An intriguing feature of CIE that sets it apart from CDE is that it can operate constitutively in “resting” cells but, upon stimulation, the architecture of the pathway can change to internalization by macropinocytosis during PM ruffling (). In HeLa and COS cells, this switch to macropinocytosis involves the same cargo proteins and membranes involved in CIE. We have found that this transformation can be initiated through the activation of a number of those signaling molecules that travel along the CIE pathway. Activation of Arf6, Rac, Ras, and Src all lead to PM ruffling and corresponding macropinocytosis. Here we will consider how Arf6, Ras and Src in particular can shift this endocytic pathway into a stimulated one.
We initially encountered macropinocytosis years ago when we were examining the effect of “activation” of Arf6 by treatment of Arf6 overexpressing cells with aluminum fluoride [35
]. Such cells appear similar to untransfected cells in the absence of stimulation but upon aluminum fluoride treatment exhibit actin-driven protrusive structures that internalize fluid phase tracers [35
] and CIE cargo proteins [7
] into large macropinosomes. Most of the fluid and membrane internalized is returned back to the PM. The same phenotype of protrusions and macropinocytosis could be observed by expressing a guanine nucleotide exchange factor (GEF), EFA6, in cells that activates endogenous Arf6 [7
]. Furthermore, expression of the downstream effector of Arf6, PIP5-kinase, and resultant generation of PIP2
contributes to these changes in PM dynamics. Further examination of the changes in phosphoinositide composition during EFA6 induced macropinocytosis revealed that in addition to PIP2
, phosphatidylinositol 3,4,5 trisphosphate (PIP3
) was also present at the PM and on the incoming macropinosome [34
]. During maturation of the macropinosome, PIP2
is lost from the macropinosome prior to the loss of PIP3
]. For the cell to continue PM ruffling, Arf6 needs to be inactivated and the PIP2
lost for the membrane to move further into the cell and then recycle back out to the PM. Expression of either Arf6Q67L or overexpression of PIP5-kinase leads to a block in recycling and accumulation of vacuolar membranes. These vacuoles, induced by Arf6Q67L, represent trapped macropinosomes.
Another way that Arf6 can lead macropinocytosis is through the activation of Rac (), which has itself been linked to ruffling and macropinocytosis [36
]. Expression of the Arf GEFs EFA6 [37
] and ARNO [38
], which activate Arf6, subsequently lead to the activation of Rac. 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
Downstream effectors of Arf6, Ras and Src that contribute to PM ruffling, macropinocytosis, and membrane recycling. A partial list of effector proteins involved in macropinocytosis emphasizing overlapping targets. See text for details.
In addition to activating Rac, we recently found that Arf6-GTP can amplify its signal at the PM by activating “Golgi” Arfs, such as Arf1 at the PM. Arf6-GTP binds directly to the pleckstrin homology (PH) domains of ARNO/Cytohesin GEFs and thereby recruits these GEFs to the PM where they can activate Arf1. Although ARNO GEFs can activate Arf6 to some extent, in cells and in in vitro biochemical assays Arf1 is the preferred substrate [44
]. The binding of the PH domains to Arf6 is also dependent upon the presence of phosphosphoinositides, either PIP2
, at the PM and thus recruitment of ARNO is sensitive to both Arf6-GTP and the presence of PIPs [44
]. Recently the structure of Grp1, an ARNO/Cytohesin family member, was solved and revealed that the PH domain acts to autoinhibit the Sec7 catalytic domain and that the inhibition is relieved by Arf6-GTP binding to the PH domain [46
The sequential activation of Arf6 and then Arf1 at the PM amplifies Arf signaling at the PM and possibly on macropinosomes. Since Arf6 is of low abundance in most cell types, the reserve of Arfs that cycle on and off of Golgi membranes are available to function transiently at the PM. Indeed, the activation of Arf1 can itself lead to generation of PM ruffling and macropinocytosis [44
]. Arf6 had previously been associated with Fc-mediated phagocytosis, a related, actin-dependent process. A recent study showed that during phagocytosis there is a sequential activation of Arf6 to initiate phagocytic cup formation followed by Arf1 to induce closure [47
]. The use of tandem activation provides acute control of signal amplification since Arf6 remains associated with membranes regardless of nucleotide status, whereas ARNO and Arf1 recruitment are dependent upon the presence of Arf6-GTP and PIP2
]. Following Arf6 inactivation, ARNO and Arf1 will return to the cytoplasm.
