Abstract
Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling.
Highlights
Ras proteins are highly similar in sequence and vary mostly in their C-terminal hypervariable region
We found a significant increase in Förster Resonance Energy Transfer (FRET) upon incubation of ATTO488-labelled GTPγ S-H-ras with the DY-547-labelled Ras Binding Domain of the Ras effector C-Raf, which is known to bind to Ras and here served as a positive control
Both the C-Raf-Ras binding domain (RBD) and the same extended by the cysteine rich domain (CRD) showed identically high FRET with Gal-1 when expressed in BHK21 cells (Fig. 3A)
Summary
Ras proteins are highly similar in sequence and vary mostly in their C-terminal hypervariable region (hvr). While purified A1-labelled GTPγ S-H-ras showed high FRET with lysates from BHK21 cells expressing mRFP-labelled C-Raf-RBD, no FRET was observed with mRFP-Gal-1 lysates (Fig. 1D). Whether residue D38A, which is at the centre of the Ras/RBD interface[48], affects the FRET between mRFP-Gal-1 and mGFP-H-rasG12V in HEK293-EBNA cells.
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