This suggests that the PKA-mediated phosphorylation on GluR1 appears crucial in AMPA receptor trafficking. of neuronal circuits, and behavioral effects of drug addiction (Esteban et al., 2003;Boudreau and Wolf, 2005;Sun et al., 2005;Zhang et al., 2007). AMPA receptors are tetramers comprised of various combinations of four subunits, GluR1-GluR4 (Barry and Ziff, 2002). Among various mechanisms, phosphorylation of the GluR1 subunit at the Ser831, Ser845, and Ser818residues in its intracellular carboxy-terminal domain plays a major role in regulating AMPA neurotransmission. Cell-based studies suggest that PKA-mediated phosphorylation of Ser845can stabilize GluR1 in the plasma membrane, enhance the incorporation of GluR1-containing AMPA receptors into the synaptic membrane (Lee et al., 2000;Havekes et al., 2007) and decrease AMPA receptor internalization (Man et al., 2007), thus allowing for dynamic regulation of AMPA receptor trafficking. However, whether specific phosphorylation of the Ser845residue serves the same functionin vivo(intact animals) has not been directly addressed. Recent studies have demonstrated that phosphorylation at Ser845of GluR1 subunit, but not Ser831, is significantly increased in the striatum and prefrontal cortexin vivoin response to the psychostimulant cocaine (Snyder et al., 2000;Edwards et al., 2007;Tropea et al., 2008). We have also demonstrated that: (1) long-term behavioral sensitization established by daily injections of high-dose cocaine and MRPS5 a chronic withdrawal increases phosphorylation of GluR1 Ser845but not Ser831residue in the prefrontal cortex and nucleus accumbens; and (2) these increases are normalized when the established behavioral sensitization is reversed (Zhang et al., 2007). Furthermore,Boudreau and Wolf (2005)have shown that cocaine behavioral sensitization is associated with increased surface/intracellular ratios of GluR1 BYK 204165 subunit in the nucleus accumbens (NAc). However, whether Ser845phosphorylation of GluR1 subunit plays direct roles in cocaine sensitization remains to be elucidated. One of the main reasons for this shortcoming is that effective methodologies for selective suppression ofin vivophosphorylation at single amino acid residues have not been established. Thus, a protocol for specifically inhibiting single residue phosphorylation of GluR1 subunit or its functional consequences is expected to provide the first step toward elucidating roles of BYK 204165 this and other protein phosphorylation events in physiology. A technology has recently emerged that can evaluate the role of single residue phosphorylation events. Aptamers have been shown to possess the ability to bind targeted proteins with high affinity and specificity (Ellington and Szostak, 1990,1992; Sullenge et al., 1990;Hermann and Patel, 2000), and, as nucleic acids, they exhibit increased stability, ease of generation, and simple modification which offers advantages over antibodies to artificially interfere with target gene function (Murphy et al., 2003;Hirao et al., BYK 204165 2004). More importantly, aptamers can be selectedin vitroand the process can be tightly controlled (Griffin et al., 1993). In the present study we have developed and identified aptamers to selectively inhibit BYK 204165 GluR1 phosphorylation at Ser845and GluR1-containing AMPA receptor trafficking to the cell surface. == Materials and Methods == == Production of a GST-fused C-terminal GluR1 == The intracellular website of the rat GluR1 subunit cDNA (deduced amino acids 808 to 889) was amplified by reverse transcriptase PCR from a rat RNA library with the ahead primer: 5-CGGGATCCGGTGGCGGAGGGTCTGGAGAGTTCTGCTACAAATCCCG-3 and reverse primer: 5-GGAATTCCTTACAATCCTGTGGCTCCCAAG-3. A 6-glycine hinge sequence (italic) and aBamHIsite (underline) were designed in the ahead primer and anEcoRIsite (underline) in the reverse primer. The amplified fragment was cloned into pGEX-2T vector at theBamHI/EcoRIsites downstream of the GST, producing.