Mammals, including humans, use signaling molecules called hormones to carry information from one cell to another. Insulin-like growth factor (or IGF for short) is a hormone that is essential throughout an animal’s lifetime. It is needed for growth and for many of the chemical processes that must occur to maintain life (which are collectively referred to as an animal’s metabolism). IGF binds to and activates a protein found on the surface of cells, which then transmits the signal inside the cells. This surface protein is known as the IGF-I receptor, and once it is activated by IGF binding, it is removed from the cell surface and then incorporated inside the cell to switch off the signal. The IGF signal in cells needs to be properly balanced to prevent disorders of growth and metabolism.

How long the activated IGF-I receptor remains at the cell surface and when the IGF-I receptor starts to enter inside the cells after cells receive IGF influence the signals within the cell. Often IGF signaling must be activated for long periods, for example when cells maintain their balance between making and breaking proteins. However, it remains poorly understood how the IGF-I receptor produces a sustained signal.

Yoneyama et al. have now focused on a protein called IRS-1, which was known to act downstream of the receptor. The experiments revealed that this protein determines how long activated IGF-IR remains at the cell surface before it enters inside cells. It achieves this by binding to a complex of proteins, known as AP2, which normally internalizes the IGF-I receptor. However, when IRS-1 binds, it inhibits AP2. This means that the receptor is no longer rapidly removed from the cell surface and can continue signaling for long periods of time.

The findings of Yoneyama et al. help to explain how long-term IGF signaling is regulated. Further work that builds on these findings could help scientists to understand how uncontrolled IGF signals cause the development of diseases including cancer and metabolic disorders.

Insulin-like growth factor (IGF)-I receptor (IGF-IR) is an important receptor tyrosine kinase (RTK) that regulates a variety of biological processes including proliferation, cell survival, and control of metabolism in a wide range of mammalian tissues by binding the ligands IGF-I and IGF-II (Nakae et al., 2001). Ligand binding to the IGF-IR extracellular domain causes conformational changes of the intracellular region, inducing the tyrosine kinase domain to autophosphorylate multiple Tyr residues and activate intrinsic RTK activity (Kavran et al., 2014; Favelyukis et al., 2001). IGF-IR then initiates downstream signaling through tyrosine phosphorylation of insulin receptor substrate (IRS) adaptor proteins to activate the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and its various biological responses (Myers et al., 1996; Sun et al., 1993; White, 2002).

IGF/IGF-IR stimulates the PI3K-Akt pathway in a stereotypical way – sustained tonal induction. Sustained induction is thought to define the specific biological outcomes of IGF signaling, and distinguish the function of the IGF ligand from other RTKs/ligands that access the Akt cascade (Gross and Rotwein, 2016; Kubota et al., 2012). In particular, sustained activation of the PI3K-Akt pathway, mediated by IGF-IR, induces cell proliferation in multiple types of cells, cell survival in neural cells, and protein homeostasis in skeletal muscle cells (Fernandez and Torres-Alemán, 2012; Fukushima et al., 2012; Ness and Wood, 2002; Sacheck et al., 2004; Stewart and Rotwein, 1996). To date, the mechanism by which IGF-IR produces sustained signaling remains poorly understood.

Clathrin-mediated endocytosis (CME) is a major regulator of RTKs (Goh and Sorkin, 2013) involving the heterotetrameric AP2 complex composed of large α and β2, medium μ2, and small σ2 subunits (Collins et al., 2002). AP2 binds to transmembrane cargo proteins that contain specific motifs such as YxxΦ (Y denotes Tyr; x, any amino acid; and Φ, bulky hydrophobic residue) serving as μ2 binding sites (Owen and Evans, 1998; Traub and Bonifacino, 2013). In addition, AP2 associates with clathrin and with endocytic accessory proteins at the plasma membrane to coordinate clathrin-coated pit (CCP) formation (Schmid and McMahon, 2007). Ligand-bound RTKs enter the endocytic process through CME, but perhaps with different signaling consequences. If endocytosed RTKs are sorted to lysosomes for degradation, this process down-regulates signaling as exemplified by the model RTKs including epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (Goh and Sorkin, 2013). On the other hand, some RTKs continue to signal locally across the endosome membrane even after endocytosis (Schenck et al., 2008; Villaseñor et al., 2015; Lin et al., 2006). In either case, RTK internalization strongly impacts its signaling outputs. Thus, the duration at the cell surface of ligand-bound RTKs, which is tightly regulated by CME, critically fine-tunes their signaling and biological functions. Accordingly, we hypothesize that ligand-bound IGF-IR, which exhibits sustained activation and slow degradation (Fukushima et al., 2012; Mao et al., 2011; Zheng et al., 2012), undergoes slow or delayed CME (Martins et al., 2011; Monami et al., 2008). To evaluate this idea, here we study the molecular components regulating perdurance of IGF-IR at the cell surface through its interactions with CME, and elucidate how this dictates IGF signaling and outputs.

