Dynamics simulation, energetics calculation and experimental analysis of the intermolecular interaction between human neonatal ABL SH3 domain and its N-substituted peptoid ligands.

Non-receptor tyrosine kinase of neonatal ABL (nABL) is distributed in the nucleus and cytoplasm of proliferating cells in embryo and neonate, and has been implicated in the pathogenesis of neonatal leukemia and other hematological diseases. The kinase contains a regulatory Src homology 3 (SH3) domain that can specifically recognize proline-rich peptide segments on its partner protein surface. In this study, we systematically investigated the N-substitution effect on the binding of an empirically designed proline-rich peptide p9 to nABL SH3 domain by integrating dynamics simulations, energetics calculations and fluorescence affinity assays. The p9 is an almost all proline-composed decapeptide, with only a sole tyrosine at its residue 4, which has been found to bind nABL SH3 domain at a micromolar level in a class I mode. Here, the non-key residues of p9 peptide were independently replaced by various N-substituted amino acids to create a systematic N-substitution profile, from which we can identify those favorable, neutral and unfavorable substitutions at each peptide residue. On this basis a combinatorial peptoid library was rationally designed by systematically combining the favorable N-substituted amino acids at non-key residues of p9 peptide, thus resulting in a number of its peptoid counterparts. The binding affinity of top peptoid hits was observed to be comparable with or improved moderately relative to p9 peptide, with Kd ranging between 3.1 and 76 µM. Structural analysis revealed that the peptoids can be divided into exposed, polar and hydrophobic regions from N- to C-termini, in which the polar and hydrophobic regions confer specificity and stability to the domain-peptoid interaction, respectively. In addition, a designed peptoid was also observed to exhibit 5.3-fold SH3-selectivity for nABL over cSRC, suggesting that the N-substitution can be used to improve not only binding affinity but also recognition specificity of SH3 binders.Communicated by Ramaswamy H. Sarma.

D(1) dopamine receptor stimulation increases GluR1 phosphorylation in postnatal nucleus accumbens cultures.

Postsynaptic interactions between dopamine and glutamate receptors in the nucleus accumbens are critical for acute responses to drugs of abuse and for neuroadaptations resulting from their chronic administration. We tested the hypothesis that D(1) dopamine receptor stimulation increases phosphorylation of the AMPA receptor subunit GluR1 at the protein kinase A phosphorylation site (Ser845). Nucleus accumbens cell cultures were prepared from postnatal day 1 rats. After 14 days in culture, GluR1 phosphorylation was measured by western blotting using phosphorylation site-specific antibodies. The D(1) receptor agonist SKF 81297 increased Ser845 phosphorylation in a concentration- dependent manner, with marked increases occurring within 5 min. This was prevented by the D(1) receptor antagonist SCH 23390 and the protein kinase A inhibitor H89, and reproduced by forskolin. The D(2) receptor agonist quinpirole attenuated the response to D(1) receptor stimulation. Neither D(1) nor D(2) receptor agonists altered GluR1 phosphorylation at Ser831, the site phosphorylated by protein kinase C and calcium/calmodulin-dependent protein kinase II. In other systems, phosphorylation of GluR1 at Ser845 is associated with enhancement of AMPA receptor currents. Thus, the present results suggest that AMPA receptor transmission in the nucleus accumbens may be augmented by concurrent D(1) receptor stimulation.

Repeated administration of amphetamine or cocaine does not alter AMPA receptor subunit expression in the rat midbrain.

We previously reported that ventral tegmental area (VTA) dopamine neurons are supersensitive to AMPA when recorded three days after discontinuing repeated amphetamine or cocaine administration. By increasing dopamine cell activity, this may contribute to the induction of behavioral sensitization. The goal of this study was to determine if increased sensitivity to AMPA reflects increased AMPA receptor expression in the midbrain. Immunolabeling for GluR1, GluR2, GluR2/3, and GluR4 was quantified by immunohistochemistry with 35S-labeled secondary antibodies in VTA, substantia nigra, and a transitional area. First, rats were treated for five days with saline or amphetamine (5 mg/kg) and killed three or 14 days after the last injection. No significant changes in immunolabeling were observed for any subunit at either withdrawal time. GluR1 immunolabeling was further examined in rats killed 16-18 hrs or 24 hrs after a single injection of amphetamine or repeated injections of saline, amphetamine (5 mg/kg x 5 days) or cocaine (20 mg/kg x 7 days). No significant differences were observed in any region. Finally, neither repeated amphetamine or cocaine administration significantly altered GluR1 mRNA levels as quantified by reverse transcriptase-polymerase chain reaction. Our results suggest that enhanced responsiveness of VTA dopamine neurons to AMPA after withdrawal from repeated stimulant administration involves mechanisms more complex than increased expression of AMPA receptor subunits.