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First published online 24 October 2006
doi: 10.1242/jcs.03249

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GRIF1 binds Hrs and is a new regulator of endosomal trafficking

Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322, USA

View larger version (82K): [in a new window]   Fig. 1. The identified Hrs interactor, GRIF1, is a member of a new family of coiled-coil domain proteins. (A) Domain structure of GRIF1 (top) and the Hrs-interacting clone (clone 17) isolated from the yeast two-hybrid screen (bottom). (B) Domain structure of GRIF1 and its homologues. Accession numbers for the sequences are as follows: Rn GRIF1, NP_598244; Hs GRIF1, NP_055864; Gg GRIF1, XP_421937; Rn Trak1, XP_236715; and Rn HAP1, P54256. The amino acid identity and similarity of each protein relative to the protein sequence of rat GRIF1 are indicated. Each protein contains two or three predicted coiled-coil domains shown as white boxes. Rn GRIF1: 132-188, 202-354; Hs GRIF1: 132-177, 198-354; Gg GRIF1: 113-159, 187-334; Rn Trak1: 130-173, 206-354; Rn HAP1: 169-205, 262-305, 328-370. The bracket indicates the location of the HAPN domain. (C) Alignment of the HAPN domain in GRIF1 and related proteins. Numbers indicate the position of the HAPN domain in each sequence. Rn, Rattus norvegicus; Hs, Homo sapiens; Gg, Gallus gallus.

View larger version (34K): [in a new window]   Fig. 2. Preferential expression of GRIF1 mRNA and protein in brain and heart. (A) Northern blot analysis of GRIF1 mRNA expression. A rat multi-tissue northern blot was hybridized with a 32P-labeled partial cDNA probe of GRIF1 (top). Equal loading was confirmed by using a ß-actin cDNA probe (bottom). Sk., Skeletal. (B) Specificity of anti-GRIF1 antibodies, GF1 and GF2. Western blot analysis of cell lysates from untransfected PC12 and HeLa cells, and pCHA-GRIF1-transfected HeLa cells using anti-GRIF1 antibodies GF1, GF2, or the corresponding pre-immune serum. The asterisks indicate non-specific immunoreactive bands, and the open arrowheads indicate bands that appear specific to the GF2 antibody. (C) Equal amounts of homogenates (100 µg) from the indicated rat tissues were analyzed by immunoblotting using anti-GRIF1 (GF1) and anti-ß-actin antibodies. (D) NGF-differentiated PC12 cells were immunostained using purified anti-GRIF1 antibodies GF1 or GF2 (green); Hoechst-stained nuclei (blue). Insets: enlarged views of boxed region showing punctate staining. Bars, 10 µm.

View larger version (32K): [in a new window]   Fig. 3. GRIF1 and Hrs associate in vivo. (A) Co-immunoprecipitation of GRIF1 with Hrs in transfected HeLa cells. HeLa cells were co-transfected with pEGFP-GRIF1 in combination with pCHA-Hrs or pCHA-vector. Lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-GFP and anti-HA antibodies. (B) Co-immunoprecipitation of endogenous Hrs with GRIF1 in PC12 cells. PC12 cell lysates were immunoprecipitated with anti-GRIF1 antibody or pre-immune serum, followed by immunoblotting for Hrs and GRIF1.

View larger version (27K): [in a new window]   Fig. 4. Identification of Hrs and GRIF1 interaction domains by deletion analysis. (A) Domain structure of GRIF1 and its deletion mutants encoded by HA-tagged cDNA constructs. (B) Interaction of full-length and truncated GRIF1 proteins with Hrs. HeLa cells were co-transfected with the indicated HA- and GFP-tagged constructs. Lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-GFP and anti-HA antibodies. The asterisk indicates a non-specific band that probably represents undissociated IgG. (C) Schematic illustrating Hrs and its deletion mutants encoded by HA-tagged cDNA constructs. The following domains are indicated: VHS (Vps27p, Hrs and STAM), FYVE (Fab1b, YOTB, Vac1p and EEA1), UIM (ubiquitin-interacting motif), P (proline-rich), CC (coiled-coil), and Q/P (glutamine- and proline-rich region). (D) Interaction of full-length and truncated Hrs proteins with GRIF1. Co-immunoprecipitation experiments were performed as in B.

View larger version (47K): [in a new window]   Fig. 5. Colocalization of GRIF1 with Hrs on early endosomes. NGF-differentiated PC12 cells were double immunostained using purified anti-GRIF1 antibody GF2 (green; A-D) and anti-Hrs (red; A), anti-EEA1 (red; B), anti-KDEL (red; C), and anti-ß-tubulin (red; D) antibodies. Bars, 10 µm.

