Membrane targeting of GTPases that, like Rap1 and Ras, contain a CAAX motif is determined by posttranslational processing of the CAAX sequence (Casey et al., 1989
), secondary membrane-targeting sequences adjacent to the CAAX motif (Hancock et al., 1991
), and the capacity to interact after processing with cytosolic chaperones such as RhoGDI (Michaelson et al., 2001
). Unlike the CAA(S/M) motif of Ras proteins, Rap1 ends in a CAAL motif that becomes geranylgeranylated, a modification that is more hydrophobic than that of farnesylated Ras proteins (Silvius and l'Heureux, 1994
). The Rap1 CAAL motif is flanked by a relatively strong polybasic region (net charge +5) similar to that of K-Ras4B. In this regard, Rap1 is most similar to Rac1, a geranylgeranylated Rho family GTPase that is targeted to the PM (Michaelson et al., 2001
). However, unlike processed Rac1 that is sequestered in the cytosol by its interaction with RhoGDI, Rap1 has no known cytosolic binding protein and is therefore predicted to have a strong affinity for the PM.
Despite its Rac1-like membrane-targeting sequence, Rap1 has not been previously localized to the PM of cultured cells. Originally localized to the Golgi complex (Beranger et al., 1991
), both endogenous and overexpressed Rap1 were later found to be associated exclusively with late endosomes/lysosomes (Pizon et al., 1994
). Thus, whereas our localization of intracellular GFP-tagged Rap1 is consistent with earlier reports, our observation of GFP-Rap1 on the PM of cultured cells is new but not unexpected. That GFP-Rap1N17 did not localize, like GFP-Rap1, to the PM argues against a simple overexpression artifact for the PM localization of the wild-type protein. Our confirmation by subcellular fractionation, including affinity purification of biotinylated surface membrane, that endogenous Rap1 is expressed on PM suggests that the inability to visualize PM-associated Rap1 by indirect immunofluorescence of fixed and permeabilized cultured cells is a result of low sensitivity of the assay, and that the localization of overexpressed GFP-Rap1 on the PM reflects the true subcellular distribution of the GTPase. This view is supported by the observation that in primary myeloid cells, Rap1 has been localized to the PM (Quinn et al., 1992
). In lymphoid cells, a recently identified effector of Rap1, RapL, has been shown to associate with the surface adhesion molecule LFA-1 and mediate in a Rap1-dependent fashion its relocalization to the leading edge of the cell (Katagiri et al., 2003
), confirming a functional role for Rap1 on the PM.
More intriguing than the PM localization of Rap1 is the rapid up-regulation of the GTPase on this compartment that we observed after growth factor stimulation. The rapidity of the increase in GFP-Rap1 surface expression rules out new protein synthesis as a source. Cytosolic pools of processed GFP-Rap1 were not observed, which is consistent with the absence of a GDI-like binding partner. Thus, the additional GFP-Rap1 that appeared on the PM is most likely derived from an intracellular membrane compartment. Indeed, the source of Rap1 that rapidly appears on the PM of terminally differentiated myeloid cells in response to inflammatory agonists is a pool associated with two classes of specialized vesicles known as secondary (or specific) and tertiary granules that serve as intracellular reservoirs of PM (Maridonneau-Parini and de Gunzburg, 1992
; Mollinedo et al., 1993
). Although the cultured epithelial cells and fibroblasts used in this work do not contain specialized secretory granules, the large pool of intracellular Rap1 present on endosomes is a potential source of rapidly mobilizable protein. The sensitivity of Rap1 surface up-regulation to NEM suggests that membrane fusion events are required, and the inhibition by dominant-negative Rab11BP implicates recycling endosomes as the source of additional PM Rap1. Thus, specialized secretory organelles are not required for the regulation of Rap1 surface expression by exocytosis.
Given the large pool of intracellular Rap1 and the recent observation that intracellular Ras is activated in situ by growth factor signaling (Chiu et al., 2002
), it was somewhat surprising to observe Rap1 activation only at the PM. This suggests that the GEFs that activate Rap1 after EGFR ligation are localized at the PM. EGFR stimulation causes recruitment to the PM of the GEF SOS, which activates several Ras-related GTPases including M-Ras (Quilliam et al., 1999
). We observed that GTP-bound M-Ras71L stimulated activation of Rap1 at the PM. Because activation of M-Ras recruits to the PM RA GEF 2, a Rap-specific GEF (Gao et al., 2001
), we hypothesize that M-Ras links EGFR stimulation with Rap1 activation at the PM.