Among IRS family proteins IRS-1 and IRS-2 are well known as major substrates of IGF-IR (Taniguchi et al., 2006; White, 2002). We and others have shown that IRS-associated proteins contribute to the regulation of IRS-1/IRS-2 function through distinct mechanisms (Ando et al., 2015; Hakuno et al., 2015, 2007; Lee et al., 2013; Shi et al., 2011; Fukushima et al., 2015; Yoneyama et al., 2013). In this study, we discovered AP2 is also an IRS-1-associated protein. Unexpectedly IRS-1 promotes the surface retention of activated IGF-IR through inhibiting AP2-dependent internalization of IGF-IR, and this is independent of IRS’s classic role as an adaptor protein in IGF-IR and insulin receptor signaling. The ability of IRS-1 to prolong surface retention of IGF-IR is essential for long-term PI3K-Akt signaling. Our results establish a novel role of IRS-1 in ensuring the sustained effects of IGFs via its direct control of IGF-IR internalization.

The canonical function of IRS proteins is to mediate signaling of IGF-IR to the PI3K-Akt pathway through Tyr phosphorylation (Figure 9A) (White, 2002). The present results reveal a new role of IRS-1 independent of its Tyr phosphorylation: IRS-1 regulates IGF-IR internalization to produce sustained activation of IGF signaling (Figure 9B). IRS-1 binds with AP2 to prevent IGF-IR recruitment into clathrin-coated structures and thus enhance surface retention of activated IGF-IR. This function of IRS-1 in prolonging IGF-IR activity is critical for sustained activation of the PI3K-Akt pathway, and provides a key mechanism for how IGF-IR signaling induces specific biological actions of IGF (Sacheck et al., 2004; Ness and Wood, 2002; Bailey et al., 2006; Stewart and Rotwein, 1996). Thus, IRS-1 plays a dual role as a signaling adaptor of IGF-IR and an endocytic regulator of IGF-IR.

Figure 9

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The first key finding of the present study is that IRS-1 interacts with AP2 thereby regulating the rate of ligand-dependent internalization of IGF-IR. AP2-mediated recognition of YxxΦ motif in cargos is a critical step for CCP formation (Traub and Bonifacino, 2013; Kadlecova et al., 2017). Our results indicate that IRS-1 inhibits the recruitment of IGF-IR to CCPs through YxxΦ motifs in IRS-1. We have previously reported that another clathrin adaptor complex AP1 also binds to the same sites of IRS-1 as AP2 (Yoneyama et al., 2013). Since AP1 depletion did not prevent the down-regulation of activated IGF-IR (Figure 3—figure supplement 1A), the inhibitory effect of IRS-1 on IGF-IR internalization is based on its interaction with AP2. In addition, IRS-1-depleted cells show the fast onset of IGF-IR internalization in response to IGF-I and the partial decrease in IGF-IR levels, both of which are presumably caused by the promotion of AP2-dependent IGF-IR internalization and subsequent degradation (Figure 5). These results suggest that IRS-1 is an inhibitory upstream regulator for AP2-dependent internalization of IGF-IR (Figure 9B). EGFR and some G-protein coupled receptor/β-arrestin complexes are known to be recruited into pre-existing CCPs after the ligand stimulation (Rappoport and Simon, 2009; Scott et al., 2002). We observed the similar behavior of IGF-IR in live-cell TIRF-M (Figure 3—figure supplement 4D). Notably, less IGF-IR was recruited to AP2-positive spots in the cells ectopically expressing IRS-1 WT, but not 3YA mutant, suggesting that IRS-1 interferes with the recruitment step of IGF-IR to clathrin-coated structures through competing out AP2 from IGF-IR. This will need to be tested by more detailed observation at higher resolution.

The second key finding of this study is that the ability of IRS-1 to promote surface retention of IGF-IR can be separable from Tyr phosphorylation-mediated signaling function of IRS-1. The Tyr residues of the YxxΦ motifs of IRS-1 (Tyr608, 628, and 658) critical for the binding to AP2 are part of phosphorylation sites among multiple Tyr residues in the C-terminus of IRS-1 that mediate the interaction of IRS-1 with PI3K and subsequent activation of PI3K (Myers et al., 1996; Sun et al., 1993). We showed that ectopic expression of the IRS-1 mutant ΔPTB led to the accumulation of active IGF-IR at cell surface to the same degree as that of IRS-1 WT (Figure 2), indicating that IRS-1 inhibits the internalization of IGF-IR in a manner independent of its Tyr phosphorylation. In addition, AP2 would preferentially bind non-phosphorylated IRS-1 since AP2 cannot recognize phosphorylated YxxΦ sequence due to its limited capacity (Kittler et al., 2008; Owen and Evans, 1998). In line with this, our biochemical analyses support the notion that non-phosphorylated IRS-1 acts as an inhibitory factor for IGF-IR internalization via its interaction with AP2 (Figure 9B).

