Sulfation of newly synthesized POMC in wild-type and transgenic <i>Xenopus</i> intermediate pituitary cells.

<p>Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with ³⁵S-sulfate and ³H-lysine for 15 min. Newly synthesized proteins extracted from the NILs were resolved by 15% SDS-PAGE and the amount of [³⁵S]SO₄ and ³H-lysine incorporated into newly-synthesized 37K POMC was determined. Shown are the amounts of newly synthesized sulfated 37K POMC produced in the transgenic relative to wt NILs. Data are shown as means +/− SEM (wt, n = 7; transgenics, n = 5). *, p<0.05; n.s., not significant.</p

Electron microscopy analysis of wild-type and transgenic melanotrope cells.

<p>(A–C) Wild-type (wt; A1 and A2) intermediate pituitary cells showed a well-developed rough endoplasmic reticulum and extensive Golgi-ribbons. The p24α₃-transgenic cells (#55; B1 and B2) contained Golgi mini-stacks and large electron-dense structures (EDS). The p24δ₂-transgenic cells (#224; C1 and C2) showed an ultrastructure similar to that of wild-type cells. The dotted lines highlight the outline of the Golgi. (D–F) Immuno-electron microscopy analysis of intermediate pituitary melanotrope cells from wt frogs (D), and frogs transgenic for p24α₃ (#55; E) or p24δ₂ (#224; F) using an anti-POMC antiserum. Immunoreactivity was found in dense-core secretory granules (filled arrowheads) in wt and p24α₃- and p24δ₂-transgenic cells and occasionally in newly forming secretion granules still attached to the <i>trans</i>-Golgi network (open arrowheads). In addition, in the p24α₃-transgenic cells a strong POMC-immunolabeling was observed in the EDS, which were localized to the ER lumen (arrow) and occasionally in EDS newly forming within the ER lumen (open arrow). G, Golgi; L, lysosome; M, mitochondrion; N, nucleus; RER, rough endoplasmic reticulum; PM, plasma membrane; sg, immature secretory granules. Bars equal 1 µm (A–C); 500 nm (D–F).</p

The effect of p24α₃- or p24δ₂-transgene expression on POMC biosynthesis and processing in <i>Xenopus</i> melanotropes.

<p>(A–C) Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with [³⁵S]-Met/Cys for 30 min and subsequently chased for 3 hrs. Newly synthesized proteins extracted from the NILs (Cells; 5% of extract) and secreted into the incubation medium (Media; 20%) were resolved by 15% SDS-PAGE and visualized by autoradiography. (A) The analysis was performed in six independent experiments and a representative autoradiogram is shown. (B) The amount of newly synthesized 37K POMC in wild-type (n = 16) and the p24α₃-transgenic (n = 10) and p24δ₂-transgenic (n = 6) cells was quantified and is shown relative to the wild-type cells. (C) The amounts of newly synthesized 18K and 18K* POMC in wild-type (n = 12) and the p24α₃-transgenic (n = 5) and p24δ₂-transgenic (n = 6) cells were quantified and are shown relative to wild-type 18K POMC. Indicated are the 18K/18K* ratios and their statistical evaluations. Data are shown as means +/− SEM. n.s., not significant; **, p<0.01; ***, p<0.001.</p

Generation of <i>Xenopus</i> with transgene expression of p24α₃ or p24δ₂ specifically in the melanotrope cells.

<p>(A and B) Schematic representation of the linear injection fragments pPOMC-p24α₃-GFP (A) and pPOMC-p24δ₂-GFP (B) containing a <i>Xenopus</i> POMC gene promoter fragment (pPOMC) and the protein-coding sequence of p24α₃-GFP (transgenic lines #605, #55 and #602) or p24δ₂-GFP (lines #125, #115, #124 and #224); pPOMC drives transgene expression specifically to the melanotrope cells. (C) Pituitary-specific GFP-fluorescence (arrows) in living tadpoles transgenic for p24α₃ (line #55) or p24δ₂ (line #224); G, gut; E, eye; N, nose. (D) Fluorescence in the intermediate lobe (IL) and not in the anterior lobe (AL) of the pituitary of adult frogs transgenic for p24α₃ (#55) or p24δ₂ (#224).</p

Steady-state levels of secretory cargo proteins in wild-type and transgenic <i>Xenopus</i> intermediate pituitary cells.

<p>Western blot analysis of neurointermediate lobe (NIL) lysates from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) using antibodies directed against the soluble cargo proteins proopiomelanocortin (POMC) and prohormone convertase 2 (PC2), and the transmembrane cargo amyloid-β precursor protein (APP). Tubulin was used as a control for equal loading.</p

Newly synthesized 18K and 18K* POMC differ in N-glycosylation.

<p>Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with [³⁵S]-Met/Cys for 30 min and subsequently chased for 3 hrs. Newly synthesized proteins extracted from the NILs were deglycosylated with PNGaseF (F) or control-treated (C), resolved by 20% SDS-PAGE and visualized by autoradiography; the #55 lanes were exposed three times longer than the other lanes.</p