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. 2003 Apr 29;100(9):5479-84.
doi: 10.1073/pnas.1031602100. Epub 2003 Apr 21.

A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes

Erbo Dong  1 , Hector Caruncho, Wen Sheng Liu, Neil R Smalheiser, Dennis R Grayson, Erminio Costa, Alessandro Guidotti
Affiliations

A reelin-integrin receptor interaction regulates Arc mRNA translation in synaptoneurosomes

Erbo Dong et al. Proc Natl Acad Sci U S A. .

Abstract

Reelin is synthesized and secreted into extracellular matrix by cortical gamma-aminobutyric acid (GABA)ergic interneurons and binds with high affinity to the extracellular domain of integrin receptors expressed in dendritic shaft and spine postsynaptic densities (DSPSD) of pyramidal neurons. In heterozygous reeler mice, reelin bound to DSPSD, and the expression of Arc (activity-regulated cytoskeletal protein) is lower than in wild-type mice. We studied the effect of reelin on Arc and total protein synthesis in synaptoneurosomes (SNSs) prepared from mouse neocortex. Recombinant full-length mouse reelin displaces the high affinity (K(D) = 60 fM) binding of [(125)I]echistatin (a competitive integrin receptor antagonist) to integrin receptors with a K(i) of 22 pM and with a Hill slope close to 1. Echistatin (50-100 nM) competitively antagonizes and abates reelin binding. The addition of reelin (2-40 pM) to SNSs enhances the incorporation of [(35)S]methionine into Arc and other rapidly translated proteins in a concentration-dependent manner. This incorporation is virtually abolished by 50-100 nM echistatin or by 5-10 nM rapamycin, a blocker of the mammalian target of rapamycin kinase. We conclude that reelin binds with high affinity to integrin receptors expressed in SNSs and thereby activates Arc protein synthesis.

