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© The Rockefeller University Press,
0021-9525/2000//1345 $5.00
The Journal of Cell Biology, Volume 151, Number 6,
, 2000 1345-1352
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Numb Is an Endocytic Protein
pdifiore{at}ieo.it
Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of
adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.
Key Words: Numb • endocytosis • EH domain • EGFR • Eps15
© 2000 The Rockefeller University Press
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Introduction |
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 TopAbstract Introduction Materials and MethodsResults and DiscussionReferences |
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Numb physically interacts with the receptor Notch and biologically antagonizes its signaling (Frise et al. 1996; Guo et al. 1996; Zhong et al. 1996; Berezovska et al. 1999; Sestan et al. 1999). Notch is an evolutionarily conserved transmembrane receptor that specifies cell fate in a wide variety of tissues and organisms through local cell interactions (Artavanis-Tsakonas et al. 1995, Artavanis-Tsakonas et al. 1999; Greenwald 1998). Due to the asymmetric partitioning at mitosis, cells that receive Numb are rendered unresponsive to Notch signaling. Conversely, Numb-negative cells retain responsiveness to Notch and adopt the fate associated with Notch activation.
The biochemical mechanisms underlying the action of Numb are still largely obscure. An approach towards this issue is derived from the observation that Numb can interact in vivo and in vitro with Eps15, a component of the endocytic machinery (Salcini et al. 1997). Eps15 has a modular structure comprising three copies of a protein–protein interaction domain, the Eps15 homology (EH) domain (Wong et al. 1995), a central coiled coil domain, and a COOH-terminal region that binds to the clathrin adaptor complex AP2 (Benmerah et al. 1995, Benmerah et al. 1996; Iannolo et al. 1997). An Asn-Pro-Phe (NPF) motif acts as the ligand for the EH domain (Salcini et al. 1997). EH-containing and EH-binding proteins establish a complex network of interactions within the cell that regulates internalization and/or trafficking processes (Santolini et al. 1999). Numb is an EH-interacting protein that binds to Eps15, and to the related protein Eps15R, through a NPF motif contained in its COOH-terminal region. This observation led to the experimental hypothesis tested in this study that Numb might itself be an endocytic protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Protein Studies
Transfections were carried out by standard calcium phosphate protocols. Polyclonal anti-Numb antibodies were elicited against GST-Numb fusion proteins or against a peptide (encompassing aa 298–603 and 537–551 of human Numb, respectively), and affinity purified onto the same antigen used for the immunization. Other antibodies used were the anti-HA mAb 12CA5, the mAb anti–
adaptin (A4200; Sigma-Aldrich), mAbs anti– adaptin (A4325 for Western blotting from Sigma-Aldrich; A43920 for immunoprecipitation, AP6 for immunofluorescence and electron microscopy from Transduction Laboratories), anti-transferrin receptor (TfR) (K-20; Santa Cruz Biotechnology, Inc.), anti-EGFR (AB-5; Calbiochem), or anti-Eps15 (Fazioli et al. 1993). Immunoprecipitation and immunoblotting were performed as described previously (Fazioli et al. 1993). Purification of the GST fusion proteins onto agarose–glutathione and in vitro binding experiments were performed as described previously (Fazioli et al. 1993; Salcini et al. 1997).
For the competition experiment (see Fig. 4 D), agarose-immobilized GST– ear (2 µg) was incubated with 15,000 cpm of in vitro–translated Eps15 COOH-terminal region (aa 581–874) in the presence of either the purified COOH-terminal domains of Numb (aa 298–603) or Eps15 (aa 581–874), or purified BSA (Boehringer), at the indicated molar concentrations with respect to the ear. The purified COOH-terminal domains of Numb and Eps15, used as competitors, were produced as GST fusion proteins, cleaved from the GST moiety by thrombin digestion, and purified by fast performance liquid chromatography according to standard protocols. After 2 h incubation at 4°C, samples were washed and analyzed by SDS-PAGE, followed by autoradiography. The radioactive bands were excised and counted in a β counter.
