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First published online 29 January 2003
doi: 10.1242/jcs.00272

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Protein products of human Gas2-related genes on chromosomes 17 and 22 (hGAR17 and hGAR22) associate with both microfilaments and microtubules

¹ Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
² Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
³ Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA

View larger version (28K): [in a new window]   Fig. 1. hGAR17 and hGAR22 isoforms and constructs. (A) hGAR17 mRNA and protein structure. The two mRNA splice forms differ from each other by the presence or absence of exon 2B and encode proteins with deduced molecular masses of 23.5 kDa (isoform ) and 96.5 kDa (isoform ß). Exon length in bp is indicated below each exon. hGAR17ß contains a calponin homology (CH) domain and a Gas2-related (GAR) domain. The amino acids comprising each domain are indicated. hGAR17 contains only a CH domain. The last 27 amino acids of hGAR17 (hatched box) are different from the corresponding sequence of hGAR17ß owing to a frameshift introduced into the splice form by alternative splicing. The C-terminal sequence of hGAR17ß (C-tail 17) does not contain any known domains and is highly degenerate and unstructured. (B) hGAR17 constructs used in this study. All constructs contain a FLAG epitope tag at their N-terminus. The first and last amino acids of the hGAR17 sequence of each construct are indicated. (C) hGAR22 mRNA and protein structure. The two mRNA splice forms (see also Zucman-Rossi et al., 1996) differ from each other by the presence or absence of exon 5B and encode proteins with deduced molecular masses of 36.3 kDa (isoform ) and 72.6 kDa (isoform ß). Both isoforms contain a calponin homology (CH) domain and a Gas2-related (GAR) domain. The first 337 amino acids of hGAR22ß are identical to hGAR22. The additional C-terminal sequence of hGAR22ß (C-tail 22) does not contain any known domains and appears to be highly degenerate and unstructured. (D) hGAR22 constructs used in this study.

View larger version (108K): [in a new window]   Fig. 2. Localization of the isoforms of hGAR17 and hGAR22 in transfected cells. NIH 3T3 (A-F) or COS7 (G-L) cells were transiently transfected with pFLAG-hGAR17 A-C; G-I) or pFLAG-hGAR22 (D-F; J-L) and stained with a monoclonal anti-FLAG antibody (A,D,G,J), phalloidin-AlexaFluor-594 (B,E) and a polyclonal anti-tubulin antibody (H,K). (C,F,I,L) Superimposed images, with the FLAG staining in green and the phalloidin or tubulin staining in red. Both proteins colocalized with MFs (C,F) but not with MTs (I,L). Similar results were obtained with untagged proteins. Bar, 15 µm.

View larger version (98K): [in a new window]   Fig. 3. Localization of the ß isoforms of hGAR17 and hGAR22 in transfected cells. NIH 3T3 (A-F) or COS7 (G-L) cells were transiently transfected with pFLAG-hGAR17ß (A-C; G-I) or pFLAG-hGAR22ß (D-F; J-L) and stained with a monoclonal anti-FLAG antibody (A,D,G,J), phalloidin-AlexaFluor-594 (B,E) and a polyclonal anti-tubulin antibody (H,K). (C,F,I,L) Superimposed images, with the FLAG staining in green and the phalloidin or tubulin staining in red. Both ß proteins exhibited colocalization with MFs (C,F) and MTs (I,L; see insets). Bar, 15 µm.

View larger version (138K): [in a new window]   Fig. 4. Localization of the CH and GAR domains in transfected cells. NIH 3T3 (A-C) or COS7 (D-I) cells were transiently transfected with pFLAG-hCHD22 (A-C), pFLAG-hGARD17 (D-F) or pFLAG-hGARD22 (G-I) and stained with a monoclonal anti-FLAG antibody (A,D,G), phalloidin-AlexaFluor-594 (B) and a polyclonal anti-tubulin antibody (E,H). (C,F,I) Superimposed images, with the FLAG staining in green and the phalloidin or tubulin staining in red. hCHD22 protein colocalized with MFs. Both hGARD17 and hGARD22 showed colocalization with MTs (C) (F,I; see insets). Bar, 15 µm.

View larger version (104K): [in a new window]   Fig. 5. Localization of C-terminal fragments of hGAR17 and hGAR22 in transfected cells. COS7 cells were transiently transfected with pFLAG-hCt17 (A-C), pFLAG-hCt22 (D-F), pFLAG-hGARt17 (G-I) or pFLAG-hGARt22 (J-L) and stained with a monoclonal anti-FLAG antibody (A,D,G,J) and a polyclonal anti-tubulin antibody (B,E,H,K). Superimposed images, with the FLAG staining in green and the tubulin staining in red, are shown in panels C, F, I and L. Both Ct proteins accumulated in the nucleus (A,D), whereas the cytoplasmic protein showed some colocalization with MTs (C,F; see insets). The GARt fragments colocalized with and appeared to bundle MTs (I,L). Bar, 15 µm.

