A new technique to co-localise membrane proteins with Homer/vesl.
The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.
Hiroaki
Yoko
Y
Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Japan.
Nishikawa
Kouki
K
Mitsuoka
Kaoru
K
Tachibana
Taro
T
Sobue
Kenji
K
Doi
Tomoko
T
Fujiyoshi
Yoshinori
Y
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D000072233:Homer Scaffolding Proteins; D006801:Humans; D007313:Insecta; D008823:Micelles; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009479:Neuropeptides / Q000378:metabolism*; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction
D002352:Carrier Proteins; D018076:DNA, Complementary; D003902:Detergents; D000072233:Homer Scaffolding Proteins; D008823:Micelles; D009479:Neuropeptides; D011506:Proteins; D011392:Proline