博士 / 國立清華大學 / 生命科學系 / 94 / AMPA receptors (AMPARs) are major excitatory neurotransmitter receptors and are wildly expressed in the mammalian central nervous system. Through interactions with cellular proteins, the C-terminal domains of AMPAR subunits regulate membrane expression, clustering and recycling of the receptors. Due to a teleost-specific gene duplication event, both zebrafish and tilapia express 8 AMPAR subunits. The primary sequences and ion channel functions are conserved between mammalian and teleost AMPAR subunits. However, some interacting protein recognition sequences of teleost AMPAR subunits are different from that of mammalian AMPAR subunits. An AMPAR subunit tilapia tfGRIA1β C-terminal interacting protein p22 was identified by yeast two-hybrid system. The structure of p22 contains 4 EF-hand calcium binding domains and an N-myristoylation site. Disruption of the third EF-hand domain of p22 affected the interactions with tfGRIA1β C-terminal sequence, whereas disruption of the N-myristoylation site did not affect the interaction. Interaction between tfGRIA1β C-terminal domain and p22 was mapped to a stretch of 28 amino acid sequence which displayed very limited similarities to the interaction sequences found in the other known p22 binding proteins. The lack of sequence conservation among p22 interacting proteins suggests that ligand recognition by p22 is not mediated through consensus sequence. Our result indicated that amphipathic α-helix structure and its neighboring sequences are required for the interaction between p22 and tfGRIA1β subunit. Overexpressing p22 resulted in the alteration of the ratios between maximum currents induced by glutamate and by kainate (IGlu/IKA) of a chimera tfGRIA3αi/1β N21 receptor. The glutamate-induced current is subjected to desensitization, whereas kainate-induced current is not. Addition of the desensitization attenuator, cyclothiazide, abolished the p22 effects on alternation of IGlu/IKA, suggesting that p22 is involved in regulating the desensitization of tfGRIA3αi/1β N21 receptor. This is the first report of regulating AMPAR desensitization accomplished by direct protein-protein interaction.
The flip/flop region of AMPAR subunits involves in the regulation of desensitization. The flip sequence of teleost GRIA1α subunit varies from that of the other vertebrate AMPAR subunit. We demonstrated that the I740 residue of tfGRIA1αflip subunit involved in the regulation of receptor desensitization. However, mutating the T740 residue to I740 of tfGRIA3αflip subunit does not affect the desensitization, indicating that desensitization is not determined by the Ile at the position 740 alone.
| Identifer | oai:union.ndltd.org:TW/094NTHU5105019 |
| Date | January 2006 |
| Creators | Wei-Hsiang Lin, 林煒翔 |
| Contributors | Wei-Yuan Chow, 周姽嫄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 95 |
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