Identification of the endogenous ligand of SAP97 PDZ3

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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.