Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods.
mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission. The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6-8) mGluRs. In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3. Prominent among the candidate receptor interacting proteins was calmodulin, a Ca(2+) sensor known to bind identifiable sequences in group I and III mGluRs. The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs. Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs. This illustrates that certain Ca(2+)-dependent interactions are not recapitulated in yeast. Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.
Lidwell
K
K
Neuroscience Group, University of Southampton, Southampton, UK.
Dillon
J
J
Sihota
A
A
O'Connor
V
V
Pilkington
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D002147:Calmodulin; D018076:DNA, Complementary; D018094:Receptors, Metabotropic Glutamate; D005982:Glutathione Transferase; D002118:Calcium