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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: J Invest Dermatol. 2020 Feb 3;140(9):1743–1752.e4. doi: 10.1016/j.jid.2020.01.015

Flotillin and AP2A1/2 promote insulin-like growth factor receptor-1 association with clathrin and internalization in primary human keratinocytes

Duncan Hieu M Dam 1, Sophia A Jelsma 1, Jeong Min Yu 1, Haoming Liu 1, Betty Kong 1, Amy S Paller 1
PMCID: PMC7396279  NIHMSID: NIHMS1568985  PMID: 32027876

Abstract

Insulin-like growth factor-1 (IGF-1) receptor (IGF1R) signaling promotes keratinocyte proliferation, migration, and survival. However, the mechanism of IGF1R endocytosis in normal keratinocytes remains unclear. Confocal, super resolution structured illumination microscopy (SIM), total internal reflection fluorescence/TIRF microscopy, and coimmunoprecipitation studies reveal that IGF1R associates with flotillin-1, which currently has no known role in normal receptor tyrosine kinase endocytosis, under basal conditions in monolayer keratinocyte cultures. Ligand stimulation of IGF1R promotes its clathrin-dependent endocytosis, mediated by two distinct adaptors, flotillin-1 (Flot-1) in noncaveolar lipid rafts and the AP2A1/2 complex in clathrin vesicles. Concurrent (but not individual) shRNA knockdown of FLOT1/2 and AP2A1/2 reduced IGF1R association with clathrin, internalization, and pathway activation by more than 50% (of pIGF1R, pAKT, and pMEK), suggesting the complementarity of these two adaptor-specific pathways. The Flot-1 pathway is more responsive to low IGF-1 concentrations, whereas the AP2A1/2 pathway predominates at higher IGF-1 concentrations. Selective association of IGF1R-Flot-1-clathrin with Rab4, but IGF1R-AP2A1/2-clathrin with Rab11, implicates Flot-1 as the adaptor for faster recycling and AP2A1/2 as the adaptor for slower IGF1R recycling. These dual pathways, particularly flotillin-dependent, clathrin-mediated endocytosis, provides a new avenue for drug targeting in disorders with aberrant regulation of IGF1R signaling.

Keywords: insulin-like growth 1 receptor, flotillin, clathrin, endocytosis, keratinocytes

Introduction

Receptor tyrosine kinases (RTKs) are generally localized in cell membranes in a monomeric and non-phosphorylated form. Upon ligand binding to the extracellular domain, oligomerization of monomeric RTKs is induced through conformational changes that stabilize the active oligomeric form (Schlessinger, 2000). Oligomerization classically leads to RTK activation via autophosphorylation of tyrosine residues in the intracellular domain, which increases intrinsic catalytic activity and induces the formation of additional binding sites for substrate proteins (Hubbard and Till, 2000). Activated RTKs then trigger endocytosis, which is the major regulator of sustained signaling from the cell surface to the cytoplasm and nucleus (Paniagua et al., 2011). The canonical model of RTK endocytosis often involves rapid internalization of a RTK after ligand binding and subsequent sorting of internalized ligand-RTK complexes, either for receptor recycling or degradation (Goh and Sorkin, 2013).

In normal human epidermal keratinocytes (NHEKs), insulin-like growth factor-1 (IGF-1) increases proliferation, stimulates lamellipodial protrusion and cell spreading (Haase et al., 2003), and promotes cell migration via the phosphatidylinositol-3-kinase and Ras-related C3 botulinum toxin substrate (Rac1) signaling pathways (Dam et al., 2017) through activation of the IGF-1 receptor (IGF1R), a RTK of the insulin receptor (IR) family. IGFR-null keratinocytes (Sadagurski et al., 2006), but not insulin receptor (IR)-null keratinocytes (Stachelscheid et al., 2008), exhibit accelerated differentiation and decreased proliferation, and knockout of IGF1R in mice reduces epidermal thickness (Sadagurski et al., 2006, Stachelscheid et al., 2008). The important role of IGF1R activation during wound healing and its suppression in the impaired wound healing of diabetes is suggested by the: a) reduced IGF1R phosphorylation at the wound edge of diet-induced obese (DIO) diabetic mice (Wang et al., 2014); b) deficiency of IGF-1 expression in keratinocytes from human diabetic foot ulcers (Blakytny et al., 2000); and c) acceleration in wound closure when diabetic rats are treated with topical IGF-1 supplementation, which enhances protein kinase B (AKT) and extracellular signal-regulated kinases (ERK) signaling (Bitar, 1997, Korolkiewicz et al., 2000, Lima et al., 2012).