Our observation indicates that ectopic expression of IRS-1 affects endocytosis of receptors other than IGF-IR. As long as we tested, endocytosis of integrin β1 and EGFR, which could interact with IGF-IR, but not of TfR, was inhibited by IRS-1, raising the possibility that IRS-1 influences endocytosis of cargoes in the close proximity of IGF-IR. As observed in our TIRF-M observation (Figure 4—figure supplement 1D), a fraction of IRS-1 has been demonstrated to localize to membrane-associated cytoskeleton (Clark et al., 1998). IRS-1 may locally regulate the specific cargo recruitment to CCPs through association with a portion of AP2 at the actin cytoskeleton. Indeed, preferred sites of endocytosis have been observed in some cargo proteins (Grossier et al., 2014; Weng et al., 2014), although the molecular mechanisms of such spatial regulation for IGF-IR and other cargos remain unknown.

In addition to the role of IRS-1 in controlling the rate of IGF-IR internalization, we found that this ability of IRS-1 is negatively regulated by mTORC1 (Figure 6). mTORC1 has been reported to suppress IGF-IR activity via its direct substrate Grb10 (Yu et al., 2011; Hsu et al., 2011). Our findings propose another mode of IGF-IR regulation by mTORC1: mTORC1 feedback signaling leads to the degradation of IRS-1, which functions as a brake release to trigger IGF-IR internalization (Figure 9B). Hence, the time length needed for IRS-1 degradation, which is critically regulated by mTORC1, should determine the initiation timing of IGF-IR internalization.

Receptor endocytosis is now considered to play both negative and positive roles in the downstream signaling (Goh and Sorkin, 2013). Our data demonstrated that CME is required for long-term attenuation of activated IGF-IR (Figure 3). Previous studies have demonstrated that ligand-activated IGF-IR is ubiquitinated and subsequently undergoes CME for its down-regulation (Monami et al., 2008; Zheng et al., 2012). In addition, the recycling of IGF-IR has been shown to in part contribute to sustained activation of Akt in response to IGF-I (Romanelli et al., 2007). In this study we showed that stable expression of IRS-1 inhibits ligand-dependent internalization of IGF-IR, leading to sustained activation of IGF-IR kinase and the downstream Akt signaling. This effect of IRS-1 on prolonging the Akt signaling is likely based on two independent functions of IRS-1. First, the interaction of IRS-1 with AP2 is required since expression of the IRS-1 mutant 3YA could prolong neither IGF-IR phosphorylation nor Akt phosphorylation in IGF-I-stimulated cells. Second, the ability of IRS-1 to engage PI3K is also necessary because expression of the IRS-1 mutant ΔPTB could prolong phosphorylation of IGF-IR but failed to sustain Akt phosphorylation (Figure 7—figure supplement 1F). Similar signaling events were also observed in AP2-depleted cells where IRS-1 degradation, a consequence of negative feedback, was normally induced by long-term IGF-I stimulation (Figure 7—figure supplement 1D,E). Notably, the ability to interact with AP2, enhance the surface retention of IGF-IR, and prolong the Akt signaling is specific for IRS-1, but not for IRS-2. Thus, IRS-1 can act as a pivotal modulator for IGF signaling duration via its control of IGF-IR internalization while the downstream signaling activation can be mediated by either IRS-1 or IRS-2 (Figure 9B).

It is generally recognized that IGF-IR preferentially mediates growth whereas insulin receptor (IR) functions in glucose homeostasis in spite of the fact that both receptors share common signaling pathways mediated by the IRS proteins (Accili et al., 1996; Liu et al., 1993; Nakae et al., 2001). However, these functional differences between IR and IGF-IR cannot be attributed to characteristics of the receptors themselves, such as their kinetics of ligand binding or their tissue/cellular distribution (Siddle, 2012). Moreover, insulin levels fluctuate in response to the nutrients while IGF levels are constantly maintained by circulating IGF binding proteins and by paracrine/autocrine production (Jones and Clemmons, 1995). Yet, despite these differences in temporal pattern, this is unlikely to explain the specificity of IGF-IR and IR because even in cell culture these receptors mediate different bioactivities as well as gene expression profiles (Lammers et al., 1989; Palsgaard et al., 2009), including in a recent study using reconstituted model cell lines solely expressing either receptor (Cai et al., 2017). While differential substrate preference for each receptors has been proposed to explain this specificity (Cai et al., 2017), both receptors still induce signaling through the PI3K-Akt cascade and involve many IRS proteins (White, 2002; Taniguchi et al., 2006). In addition, the Akt signaling cascade itself can produce different temporal dynamics in response to specific stimuli and to induce different cellular outcomes (Gross and Rotwein, 2016; Kubota et al., 2012). Our study demonstrates that the IGF-IR pathway encodes prolonged Akt activation via IRS-1-mediated delay of IGF-IR internalization (Figure 9B). In contrast, IR has been shown to undergo rapid CME in response to insulin (Choi et al., 2016; Morcavallo et al., 2012). These observations raise the possibility that the bioactive difference between IGF-IR and IR arises in part through their differential temporal activation of the PI3K-Akt pathway governed by CME kinetics unique to each receptor. In this context, future studies could productively address whether and how the CME of IGF-IR and IR are selectively regulated, which is also a general issue in the context of CME selectivity for multiple cargos (Grossier et al., 2014; Weng et al., 2014). Notably, Choi et al. (2016) revealed that IR, but not IGF-IR, uses the receptor-associated adaptor BUBR1/MAD2 to facilitate rapid CME by recruiting AP2 to IR. We are likely to better understand the role of differential endocytic regulation of IGF-IR and IR in temporal dynamics of the PI3K-Akt pathway when we identify the specific adaptors for IGF-IR and IR that engage their CME, and determine their relationship with IRS-1.

