Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2)
Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates...
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description | Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies. |
doi_str_mv | 10.1074/jbc.M110872200 |
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Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M110872200</identifier><identifier>PMID: 11821401</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Active Transport, Cell Nucleus ; Animals ; Blotting, Northern ; Blotting, Western ; Cell Nucleus - metabolism ; Cytoplasm - metabolism ; DNA, Complementary - metabolism ; FHL2 protein ; Gene Library ; Genetic Vectors ; Homeodomain Proteins - chemistry ; Homeodomain Proteins - metabolism ; Humans ; IGFBP-5 protein ; Insulin-Like Growth Factor Binding Protein 3 - metabolism ; Insulin-Like Growth Factor Binding Protein 4 - metabolism ; Insulin-Like Growth Factor Binding Protein 5 - chemistry ; Insulin-Like Growth Factor Binding Protein 5 - metabolism ; Insulin-Like Growth Factor Binding Protein 6 - metabolism ; insulin-like growth factor-binding protein 5 ; LIM-Homeodomain Proteins ; Mice ; Microscopy, Fluorescence ; Muscle Proteins ; Plasmids - metabolism ; Precipitin Tests ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins - metabolism ; Retroviridae - genetics ; Transcription Factors ; Transfection ; Tumor Cells, Cultured ; Two-Hybrid System Techniques</subject><ispartof>The Journal of biological chemistry, 2002-04, Vol.277 (14), p.12053-12060</ispartof><rights>2002 © 2002 ASBMB. 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Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.</description><subject>Active Transport, Cell Nucleus</subject><subject>Animals</subject><subject>Blotting, Northern</subject><subject>Blotting, Western</subject><subject>Cell Nucleus - metabolism</subject><subject>Cytoplasm - metabolism</subject><subject>DNA, Complementary - metabolism</subject><subject>FHL2 protein</subject><subject>Gene Library</subject><subject>Genetic Vectors</subject><subject>Homeodomain Proteins - chemistry</subject><subject>Homeodomain Proteins - metabolism</subject><subject>Humans</subject><subject>IGFBP-5 protein</subject><subject>Insulin-Like Growth Factor Binding Protein 3 - metabolism</subject><subject>Insulin-Like Growth Factor Binding Protein 4 - metabolism</subject><subject>Insulin-Like Growth Factor Binding Protein 5 - chemistry</subject><subject>Insulin-Like Growth Factor Binding Protein 5 - metabolism</subject><subject>Insulin-Like Growth Factor Binding Protein 6 - metabolism</subject><subject>insulin-like growth factor-binding protein 5</subject><subject>LIM-Homeodomain Proteins</subject><subject>Mice</subject><subject>Microscopy, Fluorescence</subject><subject>Muscle Proteins</subject><subject>Plasmids - metabolism</subject><subject>Precipitin Tests</subject><subject>Protein Binding</subject><subject>Protein Structure, Tertiary</subject><subject>Recombinant Proteins - metabolism</subject><subject>Retroviridae - genetics</subject><subject>Transcription Factors</subject><subject>Transfection</subject><subject>Tumor Cells, Cultured</subject><subject>Two-Hybrid System Techniques</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUFvEzEQhS0EoqHlyhH5gFB72GB717F9hIpNVkpFD63Um7W2J43Lxm7tXSL-Pa4S0RNiLjOH782M3kPoAyVzSkTz5cHY-RWlRArGCHmFZmWsq5rTu9doRgijlWJcnqB3OT-QUo2ib9EJpZLRhtAZGrqQp8GHavA_AS9T3I9b3PZ2jKkyPjgf7vF1iiP4gDk-75btt-uKX-AujJAKlvHeF0WP2zgl3AdXxlU_bPC6u_orZPi8Xa3ZxRl6s-mHDO-P_RTdtt9vLlfV-seyu_y6rmwj-Vg1AhqnRL1w0iy45JRbx51parmRPTgDUkligEllrbLMUiONMgqAW8MEc_Up-nzY-5ji0wR51DufLQxDHyBOWQvKlVpI8l-w2KQaUdcFnB9Am2LOCTb6Mfldn35rSvRzELoEoV-CKIKPx82T2YF7wY_OF-DTAdj6--3eJ9DGR7uFnWZCaNpoygh_PiwPGBS_fnlIOlsPwYIrEjtqF_2_XvgDh7GfTg</recordid><startdate>20020405</startdate><enddate>20020405</enddate><creator>Amaar, Yousef G.</creator><creator>Thompson, Garrett R.</creator><creator>Linkhart, Thomas A.