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. 2000 Mar 15;19(6):1263-71.
doi: 10.1093/emboj/19.6.1263.

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain

X K Tong  1 , N K Hussain, E de Heuvel, A Kurakin, E Abi-Jaoude, C C Quinn, M F Olson, R Marais, D Baranes, B K Kay, P S McPherson
Affiliations

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain

X K Tong et al. EMBO J. .

Abstract

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.

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Figures

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Fig. 1. Identification of a 170 kDa protein (p170) that binds specifically to the SH3A domain of intersectin. Strips of rat brain cytosolic fraction (100 μg/lane) were separated by SDS–PAGE, transferred to nitrocellulose, and overlaid with GST alone or GST fused to various SH3 domains of the proteins indicated on the blots (amphi., amphiphysin; endo., endophilin; SH3A, SH3A domain of intersectin). The positions of dynamin, synaptojanin and p170, which are recognized by various SH3 domains, are indicated by arrows on the right. The identities of the additional bands in the overlays are unknown.
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Fig. 2. Tissue and subcellular distribution of p170. (A) Proteins of cytosolic fractions from various adult rat tissues (100 μg/tissue) were separated by SDS–PAGE, transferred to nitrocellulose and overlaid with a GST fusion protein encoding the SH3A domain of intersectin. (B) Proteins of brain subcellular fractions (100 μg/fraction) were separated by SDS–PAGE, transferred to nitrocellulose and overlaid with a GST fusion protein encoding the SH3A domain of intersectin. Subcellular fractions were prepared as described (McPherson et al., 1994a). H, homogenate; P, pellet; S, supernatant. For both figures, the migratory positions of dynamin, synaptojanin and p170 are indicated by arrows on the right.
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Fig. 3. Identification of consensus-binding sites for SH3A in mSos1. (A) The sequences of 12 peptides, affinity selected from a phage-displayed X6PXXPX6 peptide library (where X is any amino acid) using the SH3A domain from intersectin, are listed. The peptides define the SH3A-binding consensus sequence Pp(V/I)PPR, where p is typically proline. (B) Two putative ligand sites for the SH3A domain, PPVPPR, occur within human Sos1 at sequences between amino acids 1148 and 1161 and 1287 and 1300, and a third related site is found between amino acids 1208 and 1221, as indicated. (C) The segments of human Sos1 shown in (B) were fused to the N–terminus of secreted alkaline phosphatase and tested for binding to GST fusion protein encoding the individual SH3 domains of intersectin or to GST alone.
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Fig. 4. Conformation of p170 as mSos1. Aliquots of antiserum C23 against mSos1, as well as normal rabbit sera (NRS), were precoupled to protein A–Sepharose beads. Precoupled beads were washed, incubated overnight at 4°C with an E18 rat brain cytosolic fraction and extensively washed the next day. The material specifically bound to the beads (top two blots labeled beads) was eluted and processed, along with an aliquot of the cytosolic extract (starting material, SM), for Western blot analysis with the anti-mSos1 antibody (top blot), or for SH3A domain overlay (middle blot). The proteins that did not bind to the beads (void) were also subjected to an SH3A domain overlay assay (bottom blot).
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Fig. 5. Interaction of intersectin and mSos1 in situ. (A) mSos1 is found in puncta that are expressed throughout the cell body and neurites of hippocampal neurons maintained in culture for 1 day. The puncta are relatively homogeneous along the neurite but are enriched at growth cones (arrows). (B) A higher magnification image of the growth cone in (A) reveals that the density of mSos1 positive puncta is higher at the tip of the growth cone than along the neurite. (C) Color coding of fluorescent intensities of the area in (B) indicates that the intensity of individual mSos1 positive puncta is higher in the growth cone (red) than in other regions of the dendrite. Scale bar: (A), 10 μM; (B and C), 1.5 μM. (D) Aliquots of antisera 2173 and 2174 against intersectin, as well as pre-immune 2173 sera (NRS), precoupled to protein A–Sepharose beads, were incubated overnight at 4°C with an E18 rat brain cytosolic fraction. The beads were extensively washed and the material specifically bound to the beads was eluted and processed for Western blot analysis with polyclonal antibodies against mSos1 and intersectin. (E) As for (D) except that immuno– precipitations were performed from Triton X–100 solubilized membrane fractions. The antigens and their approximate molecular weights (kDa) are denoted with arrows on the right and left sides of the figure, respectively.
