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N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions
To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors, deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly 8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutage...
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| Published in: | The Journal of biological chemistry 1995-07, Vol.270 (30), p.18013 |
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| Main Authors: | , , , , , , , , |
| Format: | Article |
| Language: | English |
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| container_issue | 30 |
| container_start_page | 18013 |
| container_title | The Journal of biological chemistry |
| container_volume | 270 |
| creator | Ryuji Hashimoto Hiroyuki Fujiwara Nobuyuki Higashihashi Tomoko Enjoh-Kimura Hiroaki Terasawa Yoko Fujita-Yamaguchi Fuyuhiko Inagaki James F. Perdue Katsu-ichi Sakano |
| description | To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors,
deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly 8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli , and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II
were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors
remained at â¥50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to 10% and 3%, respectively, of that of rIGF-II. When the octapeptide including Leu 8 was removed prior to the Cys 9 -Cys intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased
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| doi_str_mv | 10.1074/jbc.270.30.18013 |
| format | article |
| fullrecord | <record><control><sourceid>highwire</sourceid><recordid>TN_cdi_highwire_biochem_270_30_18013</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>270_30_18013</sourcerecordid><originalsourceid>FETCH-LOGICAL-h182t-6b4d8fc5403eafedd60ae0b2f2155eadf537a7ca37daf76e9efcb2dfadfc41ae3</originalsourceid><addsrcrecordid>eNo9kDFPwzAQhS0EKqWwM97AAEOKHSdNMkIhJVIBCYrEFjnOuXFJ7Spx1F_G_yNtgVueTvfep9Mj5JLRMaNRcLsq5NiP6Jj3e0wZPyJDRmPu8ZB9HpMhpT7zEj-MT8lZ265oP0HCBmQQTfyE0WRIvl88h81aG1HDA9botDXw3DlhXAtWQWbartbGq_UXwqyxW1dBKqSzjZdlcJ3N0l5v4L2yW1hUDQhTwhw7yNYb2-wooGwD99qU2izB2T_g3rhPwxtK3PTA9j88bbTTsv9ol72r64Mxg7Qzcvdge05OlKhbvPjVEflIHxfTJ2_-Osumd3OvYrHvvEkRlLGSYUA5CoVlOaECaeErn4UhilKFPBKRFDwqhYommKCShV-q_iIDJpCPyNWBW-lltdUN5oW2ssJ13peec5rvS-c_hR12JQ</addsrcrecordid><sourcetype>Publisher</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions</title><source>ScienceDirect</source><creator>Ryuji Hashimoto ; Hiroyuki Fujiwara ; Nobuyuki Higashihashi ; Tomoko Enjoh-Kimura ; Hiroaki Terasawa ; Yoko Fujita-Yamaguchi ; Fuyuhiko Inagaki ; James F. Perdue ; Katsu-ichi Sakano</creator><creatorcontrib>Ryuji Hashimoto ; Hiroyuki Fujiwara ; Nobuyuki Higashihashi ; Tomoko Enjoh-Kimura ; Hiroaki Terasawa ; Yoko Fujita-Yamaguchi ; Fuyuhiko Inagaki ; James F. Perdue ; Katsu-ichi Sakano</creatorcontrib><description>To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors,
deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly 8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli , and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II
were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors
remained at â¥50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to 10% and 3%, respectively, of that of rIGF-II. When the octapeptide including Leu 8 was removed prior to the Cys 9 -Cys intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased
to <1% of that of rIGF-II. Substituting Gly 8 for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These
results suggest that the side chains of Thr 7 and Leu 8 may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the
mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the
relative affinity of des-(1-8)- or [Gly 8 ]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also <1% of that of rIGF-II, the relative
affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II.
These results clearly demonstrate that Thr 7 and Leu 8 are important for binding to insulin and IGF-I receptors and Leu 8 is critical for expression of all IGF-II functions.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.270.30.18013</identifier><identifier>PMID: 7629109</identifier><language>eng</language><publisher>American Society for Biochemistry and Molecular Biology</publisher><ispartof>The Journal of biological chemistry, 1995-07, Vol.270 (30), p.18013</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids></links><search><creatorcontrib>Ryuji Hashimoto</creatorcontrib><creatorcontrib>Hiroyuki Fujiwara</creatorcontrib><creatorcontrib>Nobuyuki Higashihashi</creatorcontrib><creatorcontrib>Tomoko Enjoh-Kimura</creatorcontrib><creatorcontrib>Hiroaki Terasawa</creatorcontrib><creatorcontrib>Yoko Fujita-Yamaguchi</creatorcontrib><creatorcontrib>Fuyuhiko Inagaki</creatorcontrib><creatorcontrib>James F. Perdue</creatorcontrib><creatorcontrib>Katsu-ichi Sakano</creatorcontrib><title>N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions</title><title>The Journal of biological chemistry</title><description>To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors,
deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly 8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli , and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II
were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors
remained at â¥50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to 10% and 3%, respectively, of that of rIGF-II. When the octapeptide including Leu 8 was removed prior to the Cys 9 -Cys intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased
to <1% of that of rIGF-II. Substituting Gly 8 for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These
results suggest that the side chains of Thr 7 and Leu 8 may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the
mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the
relative affinity of des-(1-8)- or [Gly 8 ]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also <1% of that of rIGF-II, the relative
affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II.