The switch from constitutive to stimulated macropinocytosis observed with activation of Arf6 can also be initiated by activation of H-Ras[48
]. Ras proteins regulate diverse cellular functions that range from cell proliferation and differentiation to apoptosis and survival. To carry out these functions activated Ras interacts with a large array of effector proteins. There are three major Ras isoforms, H-, N- and K-Ras, which differ in their carboxyl terminal amino acid sequences and lipid modification required for membrane attachment. It is speculated that Ras protein involvement in many major signaling pathways is achieved through localization to different intracellular compartments. This allows accessibility to different effectors and hence a unique signaling output [49
]. Besides the PM, Ras proteins localize and signal from the endoplasmic reticulum (ER) and Golgi membranes [50
], the mitochondria [53
], and “rasosomes” [54
]. Finally, Ras proteins were shown to signal from endosomes [55
] however, the nature of these endosomes was not clearly defined.
We found that wild type H-Ras traffics along the CIE pathway in HeLa and COS cells. Like activation of Arf6, activation of H-Ras either by EGF stimulation or expression of the constitutively active mutant of H-Ras, G12V, leads to PM ruffling and macropinocytosis. The H-Ras-induced macropinocytosis is a stimulated form of CIE that requires proper functioning of Arf6. Expression of Arf6Q67L or Arf6T27N traps RasG12V in vacuolar membranes and tubular recycling endosomes, respectively [34
During macropinocytosis stimulated by activation of either H-Ras or Arf6, there are changes to the phosphoinositide and protein composition as the macropinosome is brought into the cell (). Both PIP2
are present at the PM and on the incoming macropinosome, but as the macropinosome matures, PIP2
is lost from the membrane first, followed by the loss of PIP3
]. In COS cells where this has been examined, we believe that the PIP2
is lost due to the activity of a PIP-phosphatase or phospholipase (PLC). Following the loss of PIP2
, Rab5 is recruited onto the macropinosome whether macropinocytosis was induced by H-Ras or Arf6 activation. Rab5 is initially recruited onto the macropinosome while PIP3
is present. However, PI3P and EEA1 do not appear to be associated with these membranes. This Rab5 compartment is thus distinctly different from the Rab5-PI3P-EEA1-positive, “classical” early endosome. It resembles the APPL compartment described by Zerial and colleagues [58
] that contains EGF receptor, Rab5 and not EEA1. Interestingly, Rab5 can also recruit the PIP5-phosphatase OCRL [59
] onto this compartment which might keep PIP2
levels reduced. Following the recruitment of Rab5, the macropinosome becomes more dynamic and can extrude tubular membranes, suggesting that some type of cargo sorting event is occurring (our unpublished observations). At this point it seems that the bulk of the macropinosomal membrane is then recycled back out to the PM along the same pathway that occurs during constitutive endocytosis and recycling (see ). Consistent with this, treatment of cells expressing activated H-Ras [34
] or activated Arf6 [7
] with cytochalsin D (or other actin inhibitors) leads to accumulation of membrane in the tubular recycling endosomes.
Although the same changes in phosphoinositide content and Rab5 recruitment are observed whether macropinosomes were induced through H-Ras or Arf6, the macropinosomes were not identical. Namely, the H-Ras effector Akt is recruited onto Ras-induced macropinosomes but not onto those induced by Arf6 [34
]. In contrast, Arf1 is associated with Arf6 but not H-Ras generated macropinosomes (unpublished observations). This raises the possibility that the macropinosome could provide a platform for distinctive signaling by Ras, Arf6 and other signaling molecules.
Src activation also stimulates PM ruffling and macropinosome formation in various cell types [61
]. The Src family of tyrosine kinases is comprised of seven highly related members involved in cell division, reorganization of the actin cytoskeleton and other functions in specialized cells [62
]. In MDCK cells, macropinocytosis is dependent on PI3K, PLC, PLD and actin remodeling, all of which are Src effectors (). Furthermore, these macropinosomes are associated with a unique set of Rab5 effectors, but not with EEA1 [63
]. Similarly, the Arf6 or Ras induced macropinosomes were Rab5 positive, but mostly were not associated with EEA1 [34
What is the relationship between macropinocytosis induced by Ras, Arf6 and Src? First, it is important to note that these three important signaling molecules share some common biological outputs through the activation of common effectors (). Furthermore, all are activated in response to different growth factor and have major roles in cell transformation and metastasis. This raises the possibility that in some respects Ras, Arf6 and Src have redundant biological activities, including the induction of membrane ruffling and macropinocytosis. However, as we previously demonstrated for Arf6 and Ras, despite these common features, each can recruit unique effectors to macropinosomes [34
]. This might also be the case for Src signaling. Although we have not directly demonstrated that Src macropinocytosis utilizes the CIE membranes, we have observed that phosphorylated forms of endogenous Src are associated with CIE membranes (unpublished observations). Further studies are needed to identify these unique downstream effectors of Arf6, Ras and Src.