In this study we used surface fluorescence quenching techniques combined with immunoelectron microscopy to characterize quantitatively and spatially endocytosis and recycling of ErbB2-antibody complexes. We believe trafficking of these complexes largely reflects that of naked ErbB2 for several reasons. First, these antibodies do not stimulate (Sliwkowski et al., 1999
; Agus et al., 2002
), down-regulate (), or significantly redistribute (, , and Supplementary Table 2) surface ErbB2. Second, these antibodies have almost no influence on GA-dependent ErbB2 down-regulation kinetics (). If antibody binding were to instead mobilize a significant static pool of surface ErbB2, we should expect large differences in the down-regulation kinetics in light of our finding that GA acts at the level of endosomal sorting rather than endocytosis (see below). Third, the two antibodies used in this study show similar trafficking behavior (Figures , and , and unpublished data) yet bind to distinct epitopes (Fendly et al., 1990
) and have different mechanisms of action (Sliwkowski et al., 1999
; Agus et al., 2002
). Fourth, uptake and recycling kinetics for these antibodies were similar to that of their corresponding Fab fragments, suggesting that the influence of antibody bivalency in our system is not large ( and unpublished data). Overall our results contradict the notion that surface ErbB2 is static and instead support the idea of a dynamic equilibrium between ErbB2 endocytosis and recycling. These findings, however, do not exclude the possibility that plasma membrane retention limits to some degree ErbB2 endocytosis. Indeed in agreement with recent observations of others (Hommelgaard et al., 2004
), we found that plasma membrane ErbB2 was only occasionally (0.1–0.4%) localized to clathrin-coated pits (Supplementary Table 2). We also found that ErbB2 endocytosis is ~60-fold slower than that of TfR (), roughly corresponding to the differential occurrence of these receptors in coated pits as estimated by these authors. Hence our results suggest that if ErbB2 is indeed retained on the cell surface, such retention does not result in a static surface pool, but rather one with considerable residual dynamism.
The consequence of ErbB2 dynamism is that rapid surface down-regulation can be accomplished by diverting to degradative trafficking without altering basal endocytosis. Such appears to be the case for GA. Although GA down-regulates ErbB2 within a few hours (), it has no discernible effect on endocytosis rate per se (), while significantly reducing recycling efficiency (). More explicitly, because the basal endocytic rate is ~2% per minute and rapid recycling is ~50% efficient in the presence of GA, altered sorting alone could account for a down-regulation rate as high as ~1% per minute. This estimate indeed corresponds to the GA-induced down-regulation kinetics we observed experimentally (). Previously, it was shown that GA-induced ErbB2 degradation coincides with its ubiquitination, proteolytic fragmentation of the cytoplasmic domain, and internal redistribution of the surface pool (Mimnaugh et al., 1996
; Tikhomirov and Carpenter, 2000
). Although it might be presumed that ubiquitination induced by GA serves as an endocytosis and/or lysosomal sorting signal, the precise mechanism by which GA regulates ErbB2 endocytic trafficking is hitherto unknown. Moreover, the model in which ErbB2 is retained in a static plasma membrane pool implies that GA induces ErbB2 endocytosis. Our results however, demonstrate that GA does not influence ErbB2 endocytosis, but rather diverts basally endocytosed ErbB2 from a recycling to a degradative pathway. Because high surface distribution appears to be critical for the ability of ErbB2 to interfere with ligand-induced EGFR down-regulation and for ErbB2-associated tumorigenesis, this finding underscores the significance of endosomal recycling in ErbB2 biological function.
