Characterization of the interaction between transmembrane-agrin and the AMPA receptor auxiliary proteins Shisa6 and Shisa9 at synapses in the central nervous system

• Applicant: Professor Dr. Stephan Kröger
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells ; Developmental Neurobiology ; Molecular and Cellular Neurology and Neuropathology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 533473678

Agrin and its receptor complex are the key regulators during formation, maintenance and regeneration of the neuromuscular junction. The transmembrane form of agrin (TM-agrin) is also concentrated at interneuronal synapses in the CNS, but its function in the developing and adult CNS is still unclear. We have recently investigated potential interaction partners of TM-agrin at CNS synapses by precipitating agrin from Mouse cortex extracts followed by mass spectrometry. In this approach, we identified the AMPA receptor auxiliary proteins Shisa6 and Shisa 9 (but not Shisa7) as direct binding partners. We were able to confirm this interaction by precipitating TM-agrin and Shisa proteins from extracts of 293HEK cells coexpressing both proteins as well as from brain extracts using antibodies against both proteins. Moreover, using the “Proximity Ligation” assay we demonstrated the direct interaction of both proteins using an independent method. In this proposal, we want to characterize the interaction of TM-agrin and Shisa proteins in more detail and to start investigating potential functions of this interaction. To this end, we want to confirm the interaction using the “Bimolecular Fluorescence Complementation” assay. Moreover, using deletion constructs and site-directed mutagenesis we want to map the interaction sites in TM-agrin and both Shisa proteins. We will try to colocalize both proteins with AMPA receptors at CNS synapses and investigate if TM-agrin overexpression in the cortex in vivo changes the density of Shisa- and AMPA receptor-containing synapses. Along the same line, we will investigate if the distribution of TM-agrin at CNS synapses changes in Shisa6 knockout animals. Likewise, we will investigate of agrin affects the expression levels of Shisa proteins and if TM-agrin expression levels change in Shisa6 knockout animals using qPCR. Finally, we will analyze if the presence of TM-agrin influences the biophysical properties of AMPA receptors by expressing all three proteins in 293HEK cells and in embryonic CNS neurons using electrophysiological methods. We hope that these experiments will contribute to a better understanding of the function of agrin in the CNS and to our understanding of synaptogenesis in the CNS in general.

• DFG Programme: Research Grants