When activated by binding to its ligand (IGF-1 and, to a lesser extent, insulin), activated IGF1R forms dimers, is released from the membrane, and segregates into vesicles to enter cells (Foti et al., 2004, Morcavallo et al., 2014). In these vesicles, IGF-1 uncouples from the IGF1R, which either recycles back to the cell surface to perpetuate activation or is degraded in lysosomes, terminating the signaling cascade (Foti et al., 2004). Studies of IGF1R endocytosis have been performed only in cell lines, with no consistency in their mechanism of endocytosis. For example, in H9C2 rat cardiomyoblasts and HaCaT cells, caveolin-1 down-regulation inhibits IGFIR internalization and receptor signaling (Salani et al., 2008, Salani et al., 2010), but in Ewing sarcoma cells, inhibition of clathrin or caveolin-1 partially impairs endocytosis and reduces IGF1R signaling (Salani et al., 2009). In the human embryonic kidney (HEK293T) cell line, IGF1R localizes to noncaveolar regions (Hong et al., 2004) and associates with flotillin-1 (Jang et al., 2015).

The mechanism for endocytosis and recycling of IGF1R in normal cells, including NHEKs, has not been explored. We now demonstrate that IGF1R associates with flotillin-1 and clathrin in NHEKs for internalization and rapid recycling by an alternative, complementary mechanism to the conventional AP2 complex pathway.

RESULTS

Inactive IGF1R resides in the noncaveolar flotillin domain

To identify the membrane-based localization of IGF1R in NHEKs and the effect of IGF-1 stimulation on this localization, we used total internal reflection fluorescence (TIRF) microscopy, which allows high axial resolution of the membrane. In non-stimulated basal medium, IGF1R colocalized with flotillin-1 (Flot-1), a non-caveolar lipid raft protein marker (τ=0.68), but not with caveolin-1 (Cav-1) (τ<0.4) (p<0.001; Fig. 1a, top and bottom rows), suggesting that inactive IGF1R localizes to the flotillin domain of the lipid raft, rather than the caveolar domain. To further confirm the membrane-based association of IGF1R with Flot-1, we biotinylated IGF1R and pulled down the IGF1R in whole cell lysates with an anti-IGF1R antibody (Cell Signaling #9750) and then streptavidin-conjugated magnetic beads. This antibody strongly detected only IGF1R in blots and bound no protein when IGF1R was knocked down with lentiviral-introduced IGF1R shRNA (Fig. S1). Surface IGF1R coimmunoprecipitated (coIP’d) with Flot-1, but not with Cav-1 (Fig. 1b). When stimulated with either chronic low dose IGF-1 in complete growth medium (containing 10 ng/mL IGF-1) or acute high dose IGF-1 (overnight starvation, then 100 ng/ml IGF-1 for 10 mins), Flot-1 strongly colocalized with the IGF1R at the cell membrane based on TIRF microscopy (τ = 0.70, p<0.001), but Cav-1 did not. Furthermore, Cav-1 did not associate with surface IGF1R based on coIP (Fig. 1b). With IGF-1 stimulation, but not under basal conditions (p<0.001), the adaptor related protein complex 2 (AP2), a marker of clathrin-coated vesicles, also strongly colocalized with IGF1R at the cell membrane (τ=0.88 for the adaptor’s alpha subunit 1 and 2 (AP2A1/2) with IGF1R) (Figs. 1a, middle row) and co’IPd with IGF1R as well (Fig. 1b). We also confirmed our TIRF microscopy and protein immunoprecipitation observations using super resolution structured illumination microscopy, showing that IGF1R is associated with AP2A1/2 upon IGF-1 stimulation (100ng/ml) while Flot-1 is associated with IGR1R, regardless of IGF-1 activation status (Fig. 1c).

