Interactions between Neurogranin and Calmodulin in Vivo

Lisa Prichard,Jean Christophe Deloulme,Daniel R Storm

doi:10.1074/jbc.274.12.7689

Lisa Prichard, Jean Christophe Deloulme + Show 1 more

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https://doi.org/10.1074/jbc.274.12.7689

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Abstract

Neurogranin is a neural-specific, calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) within its IQ domain at serine 36. Since CaM binds to neurogranin through the IQ domain, PKC phosphorylation and CaM binding are mutually exclusive. Consequently, we hypothesize that neurogranin may function to concentrate CaM at specific sites in neurons and release free CaM in response to increased Ca2+ and PKC activation. However, it has not been established that neurogranin interacts with CaM in vivo. In this study, we examined this question using yeast two-hybrid methodology. We also searched for additional proteins that might interact with neurogranin by screening brain cDNA libraries. Our data illustrate that CaM binds to neurogranin in vivo and that CaM is the only neurogranin-interacting protein isolated from brain cDNA libraries. Single amino acid mutagenesis indicated that residues within the IQ domain are important for CaM binding to neurogranin in vivo. The Ile-33 --> Gln point mutant completely inhibited and Arg-38 --> Gln and Ser-36 --> Asp point mutants reduced neurogranin/CaM interactions. These data demonstrate that CaM is the major protein that interacts with neurogranin in vivo and support the hypothesis that phosphorylation of neurogranin at Ser-36 regulates its binding to CaM.

Highlights

IntroductionNeurogranin (RC3, BICKS) is a postnatal expressed brainspecific protein that is localized postsynaptically in neuronal cell bodies and dendrites of the cerebral cortex, hippocampus, and striatum [1,2,3,4,5]
Neurogranin (RC3, BICKS) is a postnatal expressed brainspecific protein that is localized postsynaptically in neuronal cell bodies and dendrites of the cerebral cortex, hippocampus, and striatum [1,2,3,4,5]. It was first identified by subtractive hybridization in a screen for mRNAs enriched in rat forebrain and was independently purified from brain as a protein kinase C (PKC)1 substrate (6 –9)
Tion within the IQ domain inhibits CaM binding, and CaM binding inhibits PKC phosphorylation at the serine within the IQ domain [21, 22]

Summary

Introduction

Neurogranin (RC3, BICKS) is a postnatal expressed brainspecific protein that is localized postsynaptically in neuronal cell bodies and dendrites of the cerebral cortex, hippocampus, and striatum [1,2,3,4,5]. Neurogranin and neuromodulin share high sequence homology within 20 amino acids designated as the IQ motif [11, 13] This sequence, AAAAKIQASFRGHMARKKIK in neurogranin, contains a binding domain for CaM and a PKC phosphorylation site [8, 14, 15]. The physiological functions of neurogranin have not been defined, its biochemical properties and postsynaptic localization have implicated it in several signal transduction pathways Both neurogranin and neuromodulin have been shown to regulate CaM-dependent nitric oxide synthase activity through sequestration of CaM [24, 25]. We utilized yeast two-hybrid technology to detect neurogranin-binding proteins and to characterize neurogranin/ CaM interactions in vivo

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