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Regulatory role of dynamin-2 in VEGFR-2/KDR-mediated endothelial signaling

Resham Bhattacharya^*, Ningling Kang-Decker, Deborah A. Hughes^*, Priyabrata Mukherjee^*, Vijay Shah, Mark A. McNiven^* and Debabrata Mukhopadhyay^*,1

^* Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Rochester, Minnesota, USA; and
GI Research Unit, Department of Medicine, Mayo Clinic Foundation, Rochester, Minnesota, USA

¹ Correspondence: Department Biochemistry and Molecular Biology, Gugg 1401A, Mayo Clinic Foundation, 200 First St. SW, Rochester, MN 55905, USA. E-mail: mukhopadhyay.debabrata{at}mayo.edu

SPECIFIC AIMS

KDR is the key receptor that initiates signaling events for VPF/VEGF causing endothelial cell migration, proliferation, and tube formation leading to angiogenesis. Currently, there are at least 80 different drugs in clinical trials targeting KDR. Nonetheless, there is a huge gap in the understanding of mechanisms that regulate compartmentalization and thus signaling of this receptor. Dynamin-2 is a signal-transducing GTPase that has been widely reported to be involved in clathrin- and caveolin-mediated receptor endocytosis. Therefore, the aim of this project was to elucidate the role of dynamin-2 in KDR endocytosis, compartmentalization, and signaling.

PRINCIPAL FINDINGS

1. KDR is present on the plasma membrane and endosomes as well as in the perinuclear region in HUVECs and colocalizes with EEA1, caveolin-1, and dynamin-2
Immunoflourescence in HUVECs, showed the presence of KDR on the membrane along with punctate staining distributed in the cytoplasm as well as distinct staining in the perinuclear area. We also performed colocalization studies of KDR with caveolin-1 and EEA-1 in control, WT and GTPase inactive K44A mutant of dynamin-2 expressing HUVECs. Most of the discrete puncta in which KDR was present colocalized with EEA-1, indicating that these were endosomes (Fig. 1 A, B). However, for the K44A mutant-expressing cells, the number of EEA-1-stained endosomes was fewer than that of the control or WT dynamin-2-expressing cells (Fig. 1C ). With caveolin-1, KDR showed some degree of colocalization at the plasma-membrane. However, a strong colocalization in the perinuclear area was observed (Fig. 1D , E). With dynamin-2, KDR colocalized in control as well as wild-type (WT) dynamin-2-overexpressing cells and was more prominent in the perinuclear area of the cells. This was further confirmed by coprecipitation of dynamin-2 and KDR in the same immune complex. Thus, KDR colocalizes with dynamin-2 and caveolin-1 and inactivation of dynamin-2 GTPase function results in loss of localization to the endosomal compartment.

View larger version (46K): [in this window] [in a new window]   Figure 1. Colocalization of KDR with EEA-1 and caveolin. HUVECs transduced without (A, D), with WT dynamin-2 (B, E), or with K44A adenovirus (C) were double stained with antibodies against KDR (green) and EEA-1 (A–C) or caveolin (D, E) (red). The arrows show area of colocalization of KDR and caveolin in the perinuclear region of the cells.

2. HUVECs overexpressing a mutant form of dynamin deficient in GTP binding (K44A) caused a selective decrease in KDR protein levels that was restored by proteasomal and lysosomal inhibitors
We observed a 60% decrease in KDR protein levels in the K44A dynamin-2 mutant-expressing cells (expression was checked using V5 tag, Fig. 2 A) as compared with that of the WT-dynamin-2 or control vector-expressing cells (Fig. 2B ). A similar decrease in the chimeric EGDR (EGFR-N-terminal and KDR-C-terminal) protein levels was also observed (Fig. 2C ). This decrease in protein level was specific to KDR as other receptors such as Flt-1, NRP-1, and FGFR-2 were not affected (Fig. 2D ). To verify that the decrease in KDR was at the protein level, we incubated the WT, K44A dynamin-2- or control vector-expressing cells with 2 cell-permeable protease inhibitors, E64d and PS1. We observed a marked increase in total as well as phosphorylated KDR levels upon treatment with either compound indicating involvement of lysosomal and proteasomal proteases.