The reversibility of Rap1 activation at the PM implicates GAP activity on this compartment. Indeed, Rap1GAP was localized to the PM (Polakis et al., 1991
). Recently, Rap1GAP has been shown to be dynamically recruited to the PM by Gz
in NGF-stimulated PC12 cells (Meng and Casey, 2002
). Because inactive Rap1 traffics through the endosomal recycling compartment, the balance of GEFs and GAPs in this compartment might favor the latter, and endocytosis may serve as a mechanism of down-regulating Rap1.
The coincidence of both up-regulated GFP-Rap1 and activated Rap1 on membrane ruffles lends further support to the view that the two processes are linked. Exocytosis to the leading edge of the cell where extending lamellipodia require rapid expansion of the surface membrane is a well established paradigm in cell biology and one that may explain the appearance of GFP-Rap1 in ruffles. Moreover, the preferential up-regulation of Rap1 in membrane ruffles suggests that the GTPase may play a role in actin-based processes such as cell motility and adhesion. Indeed, Rap1 has been implicated in the regulation of integrin-mediated adhesion in lymphoid cells downstream of the T cell receptor and of CD31 (Reedquist et al., 2000
). We confirmed the role of Rap1 in integrin-mediated T cell adhesion and showed that the Rap1-mediated regulation was sensitive to agents that block endosome recycling. This observation provides functional evidence for regulation by Rap1 of adhesive events at the PM and for modulation of that function by exocytosis. Rap1-regulated LFA-1–dependent adhesion at the ruffling leading edge of the cell and detachment at the uropod have recently been shown to depend on a residue in the LFA-1 β chain that is required for receptor internalization and recycling (Tohyama et al., 2003
). It is tempting to speculate that the endosomal compartment storing the intracellular pool of LFA-1 is the same as the one that contains a mobilizable pool of Rap1.
Our results differ substantially from those of recent works that observed Rap1 activation only on internal membranes (Mochizuki et al., 2001
; Ohba et al., 2003
). The basis for this discrepancy is not entirely clear but is undoubtedly related to the distinct methods applied. Those authors did not directly measure Rap1 activation but rather used an overexpressed chimeric FRET probe to sample, in a spatio-temporal fashion, the relative balance of GEFs and GAPs active against the chimera. Unfortunately, the Raichu-Rap1 FRET probe was not unbiased in its subcellular distribution but rather incorporated the membrane-targeting sequence of K-Ras4B, well established to target proteins exclusively to the PM (Hancock et al., 1990
; Choy et al., 1999
). Moreover, in these studies, the spatial resolution of the FRET readout was relatively low, such that specific subcellular compartments could not be distinguished and the conclusion that Rap1 was activated on endomembranes was based on a diffuse perinuclear signal. The ability of PM-targeted Raichu-Rap1 to report Rap1 activation on endomembranes has not been explained, and a Raichu-Rap1 probe with a native membrane-targeting sequence has not been reported. Interestingly, Raichu-Rap1V12, a K-Ras4B–targeted GAP-resistant probe with a constitutively high degree of GTP binding, reported activity only at the PM that was surprisingly EGF sensitive (Ohba et al., 2003
), which is consistent with our results using GFP-RBDRalGDS
. However, in our work, each Rap isoform analyzed was targeted to membranes with its native hypervariable region, and our fluorescent reporter was untargeted and thus had unbiased access to the cytosolic leaflet of all membrane compartments. Most importantly, GFP-RBDRalGDS
proved capable of reporting the spatio-temporal activation of endogenous Rap1.
In summary, our in vivo imaging of Rap1 localization and activation has provided insight into the dynamic regulation of Rap1 in response to growth factor stimulation and has highlighted important differences between Rap1 and Ras. Whereas the subcellular distribution of Ras is unaffected by growth factor stimulation and pools of Ras on intracellular compartments are activated in situ (Chiu et al., 2002
), Rap1 is up-regulated at the PM in conjunction with its activation principally on that compartment. Moreover, when up-regulation was blocked, so was Rap1 function. We propose that localization of Rap1 and Ras to different membrane compartments contributes to their distinct cellular functions.