Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.
The ionotropic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.
The glutamate receptor adaptor protein Homer is concentrated in the postsynaptic density of excitatory synapses and is critical for normal operation of synaptic transmission. In this study, we investigated the responsiveness of Homer family proteins to dopamine stimulation with the psychostimulant cocaine in rat striatal neurons both in vivo and in vitro. We found that a single dose of cocaine specifically induced a rapid and transient increase in protein levels of the Homer1a, but not Homer1b/c and Homer2a/b, isoforms in the striatum. This selective Homer1a induction was mediated primarily through activation of dopamine D1, but not D2, receptors. Both protein kinase A and Ca 2ϩ /calmodulin-dependent protein kinases are important for mediating the cocaine stimulation of Homer1a expression. At the transcriptional level, cAMP response element-binding protein serves as a prime transcription factor transmitting the signals derived from D1 receptors and associative pathways to the CaCRE sites within the Homer1a promoter. From a functional perspective, non-cross-linking Homer1a, once induced, competed with the cross-linking isoforms of Homer proteins (Homer1b/c and Homer2a/b) to uncouple the connection of group I metabotropic glutamate receptors (mGluRs) with inositol-1,4,5-triphosphate receptors. These results indicate that cocaine possesses the ability to stimulate Homer1a expression in striatal neurons through a specific synapse-to-nucleus pathway. Moreover, inducible Homer1a expression may represent a transcription-dependent mechanism underlying the dynamic regulation of submembranous macromolecular complex formation between group I mGluRs and their anchoring proteins.Synaptic Homer proteins are important for synaptic construction and function Sheng and Kim, 2002). Long-form Homer proteins (Homer1b/c, Homer2a/b, and Homer3) contain the N-terminal EVH1 (Enabled/ VASP homology 1) domain, which binds the C terminus of group I metabotropic glutamate receptors (mGluRs), whereas the C-terminal coiled-coil structure and leucine zipper motifs render a capability for self-assembly (Brakeman et al., 1997;Xiao et al., 1998;Xiao et al., 2000). Thus, as a prominent scaffolding molecule concentrated in the postsynaptic density of excitatory synapses, Homer crosslinks group I mGluRs to other targets in a specific subcellular microdomain to regulate a specific signaling activity. Emerging evidence indicates that Homer proteins play an essential role in the membrane trafficking of mGluR1␣/5 (Ango et al., 2000); the coupling of mGluR1/5 to inositol-1,4,5-triphosphate (IP 3 ) receptors and the cation (Ca 2ϩ or K ϩ ) channel (Yuan et al., 2003); the development of spines, axons, and synapses (Shiraishi et al., 2003); and drug addiction (Swanson et al., 2001;Szumlinski et al., 2004Szumlinski et al., , 2005; also see below).One distinctive member of Homer family is Homer1a (Brakeman et al., 1997;Xiao et al., 1998Xiao et al., , 2000. Unlike the long-form of Homer proteins, this short-form of Homer lacks the C-terminal coiled-co...
Extracellular signals can regulate mitogen-activated protein kinase (MAPK) cascades through a receptor-mediated mechanism in postmitotic neurons of adult mammalian brain. Both ionotropic and metabotropic glutamate receptors (mGluRs) are found to possess such an ability in striatal neurons. NMDA and AMPA receptor signals seem to share a largely common route to MAPK phosphorylation which involves first activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ influx, followed by subsequent induction of phosphoinositide 3-kinase (PI3-kinase). Through its lipid and protein kinase activity, active PI3-kinase may transduce signals to Ras-MAPK cascades via at least two distinct pathways. A novel, Ca(2+)-independent pathway is believed to mediate mGluR signals to Ras-MAPK activation. As an information superhighway between the surface membrane and the nucleus, Ras-MAPK cascades, through activating their specific nuclear transcription factor targets, are actively involved in the regulation of gene expression. Emerging evidence shows that MAPK-mediated genomic responses in striatal neurons to drug exposure contribute to the development of neuroplasticity related to addictive properties of drugs of abuse.
Shaker-type voltage-gated K + (K V 1) channels composed of K V 1.2 and K V 1.5 α-subunits are expressed in cerebral vascular smooth muscle cells (cVSMCs), where they contribute to the resting diameter and vasodilation of cerebral arteries (CAs).1,2 K V 1 channels are multiprotein structures composed of 4 K V α pore-forming subunits coassembled with intracellular K V β subunits, which may affect channel trafficking and kinetics. [3][4][5][6][7][8] In addition, post-translational modifications, such as glycosylation and protein kinase A (PKA)-mediated phosphorylation of the K V α subunits, may increase protein expression and activity of K V 1 channels. 9-15Recently, we reported the expression of a scaffolding protein, postsynaptic density protein-95 (PSD95), in rat CA. 16 Previously, PSD95 was studied primarily in neurons, where it provides an assembly platform at the plasma membrane for macromolecular signaling complexes including ion channels.17-22 However, we reported that PSD95 serves as a molecular scaffold for K V 1 channels in cVSMCs, and this interaction is required for the proper expression of K V 1 channels that exerts a tonic vasodilator influence. 16 Accordingly, antisense-mediated knockdown of PSD95 in rat CA resulted in a loss of K V 1 channel expression and caused vasoconstriction, inferring that PSD95 promotes the expression of K V 1 channels in cVSMCs. 16 Notably, the C terminus of the K V 1.2α subunit contains a structural motif that permits the channel to interact with Objective: We explored whether a specific interaction between PSD95 and K V 1 channels enables protein kinase A phosphorylation of K V 1 channels in cVSMCs to promote vasodilation. Methods and Results:Rat cerebral arteries were used for analyses. A membrane-permeable peptide (K V 1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of K V 1.2α was designed as a dominant-negative peptide to disrupt the association of K V 1 channels with PSD95. Application of K V 1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and K V 1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of K V 1-C peptide. Patch-clamp recordings confirmed that K V 1-C peptide attenuates K V 1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to K V 1-C peptide showed markedly less phosphorylation of K V 1.2α subunits. Finally, phosphatase inhibitors blunted both K V 1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. PSD95. [16][17][18][19][20] Collectively, the interactions of signaling proteins with PSD95 are enabled by 3 postsynaptic density-95, discs large, zonula occludens-1 (PDZ...
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