Figure 1. Colocalization and coimmunoprecipitation of surface IGF1R in NHEKs.

Figure 1.

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(a) Total internal reflectance fluorescence (TIRF) imaging of immunofluorescence (IF)-stained NHEKs shows IGF1R strongly colocalized with Flot-1 at the basal aspect of the cell without IGF-1 stimulation. Upon stimulation with IGF-1 through complete medium or addition of 100 ng/ml to starved cells, IGF1R colocalizes with both Flot-1 and αAP1/2, but not with caveolin-1 (Cav-1). (b) Coimmunoprecipitation of biotinylated surface IGF1R shows the association of surface IGF1R with Flot-1 under basal conditions and with both Flot-1 and AP2A1/2 (but not Cav-1) with IGF-1 stimulation. (c) Super resolution structured illumination microscopy (SIM) images indicate strong overlap of IGF1R (red signal) with Flot-1 (green) in both basal and IGF-1 stimulated conditions, but strong colocalization of IGF1R with AP2A1/2 (green) only with IGF-1 stimulation. Images and blots reflect typical results from 3 experiments for each; graphs show mean+S.E. n=30 cells. Scale bar: 20 µm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Standard confocal microscopic (i.e., not specifically focused on the membrane as in TIRF imaging) showed strong cytoplasmic colocalization of IGF1R with Flot-1 and AP2A1/2 after NHEK stimulation with 100 ng/mL IGF-1 (τ=0.7 and 0.9 respectively; Fig. 2a). IGF1R from whole lysates coIP’d with both Flot-1 and AP2A1/2 (Fig. 2b–d). These observations suggested that both AP2A1/2 and Flot-1 are recruited to IGF1R-internalized complexes during IGF1R endocytosis. Again, IGF-1 activated IGF1R did not colocalize or coIP with Cav-1 (Fig. 2a–b), confirming that cav-1 does not participate in IGF1R endocytosis in NHEKs. These data demonstrate the role of both Flot-1 and AP2A1/2, and not Cav-1, in facilitating localization and endocytosis of IGF1R, with or without IGF-1 stimulation, in normal keratinocytes.

Figure 2. Association of IGF1R with Flot-1 and AP2A1/2 in NHEK cytoplasm.

Figure 2.

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(a) Confocal imaging of IF-stained NHEKs. Under basal conditions, IGF1R strongly colocalizes only with Flot-1. With IGF-1 stimulation, IGF1R colocalizes with both Flot-1 and αAP1/2, but still not with Cav-1. Scale bar: 20 µm, mean+S.E., n=30 cells. (b-d) IGF1R coimmunoprecipitates with Flot-1 under basal conditions, but with both Flot-1 and AP2A1/2 with IGF-1 stimulation. **p<0.01, ****p<0.0001.

IGF-1 activates IGF1R internalization through clathrin-dependent endocytosis

The AP2 complex is thought to initiate formation of clathrin-coated pits and clathrin-mediated endocytosis of RTKs (Goh and Sorkin, 2013). Thus, we investigated whether IGF-1 induced an association of IGF1R with clathrin. Indeed, after exposure to IGF-1, both TIRF (Fig. 3a, top row) and confocal microscopy (Fig. 3a, bottom row) showed strong surface and cytoplasmic IGF1R-clathrin colocalization (τ = 0.85 and 0.80, respectively) (Fig. 3a). Similarly, coIP of clathrin with IGF1R and vice versa were observed (Fig. 3b and c), but very little to no colocalization or coIP was observed in basal medium without IGF-1 stimulation (Fig. 3a–c). Our results suggest that IGF-1 induces the association of clathrin with IGF1R, both at the membrane and during IGF1R internalization.

Figure 3. Association of IGF1R with clathrin in monolayer cultures.

Figure 3.

