Novel interaction of Rab13 and Rab8 with endospanins

Abstract

Rab GTPases regulate vesicular traffic in eukaryotic cells by cycling between the active GTP-bound and inactive GDP-bound states. Their functions are modulated by the diverse selection of effector proteins that bind to specific Rabs in their activated state. We previously described the expression of Rab13 in bone cells. To search for novel Rab13 interaction partners, we screened a newborn rat bone marrow cDNA library for Rab13 effectors with a bacterial two-hybrid system. We found that Rab13 binds to the C-terminus of Endospanin-2, a small transmembrane protein. In addition to Rab13 also Rab8 bound to Endospanin-2, while no binding of Rab7, Rab10, Rab11 or Rab32 was observed. Rab13 and Rab8 also interacted with Endospanin-1, a close homolog of Endospanin-2. Rab13 and Endospanin-2 colocalised in perinuclear vesicular structures in Cos1 cells suggesting direct binding also in vivo. Endospanin-2 is implicated in the regulation of the cell surface growth hormone receptor (GHR), but the inhibition of Rab13 expression did not affect GHR cell surface expression. This suggests that the Rab13-Endospanin-2 interaction may have functions other than GHR regulation. In conclusion, we have identified a novel interaction for Rab13 and Rab8 with Endospanin-2 and Endospanin-1. The role of this interaction in cell physiology, however, remains to be elucidated.

Keywords: Endospanin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GHR, growth hormone receptor; GST, glutathione-S-transferase; HA, human influenza hemagglutinin; MBP, maltose binding protein; OB-R, leptin receptor; Osteoclast; Protein interaction; Rab effector; Rab13; VPS55, vacuolar protein sorting 55.; Vesicle trafficking.

Fig. 1

Rab13 binding to the Endospanin-2 C-terminal tail is specific. (A) Bacterial transformants carrying the target vector pTRG-3D3 and the bait pBT vectors encoding Rab13Q67L, Rab13WT, Rab7 or empty pBT vector were cultured in selective minimal medium. Positive and negative controls were from Bacteriomatch II Two-Hybrid System. Positive interaction was indicated by bacterial growth in the selective medium. (B) A schematic of Endospanin-2. TM indicates transmembrane domain, dark grey and white indicate extracellular and intracellular domains, respectively. Scale bar indicates the C-terminal tail identified as Rab13 binding in bacterial two-hybrid system. Asterisk marks the insertion site of the HA tag in Endospanin-2HA overexpression construct. (C) Bacterially expressed GST-Rab13, GST-Rab7, GST-Rab11 and GST-Rab32 proteins were precipitated with C-terminal tails of Endospanin-2 (Endo-2C) or Endospanin-1 (Endo-1C) fused to MBP and subjected to immunoblotting. Negative controls were GST-Rab13 with MBP* or empty amylose resin beads and MBP-Endo-2C with GST. (D) Bacterially expressed MBP-Endo-2C and MBP-Endo-1C were bound to amylose resin beads and incubated with HeLa cell lysates. Retained proteins were analysed by western blotting. (E) The specificity of the polyclonal Rab13 antibody was validated with Cos1 cell lysates transfected with GFP-Rab8, 10 and 13 eukaryotic expression vectors. (F) MBP-Endo-2C was bound to amylose resin beads and incubated with GFP-Rab8, 10 or 13 transfected Cos1 cell lysates. Retained proteins were analysed by western blotting. Equal protein inputs were confirmed by immunoblotting and/or Coomassie blue staining. Calculated molecular mass values are: MBP-Endospanin-2 and Endospanin-1, 45 kDa; MBP* (MBP4*-β-gal α fragment from pMal-c2E plasmid with no insert), 51 kDa; GST, 26 kDa; GST-Rab7, 50 kDA; GST-Rab11, 51 kDa; GST-Rab13, 49 kDa; GST-Rab32, 52 kDa; endogenous Rab13, 23 kDa; GFP-Rab8, 50 kDa; GFP-Rab10, 49 kDa and GFP-Rab13, 49 kDa.

Fig. 2

Endospanin-2 associates with Rab13WT and Rab13Q67L in cellular systems. (A) Cos1 cells were transiently transfected with Endospanin-2HA and GFP-Rab13WT, GFP-Rab13Q67L, GFP-Rab13T22N or GFP-Rab32 constructs, lysed after 48 h, immunoprecipitated with an anti-GFP antibody and subjected to western blot analysis with anti-HA and anti-GFP antibodies. The negative control was Endospanin-2HA- and GFP-Rab13WT-transfected cell lysates without the anti-GFP antibody. (B) Densitometric analysis of three independent co-immunoprecipitation experiments (mean + standard deviation). Student's t-test shows no statistical significance for Rab13WT and Rab13Q67L binding to Endospanin-2HA. (C) Cos1 cells were co-transfected with GFP-Rab13WT and Endospanin-2HA and labelled with Ha.11 antibody. Rab13 (green) and Endospanin-2HA (red) were colocalised (yellow) in vesicles at the perinuclear area and at the cell periphery. Rab13 showed clear plasma membrane staining (white arrowheads), whereas few Endospanin-2HA-positive vesicles were detected at the cell periphery. Square box displays the magnified field. Scale bars, 10 μm. Densitometric analysis and the determination of the Pearson's correlation coefficient (n = 10 cells) were done with ImageJ software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Effect of rab13 downregulation on Endospanin-2HA localisation and cell surface expression of GHR. (A) Rab13 siRNA molecules downregulated Rab13 protein expression by 80% in HeLa cells (quantitation with ImageJ not shown). (B) The distribution of Endospanin-2HA in control siRNA- and Rab13 siRNA-transfected HeLa cells. Shown are extended focus images reconstructed from the original confocal sections by adding 20 sections taken at 0.117 μm intervals together. Scale bar, 10 μm. (C) GHR expression in LNCaP cells, as demonstrated by immunoprecipitation with an anti-GHR (mAb263) antibody. (D) Rab13 siRNA treatment reduced the expression of Rab13 protein by 80% in LNCap cells (quantitation with ImageJ not shown). (E) siRNA-transfected LNCaP cells were incubated with mAb263 (anti-GHR) and an Alexa Fuor 488-conjugated secondary antibody and analysed by flow cytometry. Data are represented as average mean fluorescent intensities with standard deviations (MFI + SD) from four independent experiments. Student's t-test shows no statistical significance.

Fig. 4

Endospanin-2 is expressed in rat osteoclasts. Total RNA was isolated from rat osteoclasts enriched by magnetic separation with a β₃-integrin antibody from newborn rat bone marrow (Osteoclasts) and the remaining cell population (Non-Osteoclasts). After reverse transcription, Endospanin-2, Cathepsin K and GAPDH were amplified with gene specific primer pairs by PCR.