June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
The presynaptic glutamate transporter in rods binds to a distinct subset of PDZ domain containing proteins
Author Affiliations & Notes
  • Sarah Hengel
    Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA
  • Joseph Laird
    Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA
  • Will Watkins
    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA
  • Randy Hall
    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA
  • Sheila Baker
    Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA
  • Footnotes
    Commercial Relationships Sarah Hengel, None; Joseph Laird, None; Will Watkins, None; Randy Hall, None; Sheila Baker, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 703. doi:
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      Sarah Hengel, Joseph Laird, Will Watkins, Randy Hall, Sheila Baker; The presynaptic glutamate transporter in rods binds to a distinct subset of PDZ domain containing proteins. Invest. Ophthalmol. Vis. Sci. 2013;54(15):703.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Visual signaling depends on removal of glutamate from the synaptic cleft between photoreceptors and bipolar cells. Excitatory amino acid transporter 5 (EAAT5) is the glutamate transporter found in rod inner segments and synapses. The location and activity of EAAT5 is likely regulated by interacting proteins, however binding partners have yet to be identified. The C-terminus of EAAT5 contains a PDZ binding motif which prompted us to screen for PDZ domain interactions.

Methods: We generated a GST fusion protein containing the 30 C-terminal amino acids of EAAT5 and probed an array of 96 PDZ domains. GST pull-downs and fluorescence anisotropy assays were used to confirm interactions. A membrane-associated GFP reporter fused to EAAT5’s C-terminus was expressed in the rods of transgenic frogs and subcellular location was visualized with confocal microscopy.

Results: The C-terminus of EAAT5 bound to 13 of the 96 PDZ domains on the array. Only three of these positive hits, β2-syntrophin, SAP97, and MAGI-1a, are known to be expressed in the outer plexiform layer of the retina along with EAAT5. All three of these interactions were validated by independent GST pull-down assays. The β2-syntrophin PDZ domain interacts with the EAAT5 C-terminal peptide with a calculated KD=4.3 +/-0.3 µM. In transgenic tadpoles the membrane reporter accumulates in the outer segment regardless of the presence of EAAT5’s C-terminus.

Conclusions: We identified multiple novel interaction partners for the glutamate transporter EAAT5. These interactions are unlikely to be necessary for the trafficking of EAAT5, suggesting they may modulate its activity.

Keywords: 648 photoreceptors • 659 protein structure/function • 740 transgenics/knock-outs  
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