These results clearly demonstrate that Thr 7 and Leu 8 are important for binding to insulin and IGF-I receptors and Leu 8 is critical for expression of all IGF-II functions.</description><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid/><recordid>eNo9kDFPwzAQhS0EKqWwM97AAEOKHSdNMkIhJVIBCYrEFjnOuXFJ7Spx1F_G_yNtgVueTvfep9Mj5JLRMaNRcLsq5NiP6Jj3e0wZPyJDRmPu8ZB9HpMhpT7zEj-MT8lZ265oP0HCBmQQTfyE0WRIvl88h81aG1HDA9botDXw3DlhXAtWQWbartbGq_UXwqyxW1dBKqSzjZdlcJ3N0l5v4L2yW1hUDQhTwhw7yNYb2-wooGwD99qU2izB2T_g3rhPwxtK3PTA9j88bbTTsv9ol72r64Mxg7Qzcvdge05OlKhbvPjVEflIHxfTJ2_-Osumd3OvYrHvvEkRlLGSYUA5CoVlOaECaeErn4UhilKFPBKRFDwqhYommKCShV-q_iIDJpCPyNWBW-lltdUN5oW2ssJ13peec5rvS-c_hR12JQ</recordid><startdate>19950728</startdate><enddate>19950728</enddate><creator>Ryuji Hashimoto</creator><creator>Hiroyuki Fujiwara</creator><creator>Nobuyuki Higashihashi</creator><creator>Tomoko Enjoh-Kimura</creator><creator>Hiroaki Terasawa</creator><creator>Yoko Fujita-Yamaguchi</creator><creator>Fuyuhiko Inagaki</creator><creator>James F. Perdue</creator><creator>Katsu-ichi Sakano</creator><general>American Society for Biochemistry and Molecular Biology</general><scope/></search><sort><creationdate>19950728</creationdate><title>N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions</title><author>Ryuji Hashimoto ; Hiroyuki Fujiwara ; Nobuyuki Higashihashi ; Tomoko Enjoh-Kimura ; Hiroaki Terasawa ; Yoko Fujita-Yamaguchi ; Fuyuhiko Inagaki ; James F. Perdue ; Katsu-ichi Sakano</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-h182t-6b4d8fc5403eafedd60ae0b2f2155eadf537a7ca37daf76e9efcb2dfadfc41ae3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ryuji Hashimoto</creatorcontrib><creatorcontrib>Hiroyuki Fujiwara</creatorcontrib><creatorcontrib>Nobuyuki Higashihashi</creatorcontrib><creatorcontrib>Tomoko Enjoh-Kimura</creatorcontrib><creatorcontrib>Hiroaki Terasawa</creatorcontrib><creatorcontrib>Yoko Fujita-Yamaguchi</creatorcontrib><creatorcontrib>Fuyuhiko Inagaki</creatorcontrib><creatorcontrib>James F. Perdue</creatorcontrib><creatorcontrib>Katsu-ichi Sakano</creatorcontrib><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ryuji Hashimoto</au><au>Hiroyuki Fujiwara</au><au>Nobuyuki Higashihashi</au><au>Tomoko Enjoh-Kimura</au><au>Hiroaki Terasawa</au><au>Yoko Fujita-Yamaguchi</au><au>Fuyuhiko Inagaki</au><au>James F. Perdue</au><au>Katsu-ichi Sakano</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions</atitle><jtitle>The Journal of biological chemistry</jtitle><date>1995-07-28</date><risdate>1995</risdate><volume>270</volume><issue>30</issue><spage>18013</spage><pages>18013-</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>To define the role of the N-terminal region of insulin-like growth factor-II (IGF-II) in its binding to insulin and IGF receptors,
deletion mutants des-(1-5)-, des-(1-7)-, and des-(1-8)-recombinant (r) IGF-II, and the Gly 8 for Leu substitution mutant of rIGF-II were prepared by site-directed mutagenesis, expressed in Escherichia coli , and purified. The binding affinity and mitogenic activity of these rIGF-II mutants as well as commercially available des-(1-6)-rIGF-II
were analyzed. While the relative affinity of des-(1-5)- and des-(1-6)-rIGF-II for purified human insulin and IGF-I receptors
remained at â¥50% levels of that of rIGF-II, the affinity of des-(1-7)-rIGF-II decreased to 10% and 3%, respectively, of that of rIGF-II. When the octapeptide including Leu 8 was removed prior to the Cys 9 -Cys intrachain bond, the relative affinity of this deletion mutant, des-(1-8)-rIGF-II, for these receptors dramatically decreased
to <1% of that of rIGF-II. Substituting Gly 8 for Leu in rIGF-II decreased the affinity of this mutant for the IGF-I and insulin receptors to about the same extent. These
results suggest that the side chains of Thr 7 and Leu 8 may play an important role in retaining all of the IGF-II functions. Decreases in the relative affinity for binding of the
mutants to these receptors paralleled the decreases in their mitogenic potency for cultured Balb/c 3T3 cells. Although the
relative affinity of des-(1-8)- or [Gly 8 ]rIGF-II for rat IGF-II/CIM6-P (cation-independent mannose 6-phosphate) receptors was also <1% of that of rIGF-II, the relative
affinities of des-(1-5)-, des-(1-6)-, and des-(1-7)-rIGF-II for these receptors was significantly greater than that of rIGF-II.
These results clearly demonstrate that Thr 7 and Leu 8 are important for binding to insulin and IGF-I receptors and Leu 8 is critical for expression of all IGF-II functions.</abstract><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>7629109</pmid><doi>10.1074/jbc.270.30.18013</doi><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1995-07, Vol.270 (30), p.18013 |
| issn | 0021-9258 1083-351X |
| language | eng |
| recordid | cdi_highwire_biochem_270_30_18013 |
| source | ScienceDirect |
| title | N-terminal Deletion Mutants of Insulin-like Growth Factor-II (IGF-II) Show Thr and Leu Important for Binding to Insulin and IGF-I Receptors and Leu Critical for All IGF-II Functions |
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