Our results demonstrate that the prolonged Akt signaling elicited by IRS-1-mediated surface retention of IGF-IR affects the FoxO-targeting gene expression. Long-term action of IGF is fundamental for various physiological aspects including growth control and neural cell survival (Ness and Wood, 2002; Gross and Rotwein, 2016; Stewart and Rotwein, 1996). Thus, IRS-1-mediated delay of IGF-IR internalization is likely to be a common mechanism for long-term IGF actions.

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Thank you for submitting your article "IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by Philip Cole as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This is an interesting study that describes a role for IRS-1 as a ligand for AP-2 adapter proteins that mediate recruitment of cell surface proteins to clathrin coated pits for endocytosis. The authors propose that IRS-1 binding prevents AP-2 recognition of IGF-1R by competition and therefore suppresses IGF-1R internalization to enable sustained IGF-1 signaling. Several lines of evidence are presented to support these conclusions. Nevertheless, there are several issues that require clarification.

Essential revisions:

1) The biotinylation assays for cell surface IGF-1R were performed following incubation with IGF-1 (Figure 1). It is therefore not clear that the maintenance of cell surface IGF-1R that is observed is not caused by recycling of internalized receptors rather than the failure to internalize. Moreover, the interpretation of the loss of pTyr from the cell surface is unclear in this assay. The authors appear to assume that this reflects internalization of cell surface pTyr-labeled receptors, but why could this not be caused by dephosphorylation? Direct assays for receptor internalization are required for the authors to draw conclusions concerning IGF-1R internalization and the role of IRS-1.

2) The authors' estimations of the rate of IGF-IR endocytosis in L6 cells is unusually long. A small decrease in the amount of pIGF-IR is observed only after 3 hours of continuous stimulation of cells with IGF1, and the total surface receptor does not change at all. This is highly unusual for a cargo internalized by CME. Technically, because surface biotinylation is used to monitor plasma membrane receptor levels at "hours" time-scale, rates of internalization, recycling, degradation and insertion of newly synthesized receptors are all contribute to the apparent rate of down-regulation in these measurements. Therefore, whether manipulations with IRS1 levels affect the AP-2 mediated CME of the receptor is not directly demonstrated. Notably, receptors are shown to accumulate in large clathrin structures on the cell-bottom membrane. These clathrin plaques are "endocytosis-passive" in many types of cells. Localization in these structures may not be as a "positive" endocytic event and is not a useful correlate of the internalization rate.

3) The authors propose that phosphotyrosine prevents the binding of the IRS-1 Yxxø motifs to AP-2. However, treatment of cells with IGF-1 does not inhibit the co-immunoprecipitation of IRS-1 with AP-2. The authors argue that this is because of low stoichiometry tyrosine phosphorylation of IRS-1. This could be tested by co-immunoprecipitation analysis.

4) The interpretation of the IRS-1 over-expression experiments is unclear. If IRS-1 binds AP-2, this should inhibit the internalization of many AP-2 cargos, but this was not observed – e.g. transferrin receptor. The authors argue that this could be because IRS-1 has some special localization within the cell; would this also be true for over-expressed IRS-1? If IRS-1 is an AP-2 competitor, why does it not inhibit internalization of other AP-2 dependent proteins when over-expressed? Moreover, if the competition for AP-2 is restricted to the local environment of the IGF-1R, does this mean that it does not affect other cell surface proteins that signal through IRS-1 signals (e.g. integrins) and if so, why not? Might this mechanism also affect transmembrane tyrosine phosphatases that dephosphorylate IGF-1R?