The abrupt deceleration in uptake kinetics at ~5 min observed in can be accounted for by commencement of rapid and efficient recycling, a phenomenon rendered less efficient by GA treatment. What explains the slow phase rise in uptake that occurs subsequently in ? Notably, our surface-quenching assays are best suited for short-term assays; prolonged incubation at 37°C would increase the influence of artifacts on the results such as intracellular antibody dissociation from ErbB2 and increased construct fluorescence upon degradation (Supplementary Figure 3B). Degradative routing alone, however, provides an incomplete explanation because it implies a trastuzumab turnover rate that is much faster than we observe (T1/2
~11 h, ). Importantly, both flow cytometric data in and quantitative immunoelectron microscopy data in indicate that only ~6% of the antibody had been internalized within 5 min. By comparison, ErbB2 label quantification reveals that at steady state, the endocytic pool comprises as much as 10–17% of total nonbiosynthetic ErbB2 (). In light of the predominantly tubulovesicular distribution pattern we see for internalized trastuzumab at 3 h (), a time when internalized antibody distribution more closely reflects that of steady-state ErbB2 (compare with ), we think a likely explanation for the slow phase rise in internalized antibody is the gradual filling of a kinetically distinct, slowly recycling internal pool. Indeed the notion of two kinetically distinct recycling routes back to the cell surface is well established (Maxfield and McGraw, 2004
). The reason for the transient plateau in uptake observed between 5 and 16 min is less clear, but might possibly be attributed to recovery from prior low-temperature incubations.
A surprising result is that trastuzumab does not down-regulate surface ErbB2 in SKBr3 cells (Figures and , ), a cell line sensitive to the growth-inhibitory effects of this antibody in vitro. Though the mechanism of action of trastuzumab in vitro is not completely understood and might involve a combination of mechanisms, surface down-regulation of ErbB2 is widely believed to be important (Sliwkowski et al., 1999
; Mendelsohn and Baselga, 2000
; Baselga and Albanell, 2001
; Yarden, 2001
). This notion in part stems from a substantial body of work demonstrating the connection between down-regulation and antitumor activity for bivalent antibodies that either have agonistic properties or recognize the activated rat or human oncogenic neu mutant version of the receptor (Drebin et al., 1985
; Harwerth et al., 1992
; Hurwitz et al., 1995
; Klapper et al., 1997
). Indeed trastuzumab was initially thought to be agonistic as well (Scott et al., 1991
), though this property was subsequently shown to be largely attributed to an in vitro artifact of nonionic detergent lysis (Sliwkowski et al., 1999
). In agreement, molecular modeling of the trastuzumab-ErbB2 complex reveals that despite its bivalency, trastuzumab is very unlikely to drive dimerization interface interactions to form ErbB2 homodimers, as one might expect for an antibody with ligand-like properties (Supplementary Figure 4). Nonetheless, several studies have reported data showing apparent ErbB2 down-regulation in SKBr3 and other cell lines after treatment with trastuzumab (Hudziak et al., 1989
; Kumar et al., 1991
; Baselga and Albanell, 2001
; Cuello et al., 2001
; Citri et al., 2002
). The kinetics of the effect in SKBr3 cells varies greatly between studies, with the fastest showing >50% down-regulation within 2 h (Citri et al., 2002
) and the slowest showing slow down-regulation commencing after 24 h and reaching a maximum at 72–96 h (Cuello et al., 2001
). Although the latter finding suggests down-regulation might be indirect and a consequence rather than a cause of growth inhibition, the former finding suggests a direct effect on the ErbB2 surface pool.
Our results contradict both of these views and instead show that the down-regulating effect of trastuzumab is not significant. The reasons for these discrepancies are unclear. Notably, our data derives from four independent techniques, three of which (immunofluorescence microscopy, flow cytometry, and quantitative immunoelectron microscopy) are unaffected by changes in sample protein resulting from growth inhibition. If trastuzumab does not down-regulate ErbB2, how does it inhibit ErbB2-dependent tumor cell growth? In vivo, studies in mice suggest that the host immune response might contribute substantially to the antitumor effect (Clynes et al., 2000
). This however does not explain the ability of trastuzumab to inhibit cell growth in vitro. Trastuzumab does not disrupt association with other ErbB family members, as does pertuzumab (Agus et al., 2002
). Other mechanisms have been proposed, including inhibition of metalloprotease-mediated cleavage of the ErbB2-ECD, resulting in a truncated constitutively active receptor (Molina et al., 2001
; See Note Added in Proof
In summary, our results reveal that surface ErbB2 in target breast cancer cells is dynamic rather than static. Trastuzumab does not down-regulate surface ErbB2 but instead passively recycles after ErbB2 endocytosis. GA by contrast rapidly down-regulates surface ErbB2 through improved degradative sorting following basal endocytosis.