Abstract
Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.
Highlights
Adenocarcinomas of the prostate are amongst the most common malignancies in men in developed countries
After interacting with Tween 20-insoluble microdomains and as soon as Prostate-specific membrane antigen (PSMA) enters the Golgi, it associates with Lubrol WX-detergent-resistant membranes (DRMs) and this interaction is either maintained or restored once it reaches the plasma membrane [21]
Dimers of PSMA are Associated with Lubrol WX-DRMs LNCaP cells were solubilized with two different detergents, Lubrol WX and Triton X-100, and DRMs were extracted
Summary
Adenocarcinomas of the prostate are amongst the most common malignancies in men in developed countries. Conventional treatment like prostatectomy or radiation can be curative only if prostate cancer is diagnosed at an early stage. Prostate-specific membrane antigen (PSMA) is a type-IItransmembrane-glycoprotein with folate hydrolase and carboxypeptidase activity [1], found initially in LNCaP cells by immunoprecipitation [2]. PSMA is expressed in epithelial cells of the prostate and at low levels in some other organs like kidney, intestine and brain [3,4]. Elevated levels of PSMA are detected in prostate cancer cells including those that are metastatic [5,6]. In neovasculature of other non prostatic tumors PSMA expression has been detected, but it is absent from healthy vasculature [8,9]
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