Serine phosphorylation of mGluR1 is elevated in FCX and hippocampi of prenatal cocaine-exposed P21 rats.

<p>(<b>A</b>) Levels of Serine (<b>S</b>), Threonine (<b>T</b>) and tyrosine (<b>Y</b>) phosphorylation on purified native mGluR1 from prenatal saline- and cocaine-exposed P21 rats were determined in immunoprecipitates of immobilized anti-mGluR1 by Western blot using specific antibodies directed against each phospho-epitope. (<b>B</b>) pS-mGluR1 levels in immobilized mGluR1 immunoprecipitates of Kreb’s-Ringer (K-R) and 162 nM PMA treated FCX synaptic membrane extracts from prenatal saline- and cocaine-exposed P21 rats. The obtained protein bands were quantified by densitometric scanning of the blots. Data are represented as means ± s.e.m. of the ratios of phosphorylated mGluR1/total mGluR1 optical intensities. <i>n</i> = 4 for each group. *<i>p</i><0.01 comparing the pS-mGluR1 levels induced by PMA vs. K-R in each treatment group. +<i>p</i><0.01 comparing the levels of pS-mGluR1 in prenatal cocaine- to saline-exposed groups by two-tailed Student’s <i>t</i> test. #<i>p</i><0.01 comparing the levels of PMA-induced pS-mGluR1 isolated from prenatal cocaine- to saline-treated rats by two-tailed Student’s <i>t</i> test. No between-group differences in the levels of mGluR1were detected.</p

Prenatal cocaine exposure did not alter the expression of various Gα proteins, Homer1 and mGluR1 in the FCX and hippocampus of P21 rats.

<p>(<b>A</b>) Representative Western blots of the Gα proteins, Homer1 and mGluR1 in the FCX and hippocampal synaptosomes of P21 rats exposed to saline or cocaine during gestation. The blots were stripped and sequentially re-probed with anti-actin (2265.5±129.9 and 2378.8±174.8 optical intensity in FCX of saline and cocaine, respectively and 2160.5±104.8 and 2125.0±131.7 optical intensity in hippocampi of saline and cocaine, respectively) to validate equal loading. (<b>B</b>) Densitometric quantification of the Gα proteins, Homer1 and mGluR1 levels in the FCX and hippocampus of P21 rats. Data are presented as means ± s.e.m. of the ratios of optical intensity of indicated protein to the optical intensity of actin. <i>N</i> = 4 for each group. There are no discernible changes in the expression levels of any of the proteins examined in either brain regions of the prenatal cocaine exposed rats.</p

The mGluR1 is associated exclusively with G_q/11 and Homer1 in the frontal cortex (FCX) and hippocampus of 10-week-old naïve rats.

<p>(<b>A</b>) The presence of mGluR1 was examined in the immobilized anti-Gα immunoprecipitates of the solubilized FCX and hippocampal synaptosomal membranes by Western blotting. The representative Western blots show exclusive detection of mGluR1 together with Gαq/11 protein in the synaptosomes prepared from FCX and hippocampus. N = 4. (<b>B</b>) Basal and glutamate (1 µM)-induced mGluR1 coupling to G_q/11 and Homer1 in synaptic membranes derived from FCX and hippocampi of 10-week-old naïve rats. The presence of mGluR1 was examined in immobilized anti-mGluR1 immunoprecipitates of the solubilized FCX and hippocampal synaptosomal membranes by Western blotting. The representative Western blots show Gαq/11 protein and Homer1 are present in anti-mGluR1 immunoprecipitates. Quantitative data are presented as means ± s.e.m. of the ratios of optical intensity of indicated protein to the optical intensity of mGluR1 (2132.8±150.8 and 2090.8±151.8 optical intensity in FCX of Kreb’s-Ringer (K-R) and glutamate treated, respectively and 2044.5±139.2 and 2000.0±114.3 optical intensity in hippocampi of K-R and glutamate treated, respectively) to validate equal loading. There is no discernible difference in the level of mGluR1-associated Homer1 indicated as Homer1/mGluR1 ratios in both brain regions (0.467±0.03 vs. 0.447±0.03 in K-R and glutamate exposed FCX and 0.468±0.03 vs. 0.488±0.02 in K-R and glutamate exposed hippocampus). Incubation with 1 µM glutamate increased the level of Gq/11 associated with mGluR1 by 343.2±28.9% and 332.7±32.1% respectively in FCX and hippocampus. <i>N</i> = 4 for each group. *<i>p</i><0.01 comparing the level of Gαq/11 in K-R and 1 µM glutamate exposed synaptosomal membranes by two-tailed Student’s <i>t</i> test.</p

Heightened PKC-mediated serine phosphorylation of mGluR1 in prenatal cocaine- exposed brains is responsible for the reduced mGluR1 coupling to G_q/11 and Homer1.

<p>(<b>A</b>) Levels of mGluR1 - G_q/11 and mGluR1 - Homer1 coupling as well as serine-phosphorylated mGluR1 (pS-mGluR1) on purified mGluR1 signaling complexes. FCX synaptosomal membranes from prenatal cocaine- and saline-exposed P21 rats were incubated with either vehicle 100 µM alkaline phosphatase, 30 µM ATP containing Kreb’s-Ringer (K-R) or 162 nM PMA (20 µg phosphoatidylserine). Following completion of dephosphorylation by addition of phosphatase inhibitors, phosphate-free mGluR1 from prenatal saline-treated rats was phosphorylated by recombinant γPKC in the presence of ATP. The reaction was terminated by specific PKC inhibitor, celestrine. The resultant synaptic membranes containing differentially phosphorylated mGluR1 were then incubated with K-R or 1 µM glutamate. The mGluR1 purified by anti-mGluR1 immunoprecipitation was analyzed for phosphoserine by Western blotting. The interaction between mGluR1 and G_q/11 or Homer1 with different phosphorylation states was assessed by the levels of Gα_q/11 and Homer1 in the anti-mGluR1 immunoprecipitates by Western blotting. (<b>B</b>) Densitometric quantification of mGluR1-associated Gα_q/11 and Homer1 as well as pS-mGluR1 levels. Data are represented as means ± s.e.m. of the ratios of Gα_q/11, Homer1 or pS-mGluR1/total mGluR1 optical intensities. <i>n</i> = 4 for each group. *<i>p</i><0.01 comparing the glutamate-induced to basal mGluR1-G_q/11 complex levels by two-tailed Student’s <i>t</i> test. #<i>p</i><0.01 comparing the respected levels in prenatal cocaine- to saline-treated rats. No between-group differences in the levels of mGluR1were detected.</p