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(a) Top: TIRF imaging of IF stained NHEKs. Bottom: Confocal imaging of IF-stained NHEKs. Representative images show strong colocalization of IGF1R with clathrin upon ligand stimulation. Scale bar: 20 µm, n=30 cells. (b-c) IGF1R coimmunoprecipitates with clathrin only with IGF-1 stimulation. d-e) Flot-1 coimmunoprecipitates and colocalizes with clathrin under IGF-1 stimulation. Scale bar: 20 µm, n=30 cells. (f-g) AP2A1/2 coimmunoprecipitates and colocalizes with clathrin under stimulated conditions. However, Flot-1 and AP2A1/2 do not colocalize or coimmunoprecipitate with each other under any conditions. Representative blots and TIRF images are shown, with graphs showing pooled results of 3 experiments (expressed as mean+S.E.). Scale bar: 20 µm, n=30 cells. All data is shown as mean+S.E. ***p<0.001, ****p<0.0001.

Flotillin-1 is an alternative adaptor for the IGF1R association with clathrin

The association of Flot-1 and IGF1R has been suggested in the epithelial human embryonic kidney cell line (Jang et al., 2015), but the potential association of Flot-1 with clathrin has not been previously explored, especially in a normal human cell that can differentiate or senesce. In NHEKs without IGF-1 stimulation, Flot-1 only weakly colocalized or coIP’d with clathrin (Fig. 3d, e), and clathrin minimally colocalized or coIP’d with IGF1R (Fig. 3b, c). In the presence of IGF-1, however, the colocalization and coIP of clathrin and Flot-1 with IGF1R was strong (Fig. 3b–c; 3d–e), suggesting that IGF-1 can stimulate the association of IGF1R, Flot-1 and clathrin, in which Flot-1could potentially serve as an adaptor for the clathrin-IGF1R association and promote IGF1R endocytosis from the flotillin-containing lipid raft.

We then examined the relationship between this putative IGF1R-Flot-1-clathrin association and the classical IGF1R-AP2-clathrin complex. Clathrin colocalized and coIP’d with both AP2A1/2 and Flot-1 (Fig. 3d–g); however, AP2A1/2 did not colocalize or coIP with Flot-1 (τ <0.30) (Fig. 3d, f–g), implying that the association of Flot-1 with IGF1R and with clathrin is separate from the AP2-clathrin complex. Furthermore, it implied that IGF1R might associate with clathrin through either the AP2 complex or one containing flotillin-1. To further investigate these possibilities, shRNA lentiviruses were used to reduce the protein expression of AP2A1/2, Flot-1 or both. This required concurrent knockdown of: i) AP2A1 and AP2A2 to reduce AP2A1/2 expression by 67.4 ± 4.7%; ii) FLOT1 and FLOT2 to reduce Flot-1 by 90.5±5.2%, (FLOT1 shRNA alone reduced Flot-1 by only 32.2±4.2%); and iii) FLOT1/2 and AP2A1/2 serially (for concurrent reduction of 50.6±2.2% for Flot-1 and 49.7±2.8% for AP2A1/2) (Fig. S2).

Individual knockdown of AP2A1/2 or FLOT1/2 did not change the association of IGF1R with clathrin (Fig. 4a–b). Similarly, blockade of translocation of clathrin and adaptor complexes from membrane to cytoplasm using 10 mM chlorpromazine (CPMZ) (Dutta and Donaldson, 2012) or dissociation of lipid rafts by 5mM methyl-β-cyclodextrin (MβCD)-induced depletion of cholesterol (Dutta and Donaldson, 2012) (Fig. S3) failed to prevent either the IGF1R-clathrin association or internalization of the complex (Fig. S4). In contrast, concurrent knockdown of AP2A1/2 and FLOT1/2 in NHEKs markedly reduced colocalization and coimmunoprecipitation of IGF1R with clathrin (p<0.01) (Fig. 4a–b). Concurrent treatment with both CPMZ and MβCD also reduced the association of IGF1R with clathrin (Fig. S4). These data suggest that the flotillin-clathrin association and the AP2 complex are part of alternative pathways, able to compensate for each other in facilitating the association of IGF1R and clathrin during IGF-1-induced IGF1R endocytosis. Furthermore, these data provide evidence for distinct pathways for clathrin-mediated endocytosis, one initiated in lipid rafts and dependent on flotillin, which uses clathrin-coated pits for endocytosis, and the other being traditional clathrin-mediated endocytosis, facilitated by the adaptor complex.