View larger version (51K): [in a new window]   Fig. 6. In vitro binding assays of hGAR17 and hGAR22 proteins. hGAR17 (A) or hGAR22 (B) proteins labeled with 35[S]-methionine were incubated with preassembled MFs (+MF) or MTs (+MT), the reaction mixtures centrifuged at high speed and the resulting supernatant (S) and pellet (P) fractions run on an SDS-PAGE gel. The gels were dried and the radioactively labeled proteins visualized by autoradiography. The presence of a protein in a pellet fraction indicates binding to the corresponding filaments. Reactions without filaments (–MF, –MT) were run to control for non-specific protein aggregation. BSA was used as a non-binding negative control; in this case the protein was visualized by Coomassie staining. The results of the assays are indicated to the right of the gels (+, binding; –, absence of binding). Since hCt22 sedimented in the absence of MFs, its sedimentation with MFs (+/–) is most probably due to non-specific aggregation rather than specific filament binding.

View larger version (56K): [in a new window]   Fig. 7. Expression of GAR17 and GAR22 mRNAs in mammalian tissues. (A) Northern blot analysis of GAR17 expression in human tissues. A human multiple-tissue northern blot was hybridized with a hGAR17-specific probe. As mRNAs of hGAR17 and hGAR17ß differ from each other by only 47 bp, they cannot be distinguished on a northern blot. (B) Northern blot analysis of GAR22 expression in human tissues. A human multiple-tissue northern blot was hybridized with a hGAR22-specific probe. hGAR22 and ß transcripts are indicated with arrows. (C) Northern blot analysis of GAR22 expression in mouse tissues. A mouse multiple-tissue northern blot was hybridized with a mGAR22-specific probe. mGAR22 and ß transcripts are indicated with arrows. The sizes of molecular mass markers are in kb.

View larger version (36K): [in a new window]   Fig. 8. Expression of GAR22 proteins in mammalian tissues and cells. (A) Western blotting of mouse tissues. Proteins extracts from various mouse tissues were resolved on an SDS-PAGE gel, transferred onto Immobilon membrane, and probed with polyclonal anti-GAR22 C15 antibody. mGAR22ß was detected in testis and, at a lower level, in brain (indicated with arrowhead). No mGAR22 protein could be detected in any of the tissues. The sizes of molecular mass markers are in kDa. (B) GAR22 immunoprecipitation from growing or arrested cultured cells. GAR22 protein was precipitated with C15 antibody from either proliferating (P) or growth-arrested (A) cells, run on an SDS-PAGE gel, transferred onto Immobilon membrane and probed with C15 antibody. Expression of GAR22ß was induced by growth arrest in all four cell lines. No GAR22a could be precipitated from any of the cell lines. CAD cells were arrested by serum starvation for 48 hours. COS7, NIH 3T3 and GC4 cells were arrested by contact inhibition for 48 hours. (C) Time course of GAR22ß induction in growth-arrested CAD cells. GAR22 protein was immunoprecipitated with the C15 antibody from CAD cells serum starved for indicated periods of time. The precipitated protein was visualized by western blotting with C15 antibody. (D) Phosphatase treatment of endogenous GAR22ß. GAR22 protein was immunoprecipitated with C15 antibody from serum-starved (48 hours) CAD or COS7 cells and treated with either the serine/threonine phosphatase PP1 or the tyrosine phosphatase TCP. The reactions were run on an SDS-PAGE gel, transferred onto Immobilon membrane and probed with C15 antibody. A mobility shift was observed only in the samples treated with PP1.

View larger version (96K): [in a new window]   Fig. 9. GAR22 immunohistochemistry of mouse testes. Testicular sections of (A) 3-week- or (B) 5-month old mice were stained with the C15 antibody and TRITC-labeled goat-anti-rabbit secondary antibody. The staining is localized predominantly in the cytoplasm of the germ cells and in the heads of the spermatozoa (indicated with arrows). The staining is likely to represent mGAR22ß since it is the only protein recognized by C15 antibody on western blots of mouse testes. Bar, 20 µm.

View larger version (128K): [in a new window]   Fig. 10. hGAR22ß bridges MFs and MTs in transfected cells. COS7 cells were transiently transfected with pFLAG-hGAR22ß and triple stained with a monoclonal anti-FLAG antibody (A), a polyclonal anti-tubulin antibody (B) and phalloidin-AlexaFluor-350 (C). (D) A superimposed image, with the FLAG staining in blue, the tubulin staining in red and the actin staining in green. Triple-stained filaments, probably resulting from crosslinking by hGAR22ß, are white colored. Bar, 10 µm.

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