</creator><creator>Chen, Shin-Tai</creator><creator>Baylink, David J.</creator><creator>Mohan, Subburaman</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TM</scope><scope>7X8</scope></search><sort><creationdate>20020405</creationdate><title>Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2)</title><author>Amaar, Yousef G. ; Thompson, Garrett R. ; Linkhart, Thomas A. ; Chen, Shin-Tai ; Baylink, David J. ; Mohan, Subburaman</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c485t-47e4d9736d8b658515cd5db438f8aedbe8980be289cc9c2c1b8b9b9ee5cb272d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Active Transport, Cell Nucleus</topic><topic>Animals</topic><topic>Blotting, Northern</topic><topic>Blotting, Western</topic><topic>Cell Nucleus - metabolism</topic><topic>Cytoplasm - metabolism</topic><topic>DNA, Complementary - metabolism</topic><topic>FHL2 protein</topic><topic>Gene Library</topic><topic>Genetic Vectors</topic><topic>Homeodomain Proteins - chemistry</topic><topic>Homeodomain Proteins - metabolism</topic><topic>Humans</topic><topic>IGFBP-5 protein</topic><topic>Insulin-Like Growth Factor Binding Protein 3 - metabolism</topic><topic>Insulin-Like Growth Factor Binding Protein 4 - metabolism</topic><topic>Insulin-Like Growth Factor Binding Protein 5 - chemistry</topic><topic>Insulin-Like Growth Factor Binding Protein 5 - metabolism</topic><topic>Insulin-Like Growth Factor Binding Protein 6 - metabolism</topic><topic>insulin-like growth factor-binding protein 5</topic><topic>LIM-Homeodomain Proteins</topic><topic>Mice</topic><topic>Microscopy, Fluorescence</topic><topic>Muscle Proteins</topic><topic>Plasmids - metabolism</topic><topic>Precipitin Tests</topic><topic>Protein Binding</topic><topic>Protein Structure, Tertiary</topic><topic>Recombinant Proteins - metabolism</topic><topic>Retroviridae - genetics</topic><topic>Transcription Factors</topic><topic>Transfection</topic><topic>Tumor Cells, Cultured</topic><topic>Two-Hybrid System Techniques</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Amaar, Yousef G.</creatorcontrib><creatorcontrib>Thompson, Garrett R.</creatorcontrib><creatorcontrib>Linkhart, Thomas A.</creatorcontrib><creatorcontrib>Chen, Shin-Tai</creatorcontrib><creatorcontrib>Baylink, David J.</creatorcontrib><creatorcontrib>Mohan, Subburaman</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Amaar, Yousef G.</au><au>Thompson, Garrett R.</au><au>Linkhart, Thomas A.</au><au>Chen, Shin-Tai</au><au>Baylink, David J.</au><au>Mohan, Subburaman</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2)</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2002-04-05</date><risdate>2002</risdate><volume>277</volume><issue>14</issue><spage>12053</spage><epage>12060</epage><pages>12053-12060</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>11821401</pmid><doi>10.1074/jbc.M110872200</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Active Transport, Cell Nucleus Animals Blotting, Northern Blotting, Western Cell Nucleus - metabolism Cytoplasm - metabolism DNA, Complementary - metabolism FHL2 protein Gene Library Genetic Vectors Homeodomain Proteins - chemistry Homeodomain Proteins - metabolism Humans IGFBP-5 protein Insulin-Like Growth Factor Binding Protein 3 - metabolism Insulin-Like Growth Factor Binding Protein 4 - metabolism Insulin-Like Growth Factor Binding Protein 5 - chemistry Insulin-Like Growth Factor Binding Protein 5 - metabolism Insulin-Like Growth Factor Binding Protein 6 - metabolism insulin-like growth factor-binding protein 5 LIM-Homeodomain Proteins Mice Microscopy, Fluorescence Muscle Proteins Plasmids - metabolism Precipitin Tests Protein Binding Protein Structure, Tertiary Recombinant Proteins - metabolism Retroviridae - genetics Transcription Factors Transfection Tumor Cells, Cultured Two-Hybrid System Techniques |
title | Insulin-like Growth Factor-binding Protein 5 (IGFBP-5) Interacts with a Four and a Half LIM Protein 2 (FHL2) |
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