Figure 4. Flotillin and the AP2 complex compensate for each other in promoting the IGF1R-clathrin association and IGF1R signaling.

Figure 4.

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(a) Coimmunoprecipitation of clathrin with IGF1R after starvation and IGF-1 stimulation with knockdown of both components of AP2A1/2 or FLOT1/2 (separately or serially) using shRNA lentiviruses; densitometry readings are shown at right. Only concurrent knockdown of AP2A1/2 and FLOT1/2 decreases the IGF1R-clathrin coimmunoprecipitation (n=3 blots). (b) Colocalization of IGF1R (red) and clathrin (green) by confocal microscopy after selective pathway knockdown. Scale bar: 20 µm, n=30 cells. (c) Representative blot of signaling alteration with selective knockdown (left) and change in signaling molecule activation (right). Only concurrent knockdown globally decreases IGF1R pathway phosphorylation by >50%. (d) Western blot showing biotinylated-IGF1R internalization (cytoplasmic IGF1R) 2–15 min after addition of 100 ng/mL IGF-1 at 37°C (4 left lanes) and total biotinylated surface IGF1R (treated at 4°C to prevent internalization; 5th lane). e) Ratio of cytoplasmic/total surface IGF1R. All blots and images represent n=3 runs. All data is shown as mean+S.E. *p< 0.05, **p < 0.01, ***p<0.001, ****p<0.0001.

IGF1R signaling is regulated by use of the Flot-1 vs. AP2 complex endocytotic pathway

When RTKs are activated by their ligands, the ability of a receptor to signal after endocytosis is important to sustain sufficient duration and intensity of signaling (Sigismund et al., 2008, Sorkin and Von Zastrow, 2002, 2009). After 15 min of IGF-1 stimulation, IGF1R, protein kinase B (AKT), and mitogen-activated protein kinase kinase (MEK) were all phosphorylated (Fig. 4c). Signaling was largely unaffected by knockdown of FLOT1/2 alone (internalization through the AP2 complex pathway) (Fig. 4c) or AP2A1/2 alone (endocytosis through the Flot-1 complex; associated with a 31.7±1.3% inhibition in AKT S437 phosphorylation and a 39.8±3.3% loss of AKT T308 phosphorylation (Fig. 4c). In contrast, knockdown of both FLOT1/2 and AP2A1/2 reduced phosphorylation of IGF1R, AKT, and MEK by 60.1±3.0%, 58.2±2.5% (57.1±1.8% pAKT S437; 58.8±2.7% pAKT T308), and 74.1±7.9%, respectively.

The rate of IGF1R endocytosis is regulated by choice of Flot-1 vs. AP2 complex

We next investigated whether the distinct Flot-1/clathrin and AP2/clathrin complex pathways could regulate the rate of IGF1R endocytosis. Biotinylated IGF1R (at the surface) was internalized within 2 min of stimulation with IGF-1, reaching a peak internalization of almost 93% at 15 min of IGF-1 stimulation (Fig. 4d). When FLOT1/2 was knocked down, the rate of internalization was greatly reduced. By 2 min, only 14% of surface biotinylated IGF1R entered the cytoplasm of Flot-1-depleted NHEKs, and only 66% of the total surface receptor population was internalized in the cytoplasm after 15 min of IGF-1 stimulation (Fig. 4d–e), lower than in vector control-treated NHEKs (Fig. 4d–e) (p<0.01). When AP2A1/2 was knocked down, forcing IGF1R to endocytose through the putative Flot-1-clathrin association, surface IGF1R internalized quickly, achieving 72% of biotinylated IGF1R in cytoplasm after 2 min and 94% after 5 min of IGF-1 stimulation (Fig. 4d–e). Detection was reduced to 67% 10 min after initiation of IGF-1 and increased to almost 99% after 15 min of stimulation, significantly higher than that observed in NHEKs with reduced Flot-1 expression (p<0.01) (Fig. 4d–e). The rapid, almost complete internalization of IGF1R after 5 min of IGF-1 stimulation through the Flot-1 pathway suggested that Flot-1-dependent IGF1R internalization is faster than AP2 complex-dependent internalization. Furthermore, reduction of internalized cytoplasmic IGF1R through the Flot-1 pathway at 10 min and recovery at 15 min implied that this pathway not only facilitates faster internalization of IGF1R, but also generates a quicker recycling of the receptor to the surface for further activation. Concurrent knockdown of FLOT1/2 and AP2A1/2 significantly reduced IGF1R endocytosis, with less than 25% of surface IGF1R entering the cytoplasm at each timepoint (Fig. 4d–e), confirming the dependence on Flot-1 and AP2 pathways for IGF1R internalization.

