The Primate-specific Protein TBC1D3 Is Required for Optimal Macropinocytosis in a Novel ARF6-dependent Pathway

Abstract

The generation of novel genes and proteins throughout evolution has been proposed to occur as a result of whole genome and gene duplications, exon shuffling, and retrotransposition events. The analysis of such genes might thus shed light into the functional complexity associated with highly evolved species. One such case is represented by TBC1D3, a primatespecific gene, harboring a TBC domain. Because TBC domains encode Rab-specific GAP activities, TBC-containing proteins are predicted to play a major role in endocytosis and intracellular traffic. Here, we show that the TBC1D3 gene originated late in evolution, likely through a duplication of the RNTRE locus, and underwent gene amplification during primate speciation. Despite possessing a TBC domain, TBC1D3 is apparently devoid of Rab-GAP activity. However, TBC1D3 regulates the optimal rate of epidermal growth factor-mediated macropinocytosis by participating in a novel pathway involving ARF6 and RAB5. In addition, TBC1D3 binds and colocalize to GGA3, an ARF6-effector, in an ARF6-dependent manner, and synergize with it in promoting macropinocytosis, suggesting that the two proteins act together in this process. Accordingly, GGA3 siRNA-mediated ablation impaired TBC1D3-induced macropinocytosis. We thus uncover a novel signaling pathway that appeared after primate speciation. Within this pathway, a TBC1D3:GGA3 complex contributes to optimal propagation of signals, ultimately facilitating the macropinocytic process. INTRODUCTIONGene duplication, exon shuffling, and retrotransposition events played crucial roles during vertebrate evolution (Long, 2001;McLysaght et al., 2002;Brosius, 2003). About 5% of the human genome is composed of duplicated segments that emerged during the past 35 million years of primate evolution, resulting in the generation of novel protein functions (Courseaux and Nahon, 2001;Taylor and Raes, 2004).One of the cellular processes, whose regulation has became more and more complex and tightly regulated during evolution is endocytosis. Endocytosis serves to maintain cellular and organismal homeostasis by mediating the uptake of fluids, solutes, and signaling molecules and their receptors. Multiple mechanisms of endocytosis operate within a single cell (Conner and Schmid, 2003). Among them, two major categories are phagocytosis and pinocytosis, which mediate the uptake of particles or of fluid, respectively (Conner and Schmid, 2003).Pinocytosis encompasses a variety of different membraneentry routes and is invariably characterized by the relatively small size (50 -150 nm) of the internalized particles. Large volumes of fluid are, instead, engulfed by extension, folding, and closure of plasma membranes (PM) through a process termed macropinocytosis (Swanson and Watts, 1995). Macropinocytosis displays many similarities to phagocytosis; the biochemical and cellular components of these endocytic mechanisms have been best characterized in hematopoietic cells, in processes such as phagocytosis by macrophages in response to Fc receptor stimulation, or ma...

ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1

“…It has been demonstrated previously that the dileucine motif (DISLLK) in the BACE1 carboxyterminal tail determines, to a significant degree, the localization and trafficking of BACE1 (34)(35)(36). This dileucine motif mediates the interaction with GGA proteins (2, 3), and among these, GGA3 is indeed involved in macropinocytotic uptake of ARF6-dependent cargo (33). In agreement with this, we could colocalize some GGA3 with BACE1 (Fig.…”

“…S8), strikingly similar to what is observed for GGA3 knockdown (6). Because GGA3 knockdown prevents BACE1 from being degraded (57) and functions as an ARF6 effector (33), this fuels the idea that the ARF6-dependent endosomal transport regulation of BACE1 is affected in both cases. Because GGA3 levels are as well downregulated in the brain of patients with AD (6), these independent findings suggest that not only GGA3 and ARF6 but likely many more effectors of ARF6 can be considered from a therapeutical point of view, with the aim of modulating APP processing through regulating BACE1 transport, recycling, and turnover in endosomes.…”

“…S9A). However, expressing TBC1D3, a Tre-2/Bub2/ Cdc16 (TBC)-containing protein that stimulates macropinocytosis through activation of the ARF6 route and recruitment of GGA3 (33), enhanced the colocalization of GGA3 and BACE1 (Fig. S9B).…”

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“…Arf6 may promote Rac activation through endosomal trafficking [39] or through interaction with Rac GEFs Dock180/ELMO [40] and Kalirin [41]. Some TBC (Tre-2, Bu2, Cdc16) domain containing proteins have also been shown to affect Arf6 activation [42] and lead to macropinocytosis [43]. …”

Genomic disorders: A window into human gene and genome evolution

“…TBC1D3 itself is derived from a segmental duplication that underwent multiple gene duplications during primate evolution (99). Interestingly, TBC1D3 underwent mutations with respect to its closest homolog, USP6NL and acquired the features of an adaptor molecule involved in the macropinocytic process (102).…”