Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application
Interakcija različitih fragmenata treće citoplazmatične petlje dopaminskog d2l receptora čoveka sa a-podjedinicom gi1 proteina - moguća terapeutska primena

2002
Authors
Ignjatović, Đurđica S.
Šukalović, Vladimir

Tasić, Bosiljka
Kostić Rajačić, Slađana

Šoškić, Vukić

Article (Published version)

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In order to find the essential structural motif of the D2L dopamine receptor necessary for the interaction with a-subunit of Gi1 protein, four fragments of the third cytoplasmic loop (CPL3) of this receptor were cloned, expressed in E. coli and purified. After that, fusion proteins with glutathione-S-transferase (GST) were prepared and the interactions quantified by a colorimetric assay for GST activity determination. The presence of D2L-CPL3 fragment-Gia1 complexes was detected by SDS-polyacrylamide gel electrophoresis (PAGE). Kd values for the interaction of the three fragments with Gia1 were similar and in nmol/L range of concentrations, while the peptide representing the insert in the long form of the dopamine D2 receptor expressed about 10-fold lower binding affinity. These results could serve to design new therapeutic agents that might act at the level of receptor/G protein interaction rather than at the level of ligand-receptor binding.
U cilju pronalaženja bitnih strukturnih motiva potrebnih za interakciju sa a podjedinicom Gi1 proteina klonirana su, eksprimirana i prečišćena 4 fragmenta treće citoplazmatične petlje (CPL3) dopaminskog D2L receptora koji su dalje pripremljeni kao fuzioni proteini sa glutation-S-transferazom (GST). Interakcije su kvantifikovane bojenom reakcijom za određivanje aktivnosti GST. Postojanje kompleksa D2L-CPL3 fragment- Gia1 je dokazano elektroforetskom analizom na SDS-poliakrilamidnom gelu (PAGE). Kd vrednosti za tri fragmenta su bile vrlo slične i u nmol/L opsegu koncentracija, dok je peptid koji predstavlja insert u dugom obliku dopaminskog D2 receptora posedovao oko 10 puta manji afinitet vezivanja za Gia1. Ovi rezultati mogu biti osnova za sintezu novih terapeutskih agenasa koji bi delovali na nivou interakcije receptora i G proteina umesto na nivou vezivanja liganda za receptor.
Keywords:
Dopamine D2L receptor / third intracellular loop / synthetic peptide / receptor/G protein coupling / dopaminski D2L receptor / treća intracelularna petlja / sintetski peptid / sprega receptor/G proteinSource:
Jugoslovenska medicinska biohemija, 2002, 21, 1, 9-14
DOI: 10.2298/JMH0201009I
ISSN: 0354-3447
WoS: 000174350800002
Scopus: 2-s2.0-0036118627
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IHTMTY - JOUR AU - Ignjatović, Đurđica S. AU - Šukalović, Vladimir AU - Tasić, Bosiljka AU - Kostić Rajačić, Slađana AU - Šoškić, Vukić PY - 2002 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/73 AB - In order to find the essential structural motif of the D2L dopamine receptor necessary for the interaction with a-subunit of Gi1 protein, four fragments of the third cytoplasmic loop (CPL3) of this receptor were cloned, expressed in E. coli and purified. After that, fusion proteins with glutathione-S-transferase (GST) were prepared and the interactions quantified by a colorimetric assay for GST activity determination. The presence of D2L-CPL3 fragment-Gia1 complexes was detected by SDS-polyacrylamide gel electrophoresis (PAGE). Kd values for the interaction of the three fragments with Gia1 were similar and in nmol/L range of concentrations, while the peptide representing the insert in the long form of the dopamine D2 receptor expressed about 10-fold lower binding affinity. These results could serve to design new therapeutic agents that might act at the level of receptor/G protein interaction rather than at the level of ligand-receptor binding. AB - U cilju pronalaženja bitnih strukturnih motiva potrebnih za interakciju sa a podjedinicom Gi1 proteina klonirana su, eksprimirana i prečišćena 4 fragmenta treće citoplazmatične petlje (CPL3) dopaminskog D2L receptora koji su dalje pripremljeni kao fuzioni