Abstract
Parathyroid hormone-related peptide (PTHrP) is an important causal factor for hypercalcemia associated with malignancy. In addition to the endocrine functions attributed to secretory forms of the peptide, PTHrP also plays a local role as a mediator of cellular growth and differentiation presumably at least in part through intracellular pathways. In studying the post-translational regulation of PTHrP, we observed that PTHrP was conjugated to multiple ubiquitin moieties. We report here that the proteasome is responsible for the degradation of the endoplasmic reticulum-associated precursor, pro-PTHrP. Cells expressing prepro-PTHrP and exposed to lactacystin accumulate pro-PTHrP assessed by anti-pro specific antibodies. Brefeldin A-treated cells also accumulate pro-PTHrP suggesting that degradation does not occur in the endoplasmic reticulum (ER) lumen. Subcellular fractionation of both lactacystin and brefeldin A-treated cells indicated that accumulated pro-PTHrP resides in microsomal fractions with a portion of the protein exposed to the cytosolic side of the ER membrane as assessed by protease protection experiments. Immunoprecipitation and Western blot analysis identified pro-PTHrP in association with the ER molecular chaperone protein BiP. We conclude that pro-PTHrP from the ER can gain access to the cytoplasmic side of the ER membrane where it can undergo ubiquitination and degradation by the proteasome.
Highlights
Parathyroid hormone-related protein (PTHrP)1 is a secretory factor that is responsible for hypercalcemia in patients with cancer [1]
Lactacystin Causes Accumulation of PTHrP—We have previously shown that the proteasome inhibitor, MG-132, caused the accumulation of ubiquitinated prepro-PTHrP when PTHrP was translated in vitro in reticulocyte lysate, expressed in transiently transfected COS-7 cells, and expressed in stably transfected E36 Chinese hamster lung fibroblasts [16]
PTHrP expressed in E36 cells but lacked the secretory prepro leader sequence (⌬P) and expressed in the cytosol, which accumulated in the presence of lactacystin (Fig. 1, compare lane 2 to lane 1)
Summary
Parathyroid hormone-related protein (PTHrP) is a secretory factor that is responsible for hypercalcemia in patients with cancer [1]. PTHrP is proposed to enhance the proliferation of chondrocytes and delay their terminal differentiation [13] This action is most likely in large part because of the secretion of PTHrP from normal proliferating and maturing chondrocytes. The ability of overexpressed PTHrP to inhibit apoptosis was shown to depend on nuclear localization of PTHrP [14] and induction of the apoptotic inhibitor, Bcl-2 [12]. Taken together, these studies suggest an intracellular function for PTHrP distinct from the classical signal transduction cascades linked to the PTH/PTHrP receptor. While studying the post-translational regulation of PTHrP, we showed that full-length endogenously overexpressed PTHrP was degraded by the ubiquitin-dependent proteolytic system [16]. The biochemical mechanisms and proteases that mediate this ER degradation remain unclear
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