RP4: Synaptic regulation of GABAA receptor turnover

GABAA受体（GABAARs）在神经元中经历广泛的内吞作用，然后再循环到表面膜或蛋白质降解。网格蛋白依赖性内吞作用似乎是启动受体内化的主要机制。网格蛋白适配蛋白2 （AP2）通过μ2亚基与GABAARs结合。PKA和pkc依赖性磷酸化调节GABAARs的表面稳定性。不同的GABAAR结合蛋白参与内化受体命运的调控。例如，亨廷顿蛋白相关蛋白（HAP1）结合GABAAR β亚基，HAP1过表达抑制GABAAR降解，从而增加受体再循环。类似地，Plic-1似乎调节受体的转换并促进GABAAR在神经元表面膜的积累。在FG885的第一个资助期内，我们的研究结果为控制突触GABAAR周转的系统增加了一个新的参与者。泛素特异性蛋白酶Usp14似乎调节含有α1亚基的GABAARs，这些GABAARs携带单/寡泛素标签。基因敲低Usp14产生共济失调表型，表现为广泛的GABAAR再分布到神经元表面。这些作用是可逆的，需要Usp14酶的催化蛋白酶活性。我们实验室最近的数据发现了两种基于肌动蛋白的肌球蛋白运动蛋白，它们与所有含亚基的GABAARs相关。myoVI具有顺行运动的性质，而myoVI具有逆行运动的性质，这表明这两个因素可能是介导质膜和膜下囊泡间室间GABAAR再分布的驱动因素。与ampa型谷氨酸受体的转换类似，本项目旨在研究gabaar -肌球蛋白相互作用的机制。由于抑制性突触的性质主要位于树突轴，而不是在局部受限的脊柱头部隔室，我们希望确定神经递质受体转换的主要机制，不同于兴奋性突触的转换机制。

GABAA receptors (GABAARs) undergo extensive endocytosis in neurons followed by either recycling to the surface membrane or protein degradation. Clathrin-dependent endocytosis appears to be the major mechanism that initiates receptor internalization. The clathrin-adaptor protein 2 (AP2) associates with GABAARs via the μ2 subunit. PKA as well as PKC-dependent phosphorylation regulate the surface stability of GABAARs. Different GABAAR binding proteins participate in the regulation of internalized receptor fate. For instance, the Huntingtin-associated protein (HAP1) binds to GABAAR β subunits and HAP1 overexpression inhibits GABAAR degradation, thereby increasing receptor recycling. Similarly, Plic-1 appears to regulate receptor turnover and facilitates GABAAR accumulation at neuronal surface membranes.In the first FG885 funding period, our results added a new player to the system controlling synaptic GABAAR turnover. The ubiquitin-specific protease Usp14 appears to regulate α1 subunit-containing GABAARs that carry a mono-/oligo-ubiquitin label. Genetic knockdown of Usp14 generates an ataxia phenotype, represented by an extensive GABAAR redistribution to the neuron surface. These effects are reversible and require the catalytic protease activity of Usp14 enzymes.Recent data from our laboratory identified two actin-based myosin motor proteins that associate with al subunit-containing GABAARs. The nature of myoV, representing an anterograde motor, as well as the nature of myoVI moving in retrograde directions, suggests that both factors might be the drivers that mediate GABAAR redistribution between plasma membrane and submembrane vesicle compartments. In analogy to the turnover of AMPA-type glutamate receptors, the proposed project aims to study the mechanisms of GABAAR-myosin interactions. Due to the nature of inhibitory synapses that mainly locate at dendrite shafts, rather than at the regionally restricted spine head compartments, we expect to identify principal mechanisms of neurotransmitter receptor turnover that differ from turnover mechanisms at excitatory synapses.