The domain-specific membrane localization and endocytosis of RTKs can depend on ligand concentration. For example, epidermal growth factor (EGF) induced endocytosis of the EGF receptor (EGFR) is primarily mediated by clathrin vesicles. However, at high concentrations of EGF ligand, EGFR is observed in caveolar domains (Knudsen et al., 2014). Indeed, after 10 min of exposure to low concentrations of IGF-1 (1–10 ng/mL), we found strong colocalization and coIP only of IGF1R with Flot-1 (τ = 0.80) and not with AP2A1/2 (τ = 0.30) (Fig. 5a–b, d). In contrast, at high concentrations of IGF-1 (500–1000 ng/mL) IGF1R colocalizes and coIP’s only with AP2A1/2 (τ = 0.81) and not Flot-1 (τ = 0.25) (Fig. 5a–b, d). Furthermore, at all IGF-1 concentrations, IGF1R still strongly colocalizes and coIP’s with clathrin (τ ~ 0.80) (Fig. 5c–d). These data further support that alternative endocytotic pathways regulate IGF1R signaling, with a Flot-1/clathrin pathway preferred at low ligand concentrations and an AP2/clathrin complex preferred at high concentrations.

Figure 5. TIRF colocalization of IGF1R with Flot-1, adaptin complex, and clathrin after exposure to different IGF-1 concentrations.

Figure 5.

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(a) Strong colocalization of IGF1R with Flot-1 at low IGF-1 concentration (0–100 ng/mL), but not at higher IGF-1 concentrations, suggesting movement out of the flotillin-containing domains. (b) In contrast, IGF1R appears to colocalize more strongly with the adaptin complex after stimulation with IGF-1 at higher concentrations (100–1000 ng/mL), but only weakly at 0–10 ng/mL IGF-1. (c) IGF1R strongly colocalizes with clathrin at 1–1000 ng IGF-1. Scale bar: 20 µm. All data is shown as mean+S.E. n=30 cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (d) Immunoprecipitation of AP2A1/2, Flot-1, and clathrin with IGF1R at different IGF-1 concentrations.

Membrane concentration of a RTK is influenced by endocytic recycling, in addition to trafficking through delivery to the membrane of newly synthesized RTK vs. receptor internalization (Miaczynska, 2013). Speed of the recycling process could deliver more receptor to the cell surface for signaling and thus can be a mechanism to further regulate IGF1R activation. Rab4 has been used as a cytoplasmic marker of fast receptor recycling, while Rab11 is a cytoplasmic marker of a slow recycling (Li et al., 2008) (Chibalina et al., 2007). Confocal analysis of IGF-1-stimulated IGF1R in vector control-treated NHEKs showed equal perinuclear colocalization of IGF1R with both Rab4 and Rab11 at 2, 5, 10, and 15 min after IGF-1 stimulation (Fig. 6a), although Rab11 was distributed more towards the cell periphery than Rab4. When AP2A1/2 was knocked down, thereby forcing a shift to flotillin-dependent endocytosis, colocalization between IGF1R and Rab4 was much greater than between IGF1R and Rab11 (p<0.0001 at all timepoints) (Fig. 6b), suggesting that flotillin promotes fast recycling of IGF1R. In contrast, when FLOT1/2 was knocked down, an increase in colocalization of IGF1R-Rab11 compared to IGF1R-Rab4 was observed (p < 0.0001 at all timepoints) (Fig. 6c), implying that IGF1R endocytosis through the αAP1/2-mediated pathway recycles IGF1R to the surface more slowly.