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Figures

Figure 1
Figure 1
Electron microscopy of SNSs. (A–C) Presynaptic endings containing vesicles and postsynaptic structures (arrows) with postsynaptic densities and scattered polysomes. (Scale bar = 0.5 μm.) (B and C) Larger magnification of SNSs with or without 0.01% Triton X-100 treatment, respectively. (Scale bar = 0.5 μm.) (D) Label-fracture replica showing the ImmunoGold (Aurion, Wageningen, The Netherlands) labeling of the α3 integrin receptor subunit (circles) in a postsynaptic membrane, E-face. (Calibration bar = 0.4 μm.) (E) Electron micrograph showing the ImmunoGold labeling of the α3 integrin subunit in unfractured SNSs. The round black areas observed in the micrograph correspond to presynaptic terminals to which remain attached postsynaptic membranes showing heavy labeling for the α3 integrin subunit (black dots). (Calibration bar = 0.4 μm.)
Figure 2
Figure 2
Biochemical characteristics of SNSs. (A) Western immunoblot with antibodies specific for reelin, 400- and 320-kDa (A1); postsynaptic density protein, 95-kDa (A2); and synaptophysin, 35-kDa proteins (A3). (B) Western immunoblot with α3 or β1 integrin receptor subunit-specific antibodies in 0.01% Triton X-100-treated SNSs. (C) Western immunoblot with reelin-specific (G10) antibody of SNSs before and after treatment with 0.01% Triton X-100. (D) RT-PCR of Arc mRNA in SNSs incubated without (lanes a and c) or with (lanes b and d) reelin (30 pM/45 min at 22°C and 30 min at 37°C). Total SNS (I) and polyribosome (II) Arc mRNA. (E) S.B. of [3H]flunitrazepam (2 nM) to SNS, myelin (My), and mitochondria (Mt) fractions. Each bar is the mean ± SE of triplicate assays. (F) S.B. of [125I]echistatin (50 fM) to the samples described in E. Each bar is the mean ± SE of triplicate assays.
Figure 3
Figure 3
Competition between echistatin and reelin binding to integrin receptors. (A) S.B. [125I]echistatin to SNS membranes treated with 0.01% Triton X-100 to remove native reelin. (Inset) Scatchard analysis of the data. (B) Recombinant reelin dose-dependent displacement of [125I]echistatin binding to integrin receptors. Culture medium from mock transfected cells [(−)reelin] was used as control. Each data point is the average of triplicate measurements. Experiments were repeated three to five times, yielding similar results.
Figure 4
Figure 4
Reelin stimulates [35S]methionine incorporation into SNS proteins. (A) Dose–response curve of reelin stimulation of [35S]methionine incorporation into proteins. SNSs treated with 0.01% Triton X-100 were preincubated with recombinant reelin [(+)reelin)] or culture medium of mock transfected cells [(−)reelin] (45 min at 22°C before adding [35S]methionine and additional 30 min at 37°C after methionine addition). Each value is the mean ± SE of triplicate assays. (B) An aliquot of reelin (30 pmol in 10 μl) was incubated with equal volume of G10 antibody (10 mg protein/ml) at 4°C overnight. After absorption with Protein-G Sepharose, an aliquot of the supernatant, diluted to obtain a final reelin concentration of 30 pM, was applied to SNSs. Western immunoblot (Inset) of reelin content in supernatants preabsorbed with or without G10 antibody. Each value is the mean ± SE of triplicate assays.
Figure 5
Figure 5
Echistatin blocks reelin but not high K+ ion-induced [35S]methionine incorporation into proteins. (A) Echistatin dose dependently antagonizes the action of reelin. (B) Echistatin (1 μM) failed to abolish K+ (40 mM)-induced [35S]methionine incorporation into SNS proteins. (C) Echistatin displaces reelin bound to SNSs. Bars represent immunofluorescence intensity ratio between reelin (r) and β-actin (a). The SNSs were first incubated with or without recombinant reelin (30 pM) for 45 min at room temperature, then echistatin was added to the mixture and incubated for another 30 min at 37°C. After two washes, reelin bound to SNS membranes was measured by Western immunoblot (Inset) with G-10 anti-reelin antibody and referred to β-actin immunoreactivity. Each bar is the average of determinations made in triplicate. Open bars, non-echistatin treatment; filled bar, echistatin treatment. Lane 1, non-treatment; lane 2, reelin; lanes 3–5, reelin plus echistatin; lanes 6–8, echistatin.
Figure 6
Figure 6
Rapamycin blocks reelin-induced [35S]methionine incorporation into proteins. (A) Rapamycin dose dependently inhibits reelin (30 pM) stimulation of [35S]methionine incorporation into SNS proteins. Each value is the mean ± SE of triplicate assays. (Inset) Western immunoblot with mTOR-specific antibody. TB, total brain. (B) Western immunoblot of reelin bound to SNSs after incubation with or without reelin in the presence or absence of different doses of rapamycin. Bars represent immunofluorescence intensity ratio between reelin (r) and β-actin (a). Lane 1, non-treatment; lane 2, reelin; lanes 3–5, reelin plus rapamycin; lanes 6–8, only rapamycin.
Figure 7
Figure 7
Reelin enhances [35S]methionine incorporation into selected proteins. (A) SNSs preincubated for 45 min at room temperature without or with reelin (30 pM) were incubated for 30 min at 37°C with [35S]methionine, and proteins were precipitated with 10% TCA. Equal amounts of proteins (10 μg) from (−)- or (+)Reelin-stimulated samples were subjected to SDS/7% PAGE and Western blotting. (A1) Western blot autoradiography of unstimulated (−) and reelin-stimulated (+) SNS samples, respectively. (A2) Western immunoblot of A1 with Arc and β-actin antibodies. No apparent differences in the amount of β-actin or Arc immunoreactivity were observed. (A3) Phosphor screen peaks (I, 150 kDa; II, 120 kDa; III, 80 kDa; and IV, 55 kDa) of the radioactive bands of A1. The areas under the peaks were corrected for the amount of β-actin measured in A2 by fluorescence intensity [i.e., (−)reelin = 2.7; (+)reelin 2.7 pixel 106]. The ratios of the area under peak I, II, III, and IV between (+)- and (−)reelin after β-actin correction were 1.7, 1.1, 1.2, and 2.2, respectively. Peak IV radioactive band corresponds to the A2 band of Arc immunoreactivity. (B) SNSs incubated as described in A were lysed and then were subjected to immunoprecipitation with anti-Arc antibody (see Materials and Methods) before SDS/7% PAGE and Western blot. The data show that reelin increases the incorporation of [35S]methionine into Arc. (B1) Western blot autoradiography showing a single radioactive band of 55 kDa in unstimulated (−) and reelin-stimulated (+) SNS samples. (B2) Western immunoblot of B1 with Arc-specific antibody, indicating the radioactive band in B1 has an electrophoretic position identical to that of Arc. (B3) Phosphor screen peaks of 55-kDa radioactive bands of B1 in (−)reelin and (+)reelin SNS-stimulated samples. The areas under the peaks were corrected for the amount of Arc measured in B2 by fluorescence intensity [i.e., (−)reelin = 3.2; (+)reelin 3.0 pixel 106]. The ratios of the area under the 55-kDa peaks between (+)- and (−)reelin was 1.55. Experiments were repeated three times, yielding similar results.
Figure 8
Figure 8
Reelin selectively stimulates [35S]methionine incorporation into Arc: inhibition by rapamycin. Open bars represent [35S]methionine incorporated into total sample (2 mg of protein per sample). Filled bars represent [35S]methionine incorporated into Arc protein (immunoprecipitated with Arc polyclonal-specific antibody, see Materials and Methods). SNSs were treated with none, basal condition (a), reelin (30 pM) (b), reelin (30 pM) and rapamycin (5 nM) (c), and rapamycin only (5 nM) (d). Each bar is the mean ± SE of triplicate assays.
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