View larger version (25K): [in a new window]   Fig. 6. GRIF1 is recruited to enlarged early endosomes in Hrs-overexpressing cells. HeLa cells were transfected with pCHA-GRIF1 alone (A) or co-transfected with pEGFP-Hrs (B). HA-GRIF1 was detected using an anti-HA antibody (red). Endogenous Hrs was detected using an anti-Hrs antibody (green; A), and GFP-Hrs was visualized by the green fluorescence emitted by the GFP tag (green; B). Bars, 10 µm.

View larger version (35K): [in a new window]   Fig. 7. GRIF1 overexpression alters the distribution of early endosomes. HeLa cells were transiently transfected with pEGFP-GRIF1 (A), pEGFP-GRIF11 (B), pEGFP-GRIF12 (C), pEGFP-GRIF13 (D), or pEGFP-GRIF14 (E). GFP-GRIF1 was visualized by the green fluorescence emitted by the GFP tag (green), and endogenous EEA1 was detected using an anti-EEA1 antibody (red). Bars, 10 µm.

View larger version (23K): [in a new window]   Fig. 8. Overexpression of GRIF1 inhibits EGF-induced EGFR degradation. (A) HeLa cells transfected with pCHA-GRIF1 or pCHA vector were incubated in the presence or absence of 100 ng/ml EGF for 1 hour at 37°C. Equal amounts of proteins from cell lysates were analyzed by immunoblotting with antibodies against EGFR (top), HA (middle), or actin (bottom). (B) The degraded EGFR is expressed as a percentage of the EGFR level in the corresponding untreated cells. Data represent mean ± s.e.m. from three independent experiments. The asterisk indicates a statistically significant difference (P<0.05) from the vector-transfected cells.

View larger version (29K): [in a new window]   Fig. 9. Overexpression of GRIF1 does not affect the endocytosis of EGF or transferrin. (A,B) HeLa cells transfected with pEGFP-GRIF1 (green) were treated with TR-transferrin for 30 minutes (red; A) or with TR-EGF for 10 minutes (red; B) at 37°C. Cells were then examined by immunofluorescence microscopy. Arrowheads and asterisks indicate transfected and untransfected cells, respectively. Bars, 10 µm. (C) Schematic representation of the GRIF1 deletion mutants used in the 125I-EGF endocytosis assays. The Hrs-binding domain (residues 359-507) and the kinesin-binding domain (residues 128-283) are indicated by brackets. (D) HeLa cells were transiently transfected with empty vector (CTL) or the following GRIF1 constructs: WT (GRIF1), 1 (GRIF11), 2 (GRIF12), 3 (GRIF13), 4 (GRIF14). Cells were incubated with 125I-EGF for 10 minutes at 37°C. The internalized 125I-EGF is expressed as a percentage of the initially bound 125I-EGF. Values are mean ± s.e.m. from three independent experiments.

View larger version (33K): [in a new window]   Fig. 10. GRIF1 overexpression inhibits trafficking of EGF-EGFR complexes from early endosomes to the lysosomal pathway. (A) HeLa cells transfected with pEGFP-GRIF1 were allowed to internalize TR-EGF for 10 minutes and then chased for 3 hours at 37°C. Cells were stained with a primary antibody against EEA1 and a CY5-conjugated secondary antibody. Arrowheads and asterisks indicate transfected and untransfected cells, respectively. Small arrows indicate EEA1-positive early endosomes that contain EGF. Merged images (bottom) compare the labeling of GRIF1 (green) with EGF (red), GRIF1 (green) with EEA1 (blue), and EGF (red) with EEA1 (blue). Bars, 10 µm. (B) HeLa cells transfected with the indicated GRIF1 constructs or the empty vector were allowed to internalize 125I-EGF for 10 minutes and then chased for 1 hour at 37°C. The degraded 125I-EGF is expressed as a percentage of the initially internalized 125I-EGF. Values are mean ± s.e.m. from three independent experiments. The asterisks indicate a statistically significant difference (P<0.05) from the vector-transfected cells.

View larger version (20K): [in a new window]   Fig. 11. GRIF1 knockdown inhibits EGF-induced EGFR degradation. (A) PC12 cells transfected with the indicated siRNAs or untransfected controls were incubated in the presence or absence of 100 ng/ml EGF for 1 hour at 37°C. Equal amounts of protein from whole cell lysates were analyzed by immunoblotting with antibodies against EGFR (top), GRIF1 (middle), or actin (bottom). (B) The degraded EGFR is expressed as a percentage of the EGFR level of the corresponding untreated cells. (C) PC12 cells transfected with the indicated siRNAs or untransfected controls were allowed to internalize 125I-EGF for 10 minutes and then chased for 1 hour at 37°C. The degraded 125I-EGF is expressed as a percentage of the initially internalized 125I-EGF. Values are mean ± s.e.m. from three (C) or six (B) independent experiments. The asterisks indicate a statistically significant difference (P<0.05) from untransfected control cells.