Identification of the endogenous ligand of SAP97 PDZ3

描述（由申请人提供）：成年神经元的复杂形态学和电生理学特性在出生前和出生后早期出现。新生儿与其环境的相互作用引起CNS内的模式化突触活动，并且这种活动在塑造神经元的最终形式和功能中起着至关重要的作用。的 活性依赖性可塑性的最佳表征的分子机制涉及NMDA受体的活化和钙触发的信号传导事件。在以前的工作中，使用脊髓神经元，我们已经发现了一个额外的分子机制，采用AMPA-R从GluA 1组装的证据。GluA 1通过其与SAP 97（分子量为97 kDa的突触相关蛋白）的细胞内结合伴侣将活性转化为树突生长。GluA 1/SAP 97形式的可塑性是NMDA-R独立的，我们认为与NMDA-R介导的事件平行运作。对于GluA 1促进树突生长，SAP 97必须共同定位在质膜上，推测在突触后密度。我们假设GluA 1/SAP 97协调组装一个多蛋白复合物，将AMPA-R的活性转化为树突生长。我们的初步数据表明，SAP 97的PDZ 3的结合伴侣在这一过程中起着至关重要的作用。为了鉴定内源性PDZ 3结合伴侣，我们将对六种最佳候选分子进行越来越严格的验证测试。首先，候选物必须结合WT SAP 97，但不结合在PDZ 3中突变的SAP 97形式。内源性蛋白质必须相互作用。第二，候选蛋白在WT中的表达，而不是GluA 1 7神经元，必须促进树突生长。GluA 17神经元具有正常的突触AMPA-Rs，但SAP 97不运输到质膜中。第三，候选蛋白的敲低必须抑制正常的树突生长。除了这项定向研究之外，还将部署使用质谱法的发现方法。总的来说，这些研究将提供一个基本的神经生物学过程的见解，并可能对正常的大脑发育以及成年人受伤后的功能恢复具有翻译意义。 神经系统

DESCRIPTION (provided by applicant): The complex morphological and electrophysiological properties of adult neurons emerge over an extended period of prenatal and early postnatal life. The interaction of neonates with their environment evokes patterned synaptic activity within the CNS and this activity plays a crucial role in sculpting the final form and function of neurons. The best characterized molecular mechanism for activity-dependent plasticity involves activation of NMDA receptors and calcium triggered signaling events. In prior work using spinal cord neurons we have found evidence for an additional molecular mechanism that employs AMPA-R assembled from GluA1. GluA1 translates activity into dendrite growth via its intracellular binding partner with SAP97 (synapse-associated protein of 97 kDa molecular weight). The GluA1/SAP97 form of plasticity is NMDA-R independent and we think operates in parallel with NMDA-R mediated events. For GluA1 to promote dendrite growth, SAP97 must be co-localized at the plasma membrane, presumably at the postsynaptic density. We hypothesize that GluA1/SAP97 coordinately assemble a multiprotein complex that translates activity of AMPA-R into dendrite growth. Our preliminary data indicate that the binding partner of PDZ3 of SAP97 plays an essential role in this process. To identify the endogenous PDZ3 binding partner we will subject the six best candidate molecules to increasingly stringent validation tests. First, the candidate must bind to WT SAP97 but not a version of SAP97 that is mutant in PDZ3. The endogenous proteins must interact. Second, expression of the candidate protein in WT, but not GluA1 7 neurons, must promote dendrite growth. GluA1 7 neurons have normal synaptic AMPA-Rs but SAP97 does not traffic into the plasma membrane. Third, knockdown of the candidate protein must inhibit normal dendrite growth. In addition to this directed study, a discovery approach using mass spectrometry will be deployed. Overall these studies will provide insight into a fundamental neurobiological process and may have translational implications for normal brain development as well as recovery of function after injury to the adult nervous system.