The small GTPase Rab5 has well-established roles in membrane trafficking — for example, in receptor endocytosis. However, as Di Fiore and colleagues now show in Nature, it seems that Rab5 is also a signalling GTPase. They show that Rab5 is essential for a form of receptor tyrosine kinase (RTK)-induced actin remodelling that produces waves/circular ruffles.

The signalling events that lead to circular ruffling have remained unclear, despite the fact that the formation of another type of ruffle — cell-edge ruffles — is known to involve an RTK–Ras–Rac linear cascade. However, from previous work, it was believed that Ras/Rac activation together with further RTK-triggered pathways might be required to induce circular ruffling.

Di Fiore and co-workers began by showing that a dominant-negative Rab5 mutant inhibited the RTK-dependent induction of circular ruffling: these effects were not the result of inhibiting endocytosis. They further dissected the signalling pathway that is involved in circular ruffling using various mutant proteins, and found that three independent signals — from Rab5, the Ras–phosphatidylinositol-3-kinase pathway and Rac — are simultaneously required to induce this ruffling.

To further understand the signals that connect Rab5 to circular ruffling, the authors carried out a structure–function analysis of the Rab5-specific GTPase-activating-protein (GAP) RN-tre. They showed that two independent functions of RN-tre — the GAP function and a function that resides in the carboxyl terminus — are linked to the formation of circular ruffles. So, what is the function of the latter region?

The authors used mass spectrometry to identify proteins that interact with the carboxyl terminus of RN-tre, and they found actinin-4 — an actin-binding protein that crosslinks filamentous (F)-actin. They showed that RNA interference against RN-tre or actinin-4 markedly impaired the circular ruffling that is induced by RTK activation. In addition, overexpressing actinin-4 could rescue the inhibitory effect of an RN-tre mutant on RTK-induced circular ruffling. It therefore seems that actinin-4 functions downstream of Rab5 and RN-tre in the RTK-dependent induction of circular ruffling.

In the final part of their study, Di Fiore and colleagues showed that RN-tre can bind to F-actin and actinin-4 simultaneously. So, this work has established Rab5 as a signalling GTPase and the authors propose “...that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4”, which might “...aid crosslinking of actin fibres into actin networks at the plasma membrane.”