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For immunoelectron microscopy, 10% gelatin–embedded, 2.3 M sucrose–infused blocks of aldehyde-fixed A172 cells were frozen in liquid nitrogen. Ultrathin cryosections were obtained with a Reichert-Jung Ultracut E with FC4E cryoattachment and collected on copper-formvar-carbon–coated grids. Single and double immunogold localization on ultrathin cryosections were performed as described previously (Schiaffino et al. 1999). In particular, for double labeling, sections immunostained with the anti-Numb antibody, followed by 10 nm protein A–gold, were incubated with 1% glutaraldehyde in 0.1 M sodium phosphate buffer, to quench free protein A. Sections were then incubated alternatively with antibodies against AP2 (AP6, mouse), AP1 (A4200 Sigma-Aldrich), EGFR (AB-5, mouse), or TfR (K20, goat). After washing, an appropriate rabbit anti-mouse or rabbit anti–goat bridging antibody was used (Dako), followed by 15 nm protein A–gold. Control sections were incubated with an unrelated antibody or without first antibodies. To determine quenching efficiency, sections incubated with anti-Numb antibodies were quenched with 1% glutaraldehyde in 0.1 M sodium phosphate buffer, and challenged with protein A. In all control sections, no labeling was detected (data not shown). Sections were examined with a ZEISS EM 902 electron microscope.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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70 kD (Fig. 1A and Fig. B). Phosphatase treatment of anti-Numb immunoprecipitates yielded a single band, comigrating with the lower band of the doublet (data not shown), suggesting that the upper band is the result of phosphorylation events.
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adaptin (data not shown). Colocalization with adaptin in the perinuclear area was also detected (not show).
To examine in detail the intracellular distribution of Numb, we performed immunogold labeling on ultrathin cryosections and found Numb associated to endosomes, clathrin-coated pits, and vesicles (Fig. 1 D and Table ). Immunoreactivity for Numb was also evidenced in locations compatible with the Golgi area and the TGN (Fig. 1 D). Double labelings with anti-Numb and anti– adaptin (Fig. 1 D) also supported the idea of presence of Numb in the TGN. A morphometric analysis revealed good colocalization of Numb with both EGF and Tf receptors in coated pits and with AP2 in coated pits and vesicles (Table ). Surprisingly, when double labeling for Numb and Eps15 was performed, we could not detect the presence of the two proteins in the same coated pit, despite their individual presence in different pits (Table ). In endosomes, however, Numb and Eps15 were found together on the same organelle (Fig. 1 E and Table ).
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Numb Binds Specifically and Directly to the Appendage of Adaptin
Numb could be recruited to endocytic organelles through interaction with Eps15. However, the lack of colocalization of Numb and Eps15 in the same coated pit posed the question as to whether Numb interacts with other component(s) of the endocytic machinery.
A GST fusion protein containing aa 63–603 of Numb could efficiently bind to adaptin from cellular lysates, but not to the subunit of the AP1 complex (Fig. 3 A). The binding was mediated through the appendage of adaptin ( ear) (Fig. 3 B), and was direct, since a GST– ear was able to bind to the COOH-terminal domain of Numb, recombinantly produced and purified (Fig. 3 C). Finally, endogenous Numb and adaptin could be readily coimmunoprecipitated from cell lysates (Fig. 3 D).
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adaptin, we tested fragments of Numb engineered as GST fusion proteins (Fig. 4 A). A region between position 564 and 588 contained the determinants necessary for the interaction (Fig. 4A and Fig. B). This region contained a DPF motif (aa positions 566–568), which has been recently shown to constitute a ligand for the ear and is present in several proteins known to bind to the appendage domain of adaptin (Owen et al. 1999). We engineered an HA-tagged mutant of Numb, in which the sequence encoding for DPF was mutagenized to one encoding DLA. This mutant could not coimmunoprecipitate adaptin (Fig. 4 C), indicating that the DPF motif in position 566–568 of Numb is responsible for its interaction with adaptin. Of interest, the DPF motif identified is conserved in evolution, being also present in dNumb (aa 503–505).