View larger version (38K): [in this window] [in a new window]   Figure 2. Effect of K44A dynamin-2 expression on KDR and other receptors on HUVECs. A) Expression of K44A in HUVECs was confirmed by presence of V5 tag. B) Western blot analysis for KDR was done from lysates prepared from HUVECs infected with control vector (CV), WT dynamin-2, or the K44A adenovirus. C)Chimeric receptor EGDR was expressed in HUVECs by retroviral infection 1 day prior to adenoviral expression of CV, WT, or the K44A mutant. Immunoprecipitation from lysates was done using N-terminal EGFR antibody followed by immunoblot analysis with C-terminal KDR antibody. D) Western blot analysis of Flt-1, NRP-1, and FGFR-2 from lysates isolated from HUVECs after CV, WT, or K44A adenovirus infection.

3. Dynamin-2 is required for EC signaling/survival because expression of the K44A mutant significantly inhibited cell division and induced cell cycle arrest by inducing p21
The growth rate of HUVECs expressing the K44A dynamin-2 mutant was substantially lower than the WT-dynamin-2 and GFP-expressing cells. Proliferation, as measured by BrdU incorporation, was also significantly inhibited in the K44A dynamin-2-expressing cells. Cell cycle analysis revealed an increase in the percentage of cells in the G1 phase and a significant decrease in the percentage of cells in the S phase in K44A dynain-2 mutant as compared with that of WT dynamin-2 and control vector-expressing cells. Western blot analysis of cell cycle inhibitor proteins indicated induction of p21.

CONCLUSIONS AND SIGNIFICANCE

Overall, our data suggest that dynamin-2 not only places KDR in the right cellular compartment but may also protect it from protein degradation pathways. We have shown that KDR is localized not only on the membrane but also in the early endosomes and in the perinuclear area of HUVECs and that it colocalizes with dynamin-2, caveolin-1, and EEA1. Fewer EEA-1-stained endosomes observed in the K44A dynamin-2 mutant expressing cells compared with the control could be due to the requirement of dynamin’s GTPase function for vesicle fission from the plasma membrane. Dynamin-2 has previously been shown to be localized and involved in transport form the trans-Golgi network to the plasma membrane. Perinuclear colocalization of caveolin, dynamin-2, and KDR suggests that the three are in a complex probably located in the trans-Golgi region.

The decrease in intracellular KDR levels in the EGDR-expressing cells suggests that protein degradation was occurring in the HUVEC cytoplasm at the C terminus of the chimeric receptor. Therefore, failure of KDR to localize to particular compartments like the endosomal vesicles in the presence of mutant dynamin-2 might have allowed its enhanced degradation by the lysosomal and proteasomal proteases.

We found that the K44A dynamin-2 mutant, by decreasing KDR levels, caused a significant growth arrest of cells by inducing the cell cycle inhibitor protein p21. Our observation is in accordance with reports that p21 can cause an inhibition in S phase in venous endothelial cells. Subsequently, proliferation was also inhibited, indicating a signaling defect. Therefore, we have been able to show a novel role of dynamin-2 in endothelial cells where it is required for maintenance of KDR protein expression, thereby allowing the endothelium to have VPF/VEGF mediated signaling.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic illustration of hypothesized interactions of KDR with dynamin-2 in HUVECs. Caveolin and dynamin-2 are in the same complex with KDR, and this interaction could be mediated by Grb-2 and PLC. KDR localizes to the endosomes, lysosomes, and trans-golgi network. Dynamin-2 by its GTPase activity, regulates compartmentalization and prevents lysosomal and proteasomal degradation of KDR.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3889fje; doi: 10.1096/fj.05-3889fje

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