Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Moser, E.
The interaction of the human cytomegalovirus encoded chemokine receptor US28 and GASP-1 and its implication on receptor trafficking and melanoma growth
[ Dissertation ] Medical University of Graz; 2010. pp. 157
[OPEN ACCESS]
FullText
- Autor*innen der Med Uni Graz:
- Moser Elisabeth
- Betreuer*innen:
- Schaider Helmut
- Waldhoer Maria
- Altmetrics:
- Abstract:
- Human cytomegalovirus (HCMV) encodes the seven transmembrane (7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here I show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). I find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Thus, GASP-1 is an important cellular determinant that regulates the post-endocytic trafficking of US28, which also affects the signaling capacities of the receptor. The interplay between the sorting of US28 and its constitutive signaling capacity might also be of clinical relevance. US28 has been shown to enhance tumorigenesis in nude mice when expressed in NIH3T3 cells. In contrast, US28 induces apoptosis in other cell lines, such as HEK293T or COS-7 cells, particularly via the activation of Caspases. Here I show evidence that the interaction of US28 and GASP-1 causes partial degradation of GASP-1 in HEK293 cells. Since GASP-1 contains a putative Caspase 3/7 cutting site, US28 may cause a partial degradation of GASP-1 and/or GASP-2 via the activation of Caspases, thus linking the GASP proteins to the apoptotic pathway. Thus, I hypothesized that US28 may reduce tumor development when expressed in melanoma cells. Indeed, I show that US28 induces apoptosis in two melanoma cell lines; i.e. Sbcl2, a slow growing melanoma cell line, and 451Lu, a fast growing, metastatic cell line. In addition, in a HFF (human foreskin fibroblast) cell model, US28 has previously been shown to sequester CC-chemokines, thereby reducing the chemokine concentration in the surrounding of a US28 expressing cell. Interestingly, the presence of US28 increases the concentration of the chemokine CCL2 in the supernatants of 451Lu cells. This effect seems to be linked to the constitutive ¿ i.e. ligand independent ¿ signaling capacities of US28. This was shown by expressing the US28?317 receptor mutant in these cells. This receptor lacks the capacity to internalize and thus displays enhanced constitutive signaling properties. In fact, US28?317 expressing cells yielded an up to 20-fold increase in CCL2 concentration when compared to control cells and additionally resulted in elevated vascular endothelial growth factor (VEGF) levels. These findings suggest that US28 diminishes growth of melanoma cells by activating the apoptotic pathway, rather than acting as a `chemokine sink¿. Summarized, US28 appears to employ differential ways to dictate whether a cell is targeted for apoptosis or proliferation, including the regulation of chemokine levels in the surrounding of a cell. However, the cellular background of a given cell e. g., differential expression of sorting proteins, seems to be crucial for the functional consequences of US28 expression.