The existence of a single binding site on ear, as determined by the resolution of its structure (Owen et al. 1999), predicts competition between Numb and other ear–associated proteins. We tested this prediction experimentally, by competing the binding of Eps15 to ear with increasing concentrations of Numb (Fig. 4 D). The COOH-terminal region of Numb was indeed able to compete the interaction between the COOH-terminal domain of Eps15 (which contains all the determinants necessary and sufficient for binding to adaptin) and a GST– ear fusion protein. However, it did so with lower efficiency than the COOH-terminal portion of Eps15 itself, suggesting that, in vivo, Eps15 binds to AP2 with higher affinity than with Numb (Fig. 4 D).
Fragments of Numb Act as Dominant Negatives on Endocytosis
To test whether perturbation of Numb function might interfere with the endocytic process, we overexpressed, in CV1 cells, COOH-terminal fragments of Numb, Numb/347–588, and Numb/347–564 and monitored the uptake of fluorochrome-conjugated EGF and Tf. Both COOH-terminal fragments inhibited internalization of EGF and Tf (Fig. 5). Control GFP did not affect internalization (Fig. 5). Of note, the Numb/347–564 mutant did not retain binding sites for either AP2 (DPF) or Eps15 (NPF), thus excluding that in vivo titration of these two known endocytic components was responsible for the effect. These data functionally establish Numb as a component of the endocytic machinery. They further identify an unexpected region of Numb (aa 347–564), which does not bind to either AP2 or Eps15 as an important determinant for Numb function in endocytosis.
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adaptin and Eps15, albeit not simultaneously, at least in coated pits, where Numb and Eps15 do not colocalize. Eps15 binds to adaptin with higher affinity than Numb, suggesting that in pits, Eps15 must be first disengaged from AP2 for Numb to bind, consistent with findings that the Eps15–AP2 is progressively disrupted upon coat assembly (Cupers et al. 1998). Thus, Numb and Eps15 might participate to the assembly of pits in a temporal hierarchy. Conversely, a Numb–Eps15 complex might function at the endosome level. We note that, after its release from coated pits, Eps15 is retargeted to endosomes (Torrisi et al. 1999), playing a yet uncharacterized role that requires, however, its EH domains (Torrisi et al. 1999). Thus, EH-mediated interactions of Eps15 with target molecules are important in endosomes, where Eps15 and Numb colocalize, raising the possibility that Numb might be an effector of Eps15 in this location. Finally, a fragment of Numb, which cannot bind to either AP2 or Eps15, acts as a dominant negative on endocytosis, suggesting interactions with other endocytic proteins. The participation of Numb to endocytosis asks whether the role of Numb in development is linked to this process. Although there is evidence (Seugnet et al. 1997; Parks et al. 2000) that Notch signaling might require its endocytosis, it is conceivable that upon prolonged stimulation, and by analogy to most signaling receptors, endocytosis of Notch would counteract its signaling. Thus, in principle, the antagonistic effect of Numb on Notch could be explained, at least in part, by Numb-dependent Notch internalization with ensuing attenuation of Notch signaling. Although this speculative scenario awaits experimental testing, our results, when viewed in the context of Numb action on development, raise the possibility that cell fate determination could be due, at least in part, to asymmetric partitioning of endocytic machinery at mitosis.
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Acknowledgments |
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This work was supported by grants from Associazione Italiana Ricerca sul Cancro, Telethon Italy (grant D.90), Istituto Superiore della Sanita (AIDS 1998), and Consiglio Nazionale delle Richerche (target project Biotechnology) to P.P. Di Fiore; and Telethon-Italy (grant E0942), Consiglio Nazionale delle Richerche (target project Biotechnology), and MURST to C. Tacchetti.
Submitted: 7 August 2000
Revised: 11 October 2000
Accepted: 16 October 2000
Abbreviations used in this paper: aa, amino acid(s); dNumb, Drosophila Numb; DPF, Asp-Pro-Phe; DLA, Asp-Leu-Ala; EH, Eps15 homology; GST, glutathione S-transferase; HA, hemagglutinin; NPF, Asn-Pro-Phe; mNumb, mammalian Numb; Tf, transferrin; TfR; Tf receptor.
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, no competition; 
, competition with Numb (aa 298-603); •, competition with Eps15 (aa 581-874); 
, control competition with BSA. For details see Materials and Methods.