proteini sa glutation-S-transferazom (GST). Interakcije su kvantifikovane bojenom reakcijom za određivanje aktivnosti GST. Postojanje kompleksa D2L-CPL3 fragment- Gia1 je dokazano elektroforetskom analizom na SDS-poliakrilamidnom gelu (PAGE). Kd vrednosti za tri fragmenta su bile vrlo slične i u nmol/L opsegu koncentracija, dok je peptid koji predstavlja insert u dugom obliku dopaminskog D2 receptora posedovao oko 10 puta manji afinitet vezivanja za Gia1. Ovi rezultati mogu biti osnova za sintezu novih terapeutskih agenasa koji bi delovali na nivou interakcije receptora i G proteina umesto na nivou vezivanja liganda za receptor. T2 - Jugoslovenska medicinska biohemija T1 - Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application T1 - Interakcija različitih fragmenata treće citoplazmatične petlje dopaminskog d2l receptora čoveka sa a-podjedinicom gi1 proteina - moguća terapeutska primena VL - 21 IS - 1 SP - 9 EP - 14 DO - 10.2298/JMH0201009I ER -
@article{ author = "Ignjatović, Đurđica S. and Šukalović, Vladimir and Tasić, Bosiljka and Kostić Rajačić, Slađana and Šoškić, Vukić", year = "2002", abstract = "In order to find the essential structural motif of the D2L dopamine receptor necessary for the interaction with a-subunit of Gi1 protein, four fragments of the third cytoplasmic loop (CPL3) of this receptor were cloned, expressed in E. coli and purified. After that, fusion proteins with glutathione-S-transferase (GST) were prepared and the interactions quantified by a colorimetric assay for GST activity determination. The presence of D2L-CPL3 fragment-Gia1 complexes was detected by SDS-polyacrylamide gel electrophoresis (PAGE). Kd values for the interaction of the three fragments with Gia1 were similar and in nmol/L range of concentrations, while the peptide representing the insert in the long form of the dopamine D2 receptor expressed about 10-fold lower binding affinity. These results could serve to design new therapeutic agents that might act at the level of receptor/G protein interaction rather than at the level of ligand-receptor binding., U cilju pronalaženja bitnih strukturnih motiva potrebnih za interakciju sa a podjedinicom Gi1 proteina klonirana su, eksprimirana i prečišćena 4 fragmenta treće citoplazmatične petlje (CPL3) dopaminskog D2L receptora koji su dalje pripremljeni kao fuzioni proteini sa glutation-S-transferazom (GST). Interakcije su kvantifikovane bojenom reakcijom za određivanje aktivnosti GST. Postojanje kompleksa D2L-CPL3 fragment- Gia1 je dokazano elektroforetskom analizom na SDS-poliakrilamidnom gelu (PAGE). Kd vrednosti za tri fragmenta su bile vrlo slične i u nmol/L opsegu koncentracija, dok je peptid koji predstavlja insert u dugom obliku dopaminskog D2 receptora posedovao oko 10 puta manji afinitet vezivanja za Gia1. Ovi rezultati mogu biti osnova za sintezu novih terapeutskih agenasa koji bi delovali na nivou interakcije receptora i G proteina umesto na nivou vezivanja liganda za receptor.", journal = "Jugoslovenska medicinska biohemija", title = "Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application, Interakcija različitih fragmenata treće citoplazmatične petlje dopaminskog d2l receptora čoveka sa a-podjedinicom gi1 proteina - moguća terapeutska primena", volume = "21", number = "1", pages = "9-14", doi = "10.2298/JMH0201009I" }
Ignjatović, Đ. S., Šukalović, V., Tasić, B., Kostić Rajačić, S.,& Šoškić, V.. (2002). Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application. in Jugoslovenska medicinska biohemija, 21(1), 9-14. https://doi.org/10.2298/JMH0201009I
Ignjatović ĐS, Šukalović V, Tasić B, Kostić Rajačić S, Šoškić V. Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application. in Jugoslovenska medicinska biohemija. 2002;21(1):9-14. doi:10.2298/JMH0201009I .
Ignjatović, Đurđica S., Šukalović, Vladimir, Tasić, Bosiljka, Kostić Rajačić, Slađana, Šoškić, Vukić, "Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application" in Jugoslovenska medicinska biohemija, 21, no. 1 (2002):9-14, https://doi.org/10.2298/JMH0201009I . .
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