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Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic

¹ Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
² Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA

View larger version (16K): [in a new window]   Fig. 1. Western blots for GH and ß-COP. Cells were transfected with wt-GH, 32-71-GH or P89L-GH as shown at the top of gels. As shown on the left, wt-, P89L- and 32-71-GH were expressed at similar levels. The predicted size for wt- and P89L-GH is 22 kDa and for 32-71-GH is 17.5 kDa. As shown on the right, transfection of cells with wt-GH or 32-71-GH does not affect expression of endogenous ß-COP, so the disappearance of ß-COP staining in cells expressing 32-71-GH was not due to decreased ß-COP expression.

View larger version (44K): [in a new window]   Fig. 2. ER marker and GH staining. Cells cotransfected with wt-GH, 32-71-GH or P89L-GH and YC3er were stained with antibody to GFP, which labels the ER marker YC3er (left panels), and with antibody to GH (middle panels). Right panels show merged images with YC3er in green and GH in red; colocalized staining appears yellow. Only 32-71-GH colocalized with the ER marker.

View larger version (28K): [in a new window]   Fig. 3. Golgi marker and GH staining. Cells cotransfected with wt-GH or 32-71-GH were stained with antibody to the Golgi markers ß-COP (top left panels), Golgi 58K (middle left panels), or membrin (bottom left panels), and antibody to GH (center column of panels). Merged images show GH in red and Golgi markers in green; colocalized staining appears yellow. Where noted, 10 µg/ml tunicamycin was added for 3 hours during transfection and then removed. Arrowheads denote cells in which the Golgi almost disappeared and arrows show cells in which the Golgi was visible but dispersed.

View larger version (81K): [in a new window]   Fig. 4. Disruption of microtubule-organizing centers in cells expressing mutant GH. Cells cotransfected with wt-GH or 32-71-GH were stained with antibody to -tubulin (left panels) and antibody to GH (middle panels). Right panels show GH in red and -tubulin in green; colocalized staining appears yellow. Microtubule polymers can be seen emanating from distinct microtubule-organizing centers in cells expressing wt-GH, which is concentrated in the Golgi apparatus, and in cells not expressing any GH. The lower panels show that 32-71-GH is retained in the ER, and cells expressing 32-71-GH show no distinct microtubule organizing centers.

View larger version (53K): [in a new window]   Fig. 5. Mislocalization of membrane TRH receptor in cells expressing mutant GH and reversal with tunicamycin treatment. Cells cotransfected with wt-GH or 32-71-GH and HA-epitope-tagged TRH receptor were stained with antibody to the HA epitope (left panels) and antibody to GH (middle panels). Right panels show GH in red and HA in green; colocalized staining appears yellow. Approximately 80% of cells were positive for TRH receptor and 32% for GH. TRH receptor is localized on the plasma membrane in cells expressing wt-GH but retained in the ER in cells expressing the GH mutant. In cells expressing the GH mutant and treated with 10 µg/ml tunicamycin, plasma membrane localization of TRH receptor was restored in 45% of cells (bottom panels). TRHR, TRH receptor.

View larger version (19K): [in a new window]   Fig. 6. Mislocalization of prolactin in cells expressing mutant GH. Cells cotransfected with wt-GH or 32-71-GH and human prolactin were stained with antibody to prolactin (left panels) and antibody to GH (middle panels). Right panels show GH in red and prolactin in green; colocalized staining appears yellow. Prolactin is found in the Golgi apparatus of cells expressing wt-GH but retained in the ER in cells expressing 32-71-GH. PRL, prolactin.

View larger version (32K): [in a new window]   Fig. 7. Northern blots for BiP. Lanes 1-4: cells were transfected with GH or empty vector and incubated without or with 10 mM DTT for 6 hours. Lanes 5-7: cells were transfected with empty vector and treated with 1 or 10 µg/ml tunicamycin for 3 hours, then switched to drug-free medium. Lanes 8 and 9: cells were transfected with empty vector or mutant preproinsulin encoding insulin with V3A and C7S in the A chain. RNA was isolated after 24 hours. Blots were probed for BiP mRNA, then stripped and reprobed for cyclophilin A (CyA).