5) The kinetics of endocytosis of pIGF-IR and signaling are on different scales. The effects of the depletion or overexpression of IRS1 are evident on pAkt only after 6-12 hours (Figure 7) or later. The effects of IRS1 level alterations can be alternatively interpreted by the increase or decrease of the amount of phosphoIRS1 capable of engaging PI3K. The lack of the effect of the blockade of the receptor endocytosis by mu2 depletion of Akt activity (Figure 7—figure supplement 1D and 1E) is puzzling. The authors' interpretation of this data is that slow endocytosis is not sufficient in order to prolong Akt activity, and that the presence of high levels of IRS1 is also required. Such interpretation is difficult to reconcile with the model.

Essential revisions:

1) The biotinylation assays for cell surface IGF-1R were performed following incubation with IGF-1 (Figure 1). It is therefore not clear that the maintenance of cell surface IGF-1R that is observed is not caused by recycling of internalized receptors rather than the failure to internalize. Moreover, the interpretation of the loss of pTyr from the cell surface is unclear in this assay. The authors appear to assume that this reflects internalization of cell surface pTyr-labeled receptors, but why could this not be caused by dephosphorylation? Direct assays for receptor internalization are required for the authors to draw conclusions concerning IGF-1R internalization and the role of IRS-1.

We have added several key experiments that address the reviewer’s points and strengthen our conclusion. We have addressed the potential reasons for the maintenance of cell surface IGF-IR in IGF-I-stimulated cells, including the recycling of internalized receptor and receptor dephosphorylation. To inhibit the recycling, we used primaquine which has reportedly inhibited the recycling of transferrin receptor and receptor tyrosine kinases. Cells were pre-treated with primaquine followed by IGF-I stimulation, and then surface-biotinylated to measure the time-dependent changes in surface IGF-IR (Figure 3C in the revised manuscript). The results showed that surface levels of transferrin receptor were reduced by primaquine treatment. Surface IGF-IR levels were reduced by IGF-I treatment within 1 hour, and phospho-IGF-IR levels followed this change (Figure 3C in the revised manuscript). These observations indicate that the recycling contributes to the apparent surface maintenance of IGF-IR, which had been observed in Figure 2A and 2B of the original manuscript. The data also support the notion that IGF-I triggers ligand-dependent endocytosis of IGF-IR in L6 cells.

In response to the reviewer’s question regarding the dephosphorylation of IGF-IR, we focused on PTP1B, an endoplasmic reticulum-resident protein tyrosine phosphatase, which has reportedly downregulated IGF-IR by dephosphorylation (Buckley et al., 2002). The PTP1B substrate-trapping mutant (D181A) binds to but cannot dephosphorylate its substrate. Phosphorylation levels of IGF-IR observed 1 hour after IGF-I treatment and the subsequent reduction in the later period (6 hours) were comparable between PTP1B D181A-expressing and non-expressing cells as revealed by immunofluorescence (Figure 3—figure supplement 2B in the revised manuscript). Although we could not rule out the involvement of other tyrosine phosphatases targeting IGF-IR, our observation indicates a negligible role of PTP1B in the down-regulation of phospho-IGF-IR in our assay.

In response to the reviewer’s suggestion, we have directly measured the internalized IGF-IR via two different approaches. First, we performed the internalization assay using the non-permeable and cleavable biotinylation reagent (please see details in Materials and methods section). In this assay, cells were surface-biotinylated followed by incubation with or without IGF-I. Before lysis, residual surface biotin was stripped off by treating cells with a non-permeable reducing agent MesNa, which cleaves the disulfide-coupled biotin. The biotinylated proteins that were internalized from the cell surface were isolated by streptavidin pull-down. It revealed that internalized IGF-IR was detected within 15 minutes after surface biotinylation (Figure 3—figure supplement 3A in the revised manuscript).

To complement this result, we developed a double-tagged IGF-IR construct (IGF-IR-HA-EGFP) that contains an extracellular HA-tag and intracellular EGFP as a second approach. Internalization can be directly monitored by following uptake of anti-HA antibody added to the media prior to ligand treatment (Figure 3—figure supplement 3B, C in the revised manuscript). In L6 cells stably expressing IGF-IR-HA-EGFP, internalized fraction of the double-tagged IGF-R was detected within 15 to 60 minutes under both IGF-I-stimulated and non-stimulated conditions (Figure 3—figure supplement 3D, E in the revised manuscript). The internalization of IGF-IR observed in the non-stimulated state was not affected by knockdown of AP2 (Figure 3—figure supplement 3F in the revised manuscript), indicating that the basal endocytosis of IGF-IR is not dependent on AP2. In contrast, phospho-IGF-IR was predominantly localized to the cell surface and did not overlap with internalized IGF-IR (HA-positive) within 1 hour in the ligand-stimulated cells while IGF-I did not affect the apparent uptake of anti-HA antibody within 1 hour (Figure 3—figure supplement 3D). At the later period (6 hours), phospho-IGF-IR was detected in LysoTracker-positive compartments (Figure 3—figure supplement 1B, left in the revised manuscript). More importantly, the phospho-IGF-IR targeting to lysosomes was abolished by knockdown of AP2 (Figure 3—figure supplement 1B, right; Figure 3—figure supplement 1C n the revised manuscript). These observations therefore demonstrate that IGF-IR undergoes endocytosis in L6 cells both in ligand-stimulated and non-stimulated conditions, and in particular that ligand-activated IGF-IR is internalized in an AP2-dependent manner.

