Competitive and Synergistic Interactions of G Protein β2 and Ca2+ Channel β1b Subunits with Cav2.1 Channels, Revealed by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Measurements
| Autor: | Rosa L. Moreno, Stefan Herlitze, Melanie D. Mark, Alexander Hümmer, Oliver Delzeith, Shannon R. Gomez |
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| Rok vydání: | 2003 |
| Předmět: |
G protein
Protein subunit Biochemistry Fluorescence Cav2.1 Cell Line SCN3A GTP-Binding Proteins Two-Hybrid System Techniques KCNJ5 Animals Humans Molecular Biology G alpha subunit biology Chemistry Cell Biology Immunohistochemistry Förster resonance energy transfer Energy Transfer biology.protein Biophysics Calcium Channels ATP synthase alpha/beta subunits Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 278:49386-49400 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m306645200 |
| Popis: | Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein betagamma subunits modulate the binding of the Ca2+ channel beta subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel beta and G protein beta subunits on the two separate binding sites, i.e. the loopI-II and the C terminus, and on the full-length P/Q-type alpha12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel beta1b subunit induces a strong fluorescent signal when interacting with the loopI-II but not with the C terminus. In contrast, the G protein beta subunit induces FRET signals on both the C terminus and loopI-II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel beta1b and G protein beta subunits bind to the alpha1 subunit at the same time. Coexpression of the G protein increases the FRET signal between alpha1/beta1b FRET pairs but not for alpha1/beta1b FRET pairs where the C terminus was deleted from the alpha1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel beta and alpha12.1 subunits, which involves the C terminus of the alpha1 subunit and may corresponds to a new conformational state of the channel. |
| Databáze: | OpenAIRE |
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