Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells.
The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.
van Rosmalen
Jos W G
JW
Department of Molecular Animal Physiology, Nijmegen Center for Molecular Life Sciences and Institute for Neuroscience, Radboud University, Nijmegen, the Netherlands.
Martens
Gerard J M
GJ
D000818:Animals; D005260:Female; D015870:Gene Expression; D008297:Male; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism; D018621:PrPC Proteins / Q000096:biosynthesis* / Q000235:genetics; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism; D045706:Proprotein Convertase 2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D019076:Transgenes; D029867:Xenopus Proteins / Q000096:biosynthesis / Q000235:genetics; D014982:Xenopus laevis / Q000033:anatomy & histology / Q000235:genetics / Q000502:physiology
D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D011333:Pro-Opiomelanocortin; D045706:Proprotein Convertase 2