2) The authors' estimations of the rate of IGF-IR endocytosis in L6 cells is unusually long. A small decrease in the amount of pIGF-IR is observed only after 3 hours of continuous stimulation of cells with IGF1, and the total surface receptor does not change at all. This is highly unusual for a cargo internalized by CME. Technically, because surface biotinylation is used to monitor plasma membrane receptor levels at "hours" time-scale, rates of internalization, recycling, degradation and insertion of newly synthesized receptors are all contribute to the apparent rate of down-regulation in these measurements. Therefore, whether manipulations with IRS1 levels affect the AP-2 mediated CME of the receptor is not directly demonstrated. Notably, receptors are shown to accumulate in large clathrin structures on the cell-bottom membrane. These clathrin plaques are "endocytosis-passive" in many types of cells. Localization in these structures may not be as a "positive" endocytic event and is not a useful correlate of the internalization rate

We agree with the issues raised by the reviewer. In the original manuscript, we have shown that phospho-IGF-IR at the cell surface started to decline 3 hours after IGF-I stimulation (Figure 2A in the original manuscript). Now we show that this was overestimated due to the contribution of recycling. We have clearly observed that in the presence of primaquine surface IGF-IR as well as phospho-IGF-IR started to reduce within 30–60 minutes after IGF-I treatment (Figure 3C in the revised manuscript). Therefore, the kinetics of IGF-IR internalization in L6 cells seems comparable with other CME cargoes.

In surface biotinylation assay described in the original manuscript, apparent changes in surface IGF-IR may reflect other events than endocytosis, including recycling and de novo synthesis of IGF-IR, as the reviewer indicated. Indeed, during long-term stimulation of IGF-IR (3 hour), we reproducibly observed the modest increase in precursor IGF-IR (Figure 3—figure supplement 2A in the revised manuscript), indicating non-negligible contribution of newly-synthesized IGF-IR in the hour-scale assay. We used cycloheximide which inhibited the increase in precursor IGF-IR observed in long-term IGF-I-stimulated cells. IGF-I reduced surface IGF-IR in the presence of cycloheximide (Figure 3—figure supplement 2A in the revised manuscript). Combined with the experiments regarding IGF-IR recycling, our data support the existence of ligand-induced IGF-IR internalization in L6 cells.

To accurately evaluate the internalization, we chose the shorter time-course than the original manuscript to avoid the contribution of de novo IGF-IR synthesis. In addition, we performed the assays in the presence of primaquine to measure the net endocytic rate of IGF-IR after the ligand exposure. To test whether ectopic expression of IRS-1 affects AP-2-mediated internalization of IGF-IR, we used L6 cells stably expressing GFP, GFP-IRS-1 WT, or GFP-IRS-1 3YA. While surface IGF-IR levels were reduced within 1 hour after ligand stimulation in cells expressing GFP and GFP-IRS-1 3YA, such reduction turned to be significantly slower in cells expressing GFP-IRS-1 WT as revealed by surface biotinylation assay (Figure 4A in the revised manuscript). The data suggest that IGF-IR internalization is inhibited by the IRS-1 binding to AP2.

As described above, assays based on direct chasing of surface-labeled IGF-IR measure both basal and ligand-dependent internalization of IGF-IR. Therefore, we consider that measuring phospho-IGF-IR distribution by confocal microscopy is a superior way to evaluate the ligand-dependent internalization among our tools. In the original manuscript, AP2 knockdown inhibited the targeting of phospho-IGF-IR into lysosomes induced by IGF-I (Figure 3—figure supplement 1B, C in both original and revised manuscript), demonstrating that the trafficking of phospho-IGF-IR from the cell surface to lysosomes depends on AP2. Notably, phospho-IGF-IR accumulated in lysosomes in IRS-1-depleted cells 1 hour after IGF-I stimulation when phospho-IGF-IR is predominantly localized to the plasma membrane in control cells (Figure 5—figure supplement 1A, B in both original and revised manuscript), indicating that knockdown of IRS-1 accelerates the targeting of phospho-IGF-IR from cell surface to lysosomes. These data support the notion that manipulation of IRS-1 levels influences the AP2-dependent internalization of IGF-IR in response to the ligand.

