Cell Type-Specific Tandem Affinity Purification of the Mouse Hippocampal CB1 Receptor-Associated Proteome

G protein coupled receptors (GPCRs) exert their effects through multiprotein signaling complexes. The cannabinoid receptor type 1 (CB1) is among the most abundant GPCRs in the mammalian brain and involved in a plethora of physiological functions. We used a combination of viral-mediated cell type-specific expression of a tagged CB1 fusion protein (CB1-SF), tandem affinity purification (TAP) and proteomics on hippocampal mouse tissue to analyze the composition and differences of CB1 protein complexes in glutamatergic neurons and in GABAergic interneurons. Purified proteins underwent tryptic digestion and were identified using deep-coverage data-independent acquisition with ion mobility separation-enhanced mass spectroscopy, leading to the identification of 951 proteins specifically enriched in glutamatergic and GABAergic CB1-SF TAP samples as compared to controls. Gene Ontology and protein network analyses showed an enrichment of single proteins and functional clusters of proteins involved in already well described domains of CB1 functions. Supported by this consistent data set we could confirm already known CB1 interactors, reveal new potentially interacting proteins and differences in cell type-specific signaling properties of CB1, thereby providing the foundation for further functional studies on differential CB1 signaling

Purification of cf-cyt. b₆ and the two cyt. b₆ halves cf-cyt. b₆-AB and cf-cyt. b₆-CD, and assembly of the cyt. b₆ holo-protein.

<p>(A) Proteins were separated on an 18% SDS gel. M: molecular mass standard; P: cell-free expressed protein; AC: protein purified by affinity chromatography. (B to E) UV/VIS absorbance spectra were acquired under oxidizing (black) and reducing (gray) conditions. The inlet shows the redox difference spectrum. (B) Cf-apo-cyt. b₆ (3 μM) was mixed with heme (6 μM. (C) cf-cyt. b₆-AB was mixed with cf-cyt. b₆-CD in a one-to-one ratio (1 μM each), and heme (2 μM) was added in total protein to a heme ratio of one to one. (D) cf-cyt. b₆-AB (2 μM) was mixed with heme (2 μM) and (E) cf-cyt. b₆-CD (2 μM) was mixed with heme (2 μM). Note that different concentrations of protein and heme were used in (B) compared to (C), (D) and (E). The spectra in (B), (C) and (D) show the typical cyt. b₆ absorbance maxima (ox: 562 nm / 531nm / 414 nm, red: 562 nm / 532 nm / 429 nm, redox: 562 nm / 532 nm for the α-/β-/γ-band).</p

Absorbance spectra of cyt. b₆ after proteolysis.

<p>(A, B) Holo-cyt. b₆ was proteolytically digested with proteinase K. At defined time points (10 min light blue, 30 min green, 1 h yellow, 2 h red, 5 h purple) proteolysis was stopped and absorbance spectra were measured under oxidizing (A) and reducing conditions (B). As a control, the absorbance spectra of free heme (black) and reconstituted, undigested cyt. b₆ (dark blue) were measured. The color legend in (A) also applies to (B). All spectra show the cyt. b₆ characteristic maxima (ox: 562 nm / 531nm / 414 nm, red: 562 nm / 532 nm / 429 nm, for the α-/β-/γ-band).</p

<i>In vitro</i> reconstitution of cyt. b₆ and its G_n-mutants containing prolonged BC loops.

<p>Absorbance spectra of <i>in vitro</i> reconstituted cyt. b₆ (black), cyt. b₆-G₅ (dark gray) and cyt. b₆-G₁₀ (light gray) under oxidizing (A) and reducing (B) conditions. G₅ and G₁₀ stands for the number of Gly residues inserted into the BC-loop. The absorbance maxima are essentially identical for all three proteins (A: 561 nm / 532 nm / 414 m; B: 562 nm / 532 nm / 429 nm for the α-/β-/γ-band). The same applies for the redox difference spectra (562 nm / 532 nm for the α-/β-band) (inlet B). (C) and (D) show titrations of cyt. b₆ (black squares), cyt. b₆-G₅ (dark gray spheres) and cyt. b₆-G₁₀ (light gray triangles) with heme (C) or SDS (D) under oxidizing conditions. Each data point represents an individual measurement. The quotient of the absorbance intensity at 414 nm to 404 nm was calculated, representing the absorbance maximum of the Soret-band of bound and unbound heme, respectively, and normalized to “0” for solely free heme and “1” for solely bound heme. (C) Increasing amounts of protein were titrated into buffer containing 5 μM heme to measure heme-binding isotherms. For all three proteins, the titration experiments show a saturation at a protein/heme ratio of 0.5 and higher. (D) To determine the proteins´ stability, increasing amounts of SDS were added to reconstituted protein. All three cyt. b₆ variants are stable up to an SDS concentration of ~4.5 mM.</p

Delayed tumor growth and tumor protection after IMI-Gel application.

<p>E.G7 thymoma cells (4×10⁵ s.c.), expressing SIINFEKL, were injected into the flank of C57BL/6 mice. After the tumor was palpable mice were immunized as indicated on two consecutive days in weekly intervals over a period of three weeks or left untreated. <b>A)</b> The tumor size and <b>B)</b> the survival were monitored. Depicted are the cumulative results of three independent experiments with n = 23 for emulsion gel and Aldara and n = 17 for untreated control. *Significant difference with p<0.05 by Mantel Cox test compared to the untreated control group.</p

In vitro characterization of IMI-Gel.

<p>To characterize IMI-Gel and Aldara, imiquimod containing formulations were analyzed in terms of <b>A)</b> presence of imiquimod crystals in IMI-Gel using electron microscopy, <b>B)</b> sizes (mean+SD) of imiquimod-particles in IMI-Gel immediately or 9 months after manufacturing (under room conditions) and <b>C)</b> flow curves defining rheological characteristics.</p

IMI-Gel and Aldara are equally potent in inducing primary CTL-responses.

<p>C57BL/6 mice were shaved on their backs and afterwards immunized on two consecutive days with either Aldara (50 mg) together with SIINFEKL (100 µg) or IMI-Gel (50 mg) and officinal cremor basalis together with SIINFEKL or left untreated (untreated control). <b>A)</b> The frequency of peptide-specific CD8⁺ T cells in the blood (mean and SD) and <b>B)</b><i>in vivo</i> cytolytic activity 24 hours (mean and SD) after transfer of peptide-loaded target cells was assessed. Depicted are the cumulative results of two independent experiments with n = 6 for the tetramer staining and three independent experiments with n = 9 for the cytotoxicity assay. *Significant difference with p<0.05 by one-way ANOVA with Bonferroni’s posttest.</p

Imiquimod passes mouse skin <i>in vitro</i> more rapidly when formulated in Aldara than in IMI-Gel.

<p><b>A)</b> Release of imiquimod as the active component was detected with a modified Franz-diffusion-cell model. <b>B)</b> Shaved skin of C57BL/6 mice (n = 6) was ablated and afterwards treated with Aldara or IMI-Gel (each 50 mg). The imiquimod concentration in the acceptor medium was determined after various time points as indicated using HPLC (UV absorption 245 nm). *Significant difference with p<0.05 by Wilcoxon signed rank test. <b>C)</b> C57BL/6 mice (n = 3) were with Aldara or IMI-Gel (each 50 mg/6 cm²) on. After 3 hours mice were sacrificed and remaining formulation was removed with gauze. 1 cm² of treated skin was prepared, fat removed and subsequently hackled with an ultra turrax. The amount of imiquimod recovered from the skin surface (left panel) and within the skin (right panel) was determined using HPLC.</p

The route of IMI-Gel application influences vaccination efficacy.

<p>C57BL/6 mice were shaved on their backs and received the following treatments: untreated (untreated control), IMI-Gel with SIINFEKL (100 µg) (IMI-Gel TCI) on two consecutive days, IMI-Gel alone (IMI-Gel without SIIN) on two consecutive days, oral IMI-Gel (50 mg) with SIINFEKL (100 µg) on two consecutive days (IMI-Gel p. o.) or s. c. once with IMI-Gel (100 mg) diluted with SIINFEKL (200 µg) and distilled water into the neck. <b>A)</b> The frequency of peptide-specific CD8⁺ T cells in the blood (mean and SD) and <b>B)</b><i>in vivo</i> cytolytic activity 24 hours (mean and SD) after transfer of peptide-loaded target cells was assessed. Depicted are the cumulative results of two independent experiments with n = 7 for IMI-Gel treated groups and n = 4 for controls. *Significant difference with p<0.05 by Students <i>t</i> test.</p

Sequence alignment of the <i>Bg</i>AChBP subunits and <i>Ls</i>AChBP (from <i>Lymnaea stagnalis</i>).

<p>The red residues are addressed in the main text in the context of ligand binding (blue boxes), inter-pentamer linkage (red boxes), N-glycan binding (black boxes), or disulfide bridges (arrow symbols). The blue residues probably form salt bridges between adjacent subunits within the same pentamer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043685#pone-0043685-g004" target="_blank">Fig. 4E</a>). Note the specific exchanges Y92→F92 in <i>Bg</i>AChBP1 and Y193→F193 in <i>Bg</i>AChBP2. Also note the strictly conserved disulfide bridges stabilizing the eponymous Cys-loop L7 and the gating C-loop L10, the putative additional disulfide bridge C16↔C64 in <i>Bg</i>AChBP1, and the single cysteine C71 in <i>Bg</i>AChBP2. (Chain-specific residue numbers are given.) The secondary structure elements predicted from the published crystal structures are also indicated (L, loop). The short helix following strand β2 and marked in blue is absent in the molecular models of the BgAChBP subunits. Genbank entries JQ814367, JQ814368, AAK64377.</p