Figure 6. Recycling of IGF1R depends on both Flot-1 and the AP2 complex.

Figure 6.

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(a) Confocal images of colocalization of IGF1R (red) with Rab4 or Rab11 (both green) at 0, 2, 5, 10 and 15 min after incubation with IGF-1. (b) AP2A1/2 knockdown (largely action of Flot-1) increases IGF1R-Rab4 colocalization and robust recycling. (c) FLOT1/2 knockdown (largely action of AP2A1/2) increases IGF1R-Rab11 colocalization, promoting slower recycling. Scale bar: 20 µm, n=30 cells. Data shown as mean+S.E*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001.

DISCUSSION

Endocytosis of RTKs often involves caveolin-1-containing lipid raft microdomains or domains marked by clathrin, which conventionally require the AP2 complex for its association with RTKs (Goh and Sorkin, 2013). Flotillin-1 and −2 are membrane-associated proteins that co-assemble into discrete lipid raft microdomains similar to caveolae but contain no caveolin-1. The flotillin microdomain has been thought to facilitate compartmentalization and functional specialization within membranes (Otto and Nichols, 2011). We have now probed the mechanism for endocytosis of IGF1R in normal keratinocytes and have shown strong colocalization of clathrin with AP2A1/2 in NHEK cytoplasm and its coIP with IGF1R after IGF-1 stimulation, suggesting a role for a clathrin-AP2-IGF1R microdomain in NHEK IGF1R endocytosis and signaling. Although Flot-1 has been hypothesized to serve as adaptor protein for clathrin (Otto and Nichols, 2011), we now provide evidence that Flot-1 associates with IGF1R and that the association of Flot-1 with clathrin can complement and compensate for the classical IGF1R-AP2-clathrin complex in mediating endocytosis, recycling, and regulation of IGF1R signaling. Furthermore, Flot-1 promotes more rapid recycling of IGF1R than the classical pathway, providing an opportunity for an alternative, more sustained response to ligand stimulation, particular at low ligand concentration. These studies suggest the importance of further comprehensive investigations using proteomic analysis to dissect the molecular complexity of the Flot-1/IGF1R/clathrin association and assessments for direct molecular interactions.

This mechanism for IGF1R endocytosis is distinct from the mechanism for IGF1R endocytosis shown in various cell lines, including in the keratinocyte-derived HaCaT cell line (Salani et al., 2010), in which the caveolar domain was implicated. We found no evidence of caveolar-based endocytosis in our normal keratinocytes based on colocalization or coimmunoprecipitation studies. HaCaT cells are immortalized human keratinocytes, and deviate in their behavior from normal cultured epidermal keratinocytes (Pastore et al., 2011, Seo et al., 2012), in addition to this different mechanism for IGF1R endocytosis. Despite the published variety of cell-specific mechanisms for RTK endocytosis (Doherty and McMahon, 2009, Polo and Di Fiore, 2006, Sorkin and Von Zastrow, 2002, 2009), it should be recognized that the possibility of Flot-1/IGF1R and clathrin/Flot-1 associations has never previously been explored.

Our investigations showed less IGF1R colocalization and coIP with Flot-1 at high IGF-1 concentrations. In addition, the reduction in pAKT (but not pMEK1/2 or pIGF1R) by knockdown of AP2A1/2, but not FLOT1/2, was surprising and raises the possibility that activation of IGF1R through the AKT pathway requires a more sustained signaling than the MEK pathway, given the more rapid recycling of IGF1R using the IGF1R-Flot-1-clathrin endocytic pathway. IGF1R activation is known to promote keratinocyte proliferation and inhibit differentiation (Ando and Jensen, 1993; Blakytny et al., 2000; Sadagurski et al., 2006; Stachelscheid et al., 2008), and its activity is increased in hyperproliferative disorders with abnormal differentiation, such as psoriasis (Krane et al., 1992). Better understanding of this regulation based on membrane compartmentalization and the requirement for specific adaptors lead to an unchartered avenue for understanding epidermal skin disorders, as well as drug discovery for cutaneous diseases with aberrant regulation of IGF1R signaling.