The reviewer raised the important issue regarding colocalization of IGF-IR with AP2/clathrin at the cell bottom membrane. The function of clathrin plaque is still much debated. Clathrin plaque has been regarded as an endocytically inactive, long-lived structures (Batchlder et al., 2010; Grove et al., 2014), whereas it has been found to be actively internalized (Saffarian et al., 2009). In addition, a recent study using super-resolution microscopy revealed that clathrin-coated pits juxtaposed to plaques are easily misclassified as plaque on cell bottom membrane in the conventional TIRF microscopy (Leyton-Puig et al., 2017). As the reviewer indicated, it is hard to conclude that the colocalization of IGF-IR and AP2 at the TIRF field well reflects the efficacy of IGF-IR internalization because of the limited spatial resolution of our TIRF microscopy. Nevertheless, our observations demonstrate that ectopic expression of GFP-IRS-1 and inhibition of IRS-1 degradation by Torin1 resulted in the diffused localization of phospho-IGF-IR at TIRF field and reduced the colocalization with AP2, suggesting that manipulation with IRS-1 levels affects the ligand-dependent changes of surface IGF-IR distribution. We agree it would be important to clarify the nature of clathrin plaque and its engagement in IGF-IR endocytosis as the reviewer has noted, but we consider that clarification would not change the overall conclusions of the paper. To avoid the over-discussion, we have tried to make the text clearer as follows.

Abstract: We changed the phrase “clathrin-coated pits” to “clathrin-coated structures.”

Results section: We deleted the sentence describing the possible engagement of IGF-IR in CCPs incorporation.

Discussion section: We changed the phrase “CCPs” to “AP2-positive spots.” In addition, we added the description about the necessity of higher-resolution analyses to conclude the IGF-IR targeting to CCPs.

3) The authors propose that phosphotyrosine prevents the binding of the IRS-1 Yxxø motifs to AP-2. However, treatment of cells with IGF-1 does not inhibit the co-immunoprecipitation of IRS-1 with AP-2. The authors argue that this is because of low stoichiometry tyrosine phosphorylation of IRS-1. This could be tested by co-immunoprecipitation analysis.

In response to the reviewer’s suggestion, we performed the pull-down assay to evaluate the stoichiometry of tyrosine phosphorylation of IRS-1 (Figure 1—figure supplement 1D). This assay is based on the molecular feature of m2 that cannot recognize the phosphorylated YxxF sequence. If majority of IRS-1 is tyrosine-phosphorylated, the fraction of IRS-1 that can be pulled down by recombinant m2 would be reduced in the lysates of IGF-I-stimulated cells. However, our data demonstrate that IGF-I did not largely influence the amount of IRS-1 fraction that was pulled down by m2 (Figure 1—figure supplement 1E, F). In addition, we never detected phosphorylated IRS-1 in the pull-down fraction when equivalent amount of immunoprecipitated IRS-1 was simultaneously compared. Therefore, we conclude the low concentration of tyrosine-phosphorylated IRS-1 in IGF-I-stimulated cells in our observation.

4) The interpretation of the IRS-1 over-expression experiments is unclear. If IRS-1 binds AP-2, this should inhibit the internalization of many AP-2 cargos, but this was not observed – e.g. transferrin receptor. The authors argue that this could be because IRS-1 has some special localization within the cell; would this also be true for over-expressed IRS-1? If IRS-1 is an AP-2 competitor, why does it not inhibit internalization of other AP-2 dependent proteins when over-expressed? Moreover, if the competition for AP-2 is restricted to the local environment of the IGF-1R, does this mean that it does not affect other cell surface proteins that signal through IRS-1 signals (e.g. integrins) and if so, why not? Might this mechanism also affect transmembrane tyrosine phosphatases that dephosphorylate IGF-1R?

The reviewer raises an important question. In our original data, GFP-fused IRS-1 localized to submembraneous actin fibers that colocalize with a portion of AP2 as revealed by TIRF microscopy (Figure 4—figure supplement 1D). Such localization of endogenous IRS-1 has also been demonstrated by the previous study (Clark et al., 1998). Since actin cytoskeleton possesses critical roles in CME (Kaksonen et al., 2006), we speculate that IRS-1 may locally regulate the specific cargo endocytosis through association with a portion of AP2 at the actin cytoskeleton.

We have tested whether overexpression of IRS-1 affects endocytosis of other receptors than IGF-IR. Ectopic expression of IRS-1 did not change the transferrin receptor endocytosis as revealed by the fluorescent-labeled transferrin uptake. We further added the data showing that transferrin receptor does not physically interact with IGF-IR (Figure 4—figure supplement 1A in the revised manuscript). As the reviewer suggested, we analyzed endocytosis of integrin b1 and EGFR, both of which physically interact with IGF-IR as validated by co-immunoprecipitation (Figure 4—figure supplement 2A, E in the revised manuscript). The transfection of mRFP-IRS-1 partially inhibited endocytosis of integrin b1 in non-stimulated condition as assessed by uptake of anti-integrin b1 antibody (Figure 4—figure supplement 2B–D in the revised manuscript). In addition, the transfection of mRFP-IRS-1 partially inhibited endocytosis of EGFR in the early period of EGF stimulation. These observations raise the possibility that IRS-1 influences endocytosis of cargoes in the close proximity of IGF-IR.

