PMC

 Human NUMB and NUMB-R cDNAs and proteins. (A) The structures of the human NUMB (hNUMB) and NUMB-R (hNUMB-R) cDNAs are depicted. The ORFs are labeled. Positions are indicated in kb. The nucleotide positions of initiator and terminator codons are also indicated. Canonical polyadenylation sites (AATAAA) are at positions 2649, 2823, 2958, and 3025, for hNUMB and hNUMB-R, respectively (not shown). (B) Predicted protein sequences and alignment of human NUMB and NUMB-R. In the hNUMB-R sequence, only nonidentical amino acids are reported, except for the NPF motifs. Dashes indicate gaps introduced to maximize the alignment. Accepted conservations, employed to calculate relatedness, are D, E, N, Q; L, I, V, M; K, R, H; F, Y, W; and A, G, P, S, T. The PTB domains and the NPF motifs are indicated in reverse print.

 EH-mediated binding to peptides and proteins in vitro and in vivo. (A) Schematic of the Eps15 and Eps15R proteins with their EH domains. Amino acids positions are indicated. (B) Predicted amino acid sequence of peptides selected by screening of a random phage-displayed peptide library with GST–EH and GST–EHR, from Eps15 and Eps15R, respectively. NPFs are in boldface type. (C) NPF-containing motifs present in the cDNAs identified by screening of a human fibroblast expression library using GST–EH as a probe. Underlined peptides were used for the in vitro binding experiments described in Figs. , , and .

 In vitro binding of NPF-containing proteins and peptides to Eps15. (A) In vitro binding of Eps15 to Gst fusions of EH-binding proteins. Total cellular lysates from NIH–3T3 cells (1 mg/lane) were incubated with the indicated GST fusion proteins (10 μg) for 1 hr at 4°C. Specifically bound Eps15 was detected by immunoblot with an anti-Eps15 antibody. (B) Binding of Eps15 to GST fusions encompassing NPF-containing peptides. GST fusion proteins were engineered to encompass the NPF-containing peptides underlined in Fig. C and are indicated with the names of the proteins from which the peptides were derived. In vitro binding experiments were performed as described in A. Lanes marked LYS in A and B were loaded with 50 μg of total cellular proteins to serve as a reference for the position of Eps15. The position of Eps15 is also indicated.

 Human RAB and RAB-R cDNAs and proteins. (A) The structures of the human RAB (hRAB) and RAB-R (hRAB-R) cDNAs are depicted. The ORFs are labeled. Positions are indicated in kb. The nucleotide positions of initatior and terminator codons are also indicated. Canonical polyadenylation sites (AATAAA) are at positions 2499, 2542, and 2556 of the RAB sequence; no polyadenylation site was found in the isolated hRAB-R cDNA (not shown). (B) Predicted protein sequences and alignment of human RAB and RAB-R. The sequence of hRAB is identical to that reported by and . In the hRAB-R sequence, only nonidentical amino acids are reported, except for the FG and NPF motifs. Dashes indicate gaps introduced to maximize the alignment. Accepted conservation, employed to calculate relatedness, are D, E, N, Q; L, I, V, M; K, R, H; F, Y, W; and A, G, P, S, T. The FG, zinc-finger, and NPF motifs are indicated in reverse print.

 Requirement for the NPF motif and surrounding positions for binding to Eps15. (A) Binding to Eps15 of mutant peptides containing mutations in the NPF sequence. Peptides engineered in GST fusion proteins are indicated on the left. GST–NPF corresponds to the sequence of a NPF-containing peptide derived from the sequence of RAB (underlined in Fig. C). Mutant peptides (GST–NGF, GST–DPF, GST–NPY) are also indicated. The in vitro bindings to Eps15, obtained as described in Fig. , are shown on the right. The lane marked RAB represents an in vitro binding obtained with the GST–RAB protein (Fig. A) to serve as a positive control. (B) Alanine scanning. Positions surrounding the NPF motif in the RAB peptide were alanine scanned as indicated on the left. The in vitro binding to Eps15, obtained as described in Fig. , are shown on the right. Lanes marked LYS were loaded with 50 μg of total cellular proteins to serve as a reference for the position of Eps15 (also indicated). Amino acids corresponding to mutagenized codons are shown in boldface type.

