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Neurogranin: Lmmunocytochemical Localization of a Brain-Specific Protein Kinase C Substrate

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Neurogranin: Lmmunocytochemical Localization of a Brain-Specific Protein Kinase C Substrate The Journal of Neuroscience, December 1990, IO(12): 3782-3792 Neurogranin: lmmunocytochemical Localization of a Brain-Specific Protein Kinase C Substrate Alfonso Repress,’ Jean Christophe Deloulme,2 Monique Sensenbrenner,2 Yeherkel Ben-Ari,’ and Jacques Baudier2 ‘INSERM Unite 29, 75014 Paris, and Xentre de Neurochimie du CNRS, INSERM Unite 44, 67064 Strasbourg, France An understanding of the exact function of the different PKC The developmental expression and the cellular localization isozymes requiresthe identification and characterization of their of neurogranin (formerly designated p17), a brain-specific in vivo substrates.In the brain, only 2 PKC substrateshave been protein kinase C (PKC) substrate, were investigated. The formally identified and characterized: neuromodulin (also called developmental expression of neurogranin was studied by GAP43, B50, Fl, ~57, and pp46; Alexander et al., 1988) and immunoblotting of rat brain and neuronal cell-culture ex- MARCKS protein (also called ~87; Stump0 et al., 1989). Neu- tracts using neurogranin polyclonal antibodies. Neurogranin romodulin is a calmodulin-binding phosphoprotein of the pre- synthesis was found to be developmentally regulated, with synaptic membrane that is associatedwith the initial establish- no expression in the embryonic and neonatal period and an ment, regeneration, and functional modulation of synaptic abrupt increase between 2 and 3 weeks of age. By immu- relationships and appearsto play a role in signal transduction nohistochemistry, neurogranin was found essentially in the mechanisms(Van Hoffet al., 1988). P87 is a protein ofunknown adult rat telencephalon, specifically located in the cell bod- function that has a widespreadtissue and subcellular distribu- ies and dendritic processes of neurons of the cerebral cor- tion (Albert et al., 1986). tex, hippocampus, striatum, and a few other discreet areas. We recently reported the purification of a new in vitro specific Neurogranin immunoreactivity was nearly absent in the thal- PKC substrate from the bovine brain, called p 17 from its ap- amus, cerebellum, and brain stem. The late developmental parent molecular mass in SDS gel (Baudier et al., 1989). A expression and the dendritic localization of neurogranin in straightfotward purification method for p 17 and neuromodulin neurons are 2 features that also characterize the type I PKC was developed using the property of these PKC substratesof isozyme. The specific localization of the protein in integra- remaining soluble in 2.5% perchloric acid (Baudier et al., 1989). tive areas of the rat brain suggests a highly specialized The biochemical characterization of the p 17 molecule has now function of neurogranin in the CNS. A possible role for neu- been extended in our laboratory, and results will be published rogranin in the transduction of the PKC activation signals at elsewhere. Significant structural and functional analogiesbe- the postsynaptic level is suggested. tween p17 and neuromodulin have been found. The purified pl7 protein has a molecular massof 7.8 kDa, as determined by Protein kinase C (PKC), a Ca*+-activated phospholipid-depen- ion-spray massspectroscopy and protein sequencing,which is dent kinase, is the receptor for tumor-promoting phorbol esters 2-fold lower than its apparent molecular masson SDS gels(Cim- and is thought to play an important role in controlling several ler et al., 1987). P17 binds to calmodulin-Sepharosecolumns cellular processes(see Nishizuka, 1988, for review). PKC is in the absenceof calcium, a feature that also characterizesneu- present ubiquitously in a variety of tissuesand is especially romodulin (Masure et al., 1986). Although the primary structure concentrated in the brain (Huang et al., 1986). Three different of p17 is unique, a striking conserved amino acid sequence, PKC isozymes, designatedtypes I, II, and III, have been de- AA(X)KIQASFRGH(X)(X)RKKL(X)K, wasfound betweenp 17 scribed (Huang et al., 1986). They are products of y-, p-, and and neuromodulin. This 18-amino acid consensussequence be- a-type genes,respectively (Coussenset al., 1986; Brandt et al., tween bovine p 17 and mammalian as well asfish neuromodulin 1987). In situ hybridization and