In this revision, we could not test endocytosis of transmembrane phosphatases targeting IGF-IR due to the time limitation. PTP1B is one of the characterized phosphatases for IGF-IR, but is localized to the endoplasmic reticulum, not to plasma membrane. Although we did not rule out the potential effect of IRS-1 on localization/endocytosis of other transmembrane phosphatases, we consider that PTP1B is not suitable target for analysis in our case. We have not had time to assess other potential phosphatases targeting IGF-IR but agree it would be interesting although as the reviewer has noted, not necessary to the overall conclusions of the paper.

5) The kinetics of endocytosis of pIGF-IR and signaling are on different scales. The effects of the depletion or overexpression of IRS1 are evident on pAkt only after 6-12 hours (Figure 7) or later. The effects of IRS1 level alterations can be alternatively interpreted by the increase or decrease of the amount of phosphoIRS1 capable of engaging PI3K. The lack of the effect of the blockade of the receptor endocytosis by mu2 depletion of Akt activity (Figure 7—figure supplement 1D and 1E) is puzzling. The authors' interpretation of this data is that slow endocytosis is not sufficient in order to prolong Akt activity, and that the presence of high levels of IRS1 is also required. Such interpretation is difficult to reconcile with the model.

We thank the reviewer for the helpful questions. As the reviewer suggested, the cause for IRS-1-mediated increase in the duration of the Akt signaling was unclear in the original manuscript. We have now performed additional experiments to assess the role of IRS-1 engaging the PI3K-Akt pathway in the sustainability of downstream signaling by using the IRS-1 mutant ΔPTB. Although ectopic expression of IRS-1 ΔPTB strongly prolonged surface phospho-IGF-IR retention (Figure 2F, G in both original and revised manuscripts), it failed to prolong phospho-Akt (Figure 7—figure supplement 1F in the revised manuscript), suggesting that the ability of IRS-1 to engage PI3K is required for prolonging the Akt signaling. The interpretation of the reviewer therefore is correct. Nevertheless, comparing the consequences of phospho-IGF-IR and phospho-Akt in cells stably expressing GFP-IRS-1 WT, IRS-1 3YA, ΔPTB, and IRS-2, our data indicate that the IRS-1 ability to bind AP2 and promote surface retention of phospho-IGF-IR is also required for prolonging the Akt signaling. In addition, AP2 knockdown prolonged phospho-IGF-IR, but failed to sustain the Akt signaling, as like the events observed in IRS-1 ΔPTB-expressing cells (Figure 7—figure supplement 1D, E in the revised manuscript). In AP2-depleted cells, IRS-1 degradation, a consequence of negative feedback, was normally induced after long-term IGF-I treatment, indicating that the IRS-1 function to activate PI3K is shut down in the later period. Collectively, the effect of IRS-1 on prolonging the Akt signaling is likely based on two independent functions of IRS-1: the first is the binding to AP2, and the second is the activating PI3K via its tyrosine phosphorylation. We have tried to make clear the discussion of this issue and focused upon the relationship between two independent functions of IRS-1 in prolonging downstream signaling in Discussion section.

Author details

1. Yosuke Yoneyama

   Department of Animal Resource Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Validation, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9170-2846

2. Peter Lanzerstorfer

   University of Applied Sciences Upper Austria, Wels, Austria

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

3. Hideaki Niwa

   1. RIKEN Systems and Structural Biology Center, Yokohama, Japan
   2. RIKEN Center for Life Science Technologies, Yokohama, Japan

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

4. Takashi Umehara

   1. RIKEN Systems and Structural Biology Center, Yokohama, Japan
   2. RIKEN Center for Life Science Technologies, Yokohama, Japan
   3. PRESTO, Japan Science and Technology Agency, Kawaguchi, Japan

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

5. Takashi Shibano

   Department of Animal Resource Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

   Present address

   Department of Oncology and Pathology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft

   Competing interests

   No competing interests declared

6. Shigeyuki Yokoyama

   1. RIKEN Systems and Structural Biology Center, Yokohama, Japan
   2. RIKEN Structural Biology Laboratory, Yokohama, Japan

   Contribution

   Resources, Data curation, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

7. Kazuhiro Chida

   Department of Animal Resource Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

   Contribution

   Resources, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

8. Julian Weghuber

   1. University of Applied Sciences Upper Austria, Wels, Austria
   2. Austrian Competence Center for Feed and Food Quality, Safety and Innovation, Wels, Austria

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

9. Fumihiko Hakuno

   Department of Animal Resource Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   ahakuno@mail.ecc.u-tokyo.ac.jp

   Competing interests

   No competing interests declared

10. Shin-Ichiro Takahashi

    Department of Animal Resource Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Writing—review and editing

    For correspondence

    atkshin@mail.ecc.u-tokyo.ac.jp

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2323-2010

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