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Fig. 5. Interaction of intersectin and mSos1 in situ. (A) mSos1 is found in puncta that are expressed throughout the cell body and neurites of hippocampal neurons maintained in culture for 1 day. The puncta are relatively homogeneous along the neurite but are enriched at growth cones (arrows). (B) A higher magnification image of the growth cone in (A) reveals that the density of mSos1 positive puncta is higher at the tip of the growth cone than along the neurite. (C) Color coding of fluorescent intensities of the area in (B) indicates that the intensity of individual mSos1 positive puncta is higher in the growth cone (red) than in other regions of the dendrite. Scale bar: (A), 10 μM; (B and C), 1.5 μM. (D) Aliquots of antisera 2173 and 2174 against intersectin, as well as pre-immune 2173 sera (NRS), precoupled to protein A–Sepharose beads, were incubated overnight at 4°C with an E18 rat brain cytosolic fraction. The beads were extensively washed and the material specifically bound to the beads was eluted and processed for Western blot analysis with polyclonal antibodies against mSos1 and intersectin. (E) As for (D) except that immuno– precipitations were performed from Triton X–100 solubilized membrane fractions. The antigens and their approximate molecular weights (kDa) are denoted with arrows on the right and left sides of the figure, respectively.
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Fig. 6. Co-immunoprecipitation of intersectin and Grb2 with mSos1. Aliquots of antiserum C23 against mSos1, as well as normal rabbit sera (NRS), were precoupled to protein A–Sepharose beads. Precoupled beads were washed, incubated overnight at 4°C with an E18 rat brain cytosolic fraction, and extensively washed the next day. The material specifically bound to the beads was eluted and processed for Western blot analysis, along with an aliquot of the cytosolic extract (starting material, SM), with polyclonal antibodies against mSos1 and intersectin, and a monoclonal antibody against Grb2. The antigens and their approximate molecular weights (kDa) are denoted with arrows on the right and left sides of the figure, respectively.
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Fig. 7. In vitro competition binding assays of intersectin SH3A domain and Grb2 to mSos1. E18 rat brain cytosolic fractions were separated on SDS–PAGE, transferred to nitrocellulose membranes, and strips of the membrane were processed for overlay assays with GST–SH3A at 200 ng/ml. The overlay assays also contained His6-tagged Grb2 at increasing molar ratios of Grb2 to SH3A ranging from 0:1 (control) to 100:1 as indicated at the bottom of the figure. An example of the overlay results is shown at the top of the graph. The intensity of the stained mSos1 band was determined by densitometry of the autoradiographs and was normalized to control. The bars represent the mean ± SEM from three separate experiments.
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Fig. 8. Co-immunoprecipitation of mSos1 with the SH3 domains of intersectin. Undifferentiated PC12 cells were infected with a recombinant adenovirus encoding the five tandom SH3 domains of intersectin fused to GFP (GFP–SH3A–E). Following protein expression, a soluble cell lysate was prepared and incubated overnight at 4°C with antisera against GFP or normal rabbit sera (NRS) precoupled to protein A–Sepharose beads. The bead samples were extensively washed the next day, and the material specifically bound to the beads was eluted and processed for Western blot analysis, along with an aliquot of the cell extract (starting material, SM), with polyclonal antibodies against mSos1 and GFP, and a monoclonal antibody against Grb2. The antigens and their approximate molecular weights (kDa) are denoted with arrows on the right and left.
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Fig. 9. Sucrose-density gradient analysis of mSos1 interactions. Proteins from a cytosolic extract of rat brain were separated on 5–20% linear sucrose-density gradients by centrifugation at 195 000 g for 2.5 h and the gradients were then collected into 19 fractions of 2 ml each from the bottom. Top panels: aliquots (100 μl) of odd gradient fractions (s.g. fraction) were analyzed by Western blotting using a variety of antibodies (the antigens and their approximate molecular masses in kDa are denoted with arrows on the right and left sides of the figure, respectively). Bottom panels: aliquots (750 μl) of the indicated gradient fractions were incubated with protein A–Sepharose beads precoupled to intersectin antibody (2173). Following an overnight incubation at 4°C, the samples were washed and the proteins specifically bound to the beads were eluted with gel sample buffer and processed for SDS–PAGE and Western blot analysis with intersectin and mSos1 antibodies. The antigens and their approximate molecular masses (kDa) are denoted with arrows on the right and left sides of the figure, respectively.
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Fig. 10. Inhibition of Ras activation by the SH3 domains of intersectin. (Top panel) HEK-293 cells were serum starved overnight and were then challenged for 2 min with serum-free media (–EGF) or with serum-free media containing 100 ng/ml EGF (+EGF). The cells were then washed, lysates prepared and incubated with a GST fusion protein encoding the Ras–GTP binding domain of Raf1 coupled to glutathione–Sepharose beads. Following incubation, the beads were washed and material bound to the beads (bead) was processed for SDS–PAGE, along with an aliquot of the cell lysate (starting material, SM) (equal to one-tenth the amount added to the beads) and an equal aliquot of the unbound material (void). The samples were processed for Western blotting with a monoclonal antibody against Ras as indicated on the figure. (Bottom panel) HEK-293 cells infected with recombinant adenovirus encoding GFP alone (GFP) or the five tandem SH3 domains of intersectin coupled to GFP (GFP–SH3A–E) were processed for Ras assays following a 2 min exposure to EGF.
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