MATERIALS AND METHODS:

All in vitro studies were performed three or more times in triplicate. Significance was determined across two independent groups using a Student’s t-test. When comparing multiple groups within a single variable (e.g. dose), one-way ANOVA with Tukey’s post-hoc adjustment was used. Multiple variable interactions (e.g. time X dose) were tested using a two-way ANOVA with a Tukey’s post-hoc adjustment. P<0.05 was considered significant. GraphPad Prism (ver. 8.2.0) was used to analyze data. See Supplementary Materials online for additional details.

Cell Culture:

NHEKs were isolated from neonatal foreskin cultured in keratinocyte serum-free medium (Cascade Biologics, Portland, OR) supplemented with 0.075 mM CaCl2. Accrual of foreskins was Institution Review Board approved without written consent as otherwise discarded tissue.

Immunoblotting:

30 µg total protein was separated on a precast gradient TGX gel (4–15%; Bio-Rad, Hercules, CA) and transferred to 0.2 µm nitrocellulose membrane. Proteins were detected using primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA or Cell Signaling, Danvers, MA) followed by HRP-tagged secondary antibody (Cell Signaling).

Immunoprecipitation:

800 µg of protein lysate was incubated with primary antibody overnight at 4°C. 40 µL mixture of protein A and protein G magnetic beads were used for pulldown studies. 20 µL of 3x elution buffer was used to elute protein from the beads at 95°C for 10 min.

Immunofluorescence:

Fixed cell samples were incubated with primary antibodies overnight at 4°C, followed by fluorescence-tagged secondary antibodies at room temperature for 1 hour.

Cell transduction:

Lentiviral shRNAs were transduced into NHEKs serially. Transduced cells were confirmed using fluorescence microscopy and selected with puromycin for 3 days.

Supplementary Material

Supplement 1

Scheme 1. Regulation of IGF1R endocytosis and recycling by two potential pathways.

Scheme 1.

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Based on our findings we propose two alternate pathways leading to IGF1R internalization and subsequent recycling: (1) Clathrin-associated, flotillin-mediated endocytosis results in fast internalization and recycling of IGF1R; and (2) Clathrin-associated, adaptor-related protein complex 2-mediated endocytosis promotes slower IGF1R internalization and recycling.

ACKNOWLEDGEMENTS

This work was supported by the National Institutes of Health Grants R01AR068375 (AP) and the Postgraduate Training in Cutaneous Biology T32 AR060710 (DD). This research also utilized Core resources provided by the Northwestern University Skin Disease Research Center (NIAMS, P30 AR057216), the Northwestern University Skin Biology and Disease Research Center (NIAMS, P30 AR075049), and Northwestern’s Center for Advanced Microscopy (NCI, P30 CA060553).

Abbreviations:

NHEK

normal human epidermal keratinocytes

RTK

receptor tyrosine kinase

IGF1R

insulin growth-like factor-1 receptor

IGF-1

insulin growth-like factor-1

IR

insulin receptor

EGFR

epidermal growth factor receptor

Flot-1

flotillin-1

Flot-2

flotillin-2

AP2

adaptor related protein complex 2

AP2A1

adaptor related protein complex 2 subunit alpha 1

AP2A2

adaptor related protein complex 2 subunit alpha 2

Cav-1

caveolin-1

AKT

Protein kinase B

MEK

Mitogen-activated protein kinase kinase

shRNA

short hairpin RNA

Footnotes

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DATA AVAILABILITY

No datasets were generated or analyzed during the current study.

CONFLICT OF INTEREST

The authors state no conflict of interest.

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Supplementary Materials

Supplement 1
NIHMS1568985-supplement-Supplement_1.pdf (185.6KB, pdf)

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