primary site of excitatory synapses, and their morphogenesis determines both the size and number of synaptic contacts (3). Although dendritic development is partly controlled by intrinsic factors, neuronal activity also plays a critical role. Indeed, the timing of afferent innervation and synapse formation coincides with the period of maximum growth and dendritic remodeling (3).The transcription factor cAMP response element-binding (CREB) protein is a key regulator of dendritic growth (4) and activity-regulated dendritic refinement in mature neurons (5). Although CREB is believed to be a critical regulator of neuronal plasticity, few CREB targets have been directly linked to plasticity. To identify these genes, we developed a novel technology, termed serial analysis of chromatin occupancy (SACO) that facilitated the genome-wide identification of CREB target regions (6). We focused on microRNAs (miRNAs) because the ability of these molecules to repress gene expression is believed to play an important role in development, differentiation, proliferation, survival, and oncogenesis (7). Interestingly, a significant fraction of miRNAs are enriched or specifically expressed in the nervous system (8), and transcription of some miRNAs changes dynamically during brain development (9, 10). miRNAs have been implicated in development of neuronal asymmetry in Caenorhabditis elegans, maturation of sensory neurons in Drosophila, and neurite outgrowth and spine homeostasis in rodents (11)(12)(13)(14). Although activity is believed to play an essential role in sculpting neuronal development, miRNAs induced by neuronal activity have not been described.Here, we show that microRNA 132 (miR132) is an a...

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...

Patterns of resistance to first-line osimertinib are not well-established and have primarily been evaluated using plasma assays which cannot detect histologic transformation and have differential sensitivity for copy number changes and chromosomal rearrangements. Experimental Design:To characterize mechanisms of resistance to osimertinib, patients with metastatic EGFR-mutant lung cancers who received osimertinib at Memorial Sloan Kettering and had next-generation sequencing performed on tumor tissue before osimertinib initiation and after progression were identified.Results: Among 62 patients who met eligibility critieria, histologic transformation, primarily squamous transformation, was identified in 15% of first-line osimertinib cases and 14% of laterline cases. Nineteen percent (5/27) of patients treated with first-line osimertinib had off-target genetic resistance (2 MET amplification, 1 KRAS mutation, 1 RET fusion, and 1 BRAF fusion) whereas 4% (1/27) had an acquired EGFR mutation (EGFR G724S). Patients with squamous transformation exhibited considerable genomic complexity; acquired PIK3CA mutation, chromosome 3q amplification and FGF amplification were all seen. Patients with transformation had shorter time on osimertinib and shorter survival compared to patients with on-target resistance. Initial EGFR sensitizing mutation, time on osimertinib treatment and line of therapy also influenced resistance mechanism that emerged. The compound mutation EGFR S768 + V769L and the mutation MET H1094Y were identified and validated as resistance mechanisms with potential treatment options. Conclusion:Histologic transformation and other off-target molecular alterations are frequent early emerging resistance mechanisms to osimertinib and are associated with poor clinical outcomes.Research.

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.