Peptidyl-prolyl Isomerization in the Regulation of Neuronal Signaling Pathways

Peptidyl-prolyl isomerases are a large class of enzymes that catalyze the cis/trans isomerization of prolyl bonds. Initially thought to function solely as chaperones and protein folding catalysts, in recent years a large number of biological substrates for these enzymes had been identified as crucial components in signaling and regulatory networks. However, little remains known about how acceleration of the isomerization rate of a Xaa-Pro bond acts as a regulatory mechanism. In the present work, the mechanisms of the Pin1-mediated regulation of the metabotropic glutamate receptor 5 (mGluR5) signaling and the FKBP mediated gating of TRPC1 channel are examined with respect to the bind-ing and catalytic interactions with the objective of elucidating the molecular mechanisms underlying the physiological function of these receptors.;Phosphorylation of the Ser1126-Pro site in mGluR5 and the subsequent interaction of this site with Pin1 have been previously shown to be required for the potentiation of the mGluR5-mediated physiological output involved in the mechanism of neural plasticity. In addition, expression of Homer1a protein was determined to be required in the context of constitutively expressed Homer1c post-synaptic density scaffolding proteins. In Chapter 2 we demonstrate that Homer1a and Homer1c proteins interact with both cis and trans isomers of mGluR5 at the Ser 1126-Pro bond in essentially identical way suggesting that Homer1a does not impart additional functionality on mGluR5. Phosphorylation increases the Homer affinity for mGluR5 ~15-fold but does not significantly affect the cis/trans distribution of pSer1126-Pro bond in the free or Homer-bound mGluR5. Experiments discussed in Chapter 3 directly demonstrate Pin1's ability to efficiently catalyze the isomerization of the pSer 1126-Pro bond. In an unexpected twist, we discovered that the catalytic domain of Pin1 binds single phosphorylated mGluR5 with ∼20-fold grater affinity than the WW domain and that the WW domain is not selective for the trans isomer as had been previously believed. Based on the experimental data we are able to pro-pose a potential model for the function of Pin1 in mGluR5-mediated signaling.;In Chapter 4 we provide direct evidence for the FKBP12 and FKBP52 catalysis of the L19-Pro and L644-Pro bonds in the C- and N-terminal domains of the TRPC1 channel believed to be involved in the regulation of the channel gating. Additionally, we identify a previously overlooked FKBP target site in the C-terminal region of TRPC1 catalyzed primarily by FKBP12 and propose that the differential catalysis of the adjacent C-terminal sites by FBP12 and FKBP52 may underlie the different physiological functions of the two isomerases.