 In vivo binding of NPF-containing proteins to Eps15. (A) Coimmunoprecipitation of Eps15 with endogenously expressed NUMB and RAB. Total cellular proteins from NIH–3T3 cells were obtained under mild lysis conditions to preserve protein–protein interactions, and 5 mg of protein was immunoprecipitated with an anti-RAB (Ip RAB), an anti-NUMB (Ip NUMB), or a control serum (C). After SDS–PAGE, coimmunoprecipitating Eps15 was detected by immunoblot with an anti-Eps15 antibody. (B) Eps15 coimmunoprecipitation with HA-tagged RAB and NUMB proteins. (Top) C33A cells were transfected with expression vectors engineered to express HA-tagged RAB or NUMB proteins (HA–RAB or HA–NUMB lanes, respectively) or mock transfected (− lanes). Total cellular lysates (100 μg) were immunoblotted with an anti-HA antibody. (Bottom) Five milligrams of total cellular proteins from HA–RAB or HA–NUMB transfectants (HA–RAB Tfx and HA–NUMB Tfx, respectively) obtained as in A were immunoprecipitated with the anti-HA antibody (HA lanes) or with a control serum (C lanes) and detected by immunoblot with an anti-eps15 antibody. (C) Eps15 coimmunoprecipitation with HA-tagged NUMB and NUMB mutant. C33A cells were transfected with expression vectors encoding HA-tagged NUMB or an HA-tagged NUMB mutant in which the NPF motif was mutagenized to NAA (lanes HA–NUMB and HA–NUMB/NAA, respectively), or mock transfected (− lane). (Top) Total cellular lysates (100 μg) were immunoblotted with an anti-HA antibody; (middle) 5 mg of total cellular proteins obtained as in A was immunoprecipitated with an anti-HA antibody and detected by immunoblot with an anti-Eps15 serum; (bottom) total cellular lysates (100 μg) were immunoblotted with an anti-Eps15 antibody. Lanes marked LYS in A–C were loaded with 50 μg of total cellular proteins to serve as a reference for the position of Eps15. The positions of Eps15, NUMB, and RAB are also indicated.

 Mapping of the minimal region of eps15 required for binding to NPF-containing proteins. (A) Secondary structure prediction of the amino-terminal region of Eps15 (containing the three EH domains) by a Chou–Fasman–Rose algorithm. (Vertical lines) α-Helices; (thick shaded bars) β-sheets; (thinner solid bars) coils; (small solid boxes) turn. Amino acid positions are also indicated. (B) Schematic representation of the Eps15 amino-terminal domain and of the GST fusion proteins engineered, with predicted turns indicated by solid boxes. The indicated fragments of Eps15 were engineered into GST fusion proteins and used for in vitro binding experiments. The EH construct contains all three EH domains. The M2 construct contains the region encompassing the second EH domain flanked by the natural regions pedicting the turns shown in A. Amino acid positions are also indicated in parentheses. (C) In vitro bindings. The GST fusions shown in B were used to bind the ³⁵S-labeled RAB protein, obtained by in vitro transcription/translation of the RAB cDNA. Detection was by autoradiography. The lane marked T/T was loaded with the primary product of the in vitro transcription/translation to serve as a reference. The positions of RAB are also indicated.

 Identification of a family of EH-containing proteins in mammals. (A) ³⁵S-Labeled lysates from NIH–3T3 (50 × 10⁶ TCA-precipitable counts) were incubated with the indicated GST fusion proteins displaying NPF-containing peptides (NPF–RAB, NPF–RAB-R, and NPF–NUMB, underlined in Fig. C), mutant peptides (NGR–RAB, DPF–RAB, and NPY–RAB, shown in Fig. A), or with GST. The first two lanes represent the same lysate immunoprecipitated with anti-eps15 antibody (EPS15) or with a control serum (PRE). Specifically bound proteins were fractionated by SDS–PAGE and visualized by autoradiography. The position of Eps15 is indicated. Molecular mass markers are indicated in kD. (B) Identification of four novel EH-containing mammalian sequences. The EST db was screened with the TBLASTN algorithm, using the three EH domains of Eps15 as a query. Only sequences displaying a P > 10⁻⁵ were considered for further analysis, and they are indicated with their database accession numbers. The alignment of these sequences to the second EH domain of Eps15 is shown, as obtained by a CLUSTAL4 algorithm modified by visual inspection. The W40735–W7520 sequence derives from a merging of two shorter EST sequences of the same gene.