Distribution of Mchr1 positive cilia in the mouse brain.

<p>The relative number of Mchr1-positive cilia in each brain region, normalized to AC3-positive cilia, is designated by: +, sparse distribution of Mchr1-positive cilia; ++, moderate distribution of Mchr1-positive cilia; +++, extensive distribution of Mchr1-positive cilia; and ++++, highest detection of Mchr1-positive cilia.</p

Mchr1 localizes to neuronal cilia throughout the mouse brain.

<p>(A–L) Representative images of multiple brain regions in 5–6 week old WT mice showing colabeling for Mchr1 (green) and AC3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for AC3 reveals numerous primary cilia present throughout the CA1 region of the hippocampus (B), amygdala (E), piriform cortex (H), and fasciolar gyrus (K). Labeling for Mchr1 (A, D, G, & J) reveals abundant Mchr1 ciliary localization in each region. Merged images (C, F, I, L) showing colocalization between Mchr1 and AC3. Scale bars represent 10 µm.</p

Mchr1 and Sstr3 colocalize in a subset of hippocampal neuronal cilia.

<p>(A–F) Representative image of the CA1 region of the hippocampus from an adult WT mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the hippocampus. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D–F) reveals a subset of neuronal cilia that are positive for Mchr1 only (arrow) and a subset that are positive for both Mchr1 and Sstr3 (arrowhead). (G–I) Colabeling of day 7 WT hippocampal neurons with antibodies to Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Merged image (I) shows Mchr1 and Sstr3 colocalization within the same cilium <i>in vitro</i>. (J) Quantification of day 7 WT hippocampal neurons colabeled for Mchr1 and AC3 or Sstr3 and AC3 reveals that Mchr1localizes to 25.73±3.53% (n = 239) of AC3-positive cilia and Sstr3 localizes to 61.46±4.12% (n = 221) of AC3-positive cilia. (K) Graphical representation of the quantification of day 7 WT hippocampal neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 54.43±5.78% (n = 67) of Mchr1-positive cilia. Scale bars represent 10 µm.</p

Mchr1 and Sstr3 interact in live cells.

<p>HEK293T cells were transiently co-transfected with constructs encoding CFP-tagged Mchr1 and YFP-tagged Sstr3. Mchr1-CFP and Sstr3-YFP colocalize at the plasma membrane and in intracellular compartments (A–C). Robust FRET signals are observed between Mchr1-CFP and Sstr3-YFP localizing in intracellular compartments and moderate FRET signals are observed between Mchr1-CFP and Sstr3-YFP localizing in the cell membrane (D). The fluorescence intensity profile along the red-dotted line within the FRET_corrected image is shown (E). The intensity scale for CFP (blue) and FRET_corrected (red) are on the left. The intensity scale for YFP (green) is on the right. AU = Arbitrary Unit.</p

Mchr1 and Sstr3 colocalize in a subset of piriform cortical neuronal cilia.

<p>(A–F) Representative image of the piriform cortex from an adult WT mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the piriform cortex. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D–F) reveals a subset of neuronal cilia that are positive for both Mchr1 and Sstr3 (arrowhead). (G) Quantification of day 7 WT piriform cortical neurons colabeled for Mchr1 and AC3 or Sstr3 and AC3 reveals that Mchr1 localizes to 55.62±3.63% (n = 186) of AC3-positive cilia and Sstr3 localizes to 14.03±1.71% (n = 159) of AC3-positive cilia. (H) Graphical representation of the quantification of day 7 WT piriform cortical neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 23.36±5.11% (n = 86) of Mchr1-positive cilia. Scale bars represent 10 µm.</p

Mchr1 and Sstr3 proteins interact.

<p>Sstr3 was co-expressed with myc-tagged glyceraldehyde-3-phosphate dehydrogenase (Gapdh-myc) or myc-tagged Mchr1 (Mchr1-myc) in HEK293T cells. (A) Cell extracts were immunoprecipitated (IP) with an anti-myc antibody. Immunoprecipitates were analyzed by western blotting (IB) with an anti-Sstr3 antibody (left). Note that Sstr3 is immunoprecipitated with Mchr1, as indicated by the 45 kDa band, but not Gapdh. (B) In the reverse experiment, cell extracts were immunoprecipitated (IP) with an anti-Sstr3 antibody and immunoprecipitates were analyzed by western blotting (IB) with an anti-myc antibody (left). Note that Mchr1 is immunoprecipitated with Sstr3, as indicated by the 37 kDa band, but not Gapdh. The input, confirming expression of each protein, is also shown (right). Sstr3 appears as a 45 kDa band, which agrees with its predicted size, and 75 kDa and 150 kDa bands, which may represent higher order oligomers. Gapdh and Mchr1 are observed as 39 kDa and 37 kDa bands, respectively. The expression pattern of GPCRs often results in a multi-band pattern due to various oligomeric combinations and post translational modifications.</p

Mchr1 and Sstr3 colocalize in a subset of hypothalamic neuronal cilia.

<p>(A–F) Representative image of the hypothalamus from an adult WT mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the hypothalamus. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D–F) reveals a subset of neuronal cilia that are positive for both Mchr1 and Sstr3 (arrowhead). (G) Quantification of day 7 WT hypothalamic neurons colabeled for Mchr1 and AC3 or Sstr3 and AC3 reveals that Mchr1localizes to 63.42±4.59% (n = 108) of AC3-positive cilia and Sstr3 localizes to 33.34±5.24% (n = 129) of AC3-positive cilia. (H) Graphical representation of the quantification of day 7 WT piriform cortex neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 20.36±4.39% (n = 85) of Mchr1-positive cilia. Scale bars represent 10 µm.</p

Mchr1 and Sstr3 interact in mouse hippocampal lysate.

<p>Membrane protein enriched cell lysate from the hippocampus of 5 week old adult WT mice was immunoprecipitated (IP) with a goat anti-Mchr1 antibody or goat IgG, as a negative control. Immunoprecipitates were analyzed by western blotting (IB) with a rabbit anti-Sstr3 antibody. Note that Sstr3 is immunoprecipitated with Mchr1 but not with the IgG negative control. The input probed with anti-Sstr3 (left) or anti-Mchr1 (right), confirms the expression of Sstr3 and Mchr1. The ∼55 kDa bands in the IP lanes may be IgG heavy chain that is detected due to cross-reactivity of the secondary antibody.</p

Practical Synthesis of MDM2 Antagonist RG7388. Part 1: A Cu(II)-Catalyzed Asymmetric [3 + 2] Cycloaddition

An efficient asymmetric synthesis of MDM2 antagonist RG7388 is reported. The highly functionalized chiral pyrrolidine carboxamide was assembled via a Cu­(OAc)₂/(<i>R</i>)-BINAP catalyzed asymmetric [3 + 2] cycloaddition, which gave the exo and endo adducts in a ratio of 10:1, with high enantiomeric excess for the exo isomer. A one-pot hydrolysis and retro-Mannich/Mannich isomerization of the cycloaddition adducts in the presence of aqueous sodium hydroxide afforded RG7388 in high chemical and enantiomeric purities and 69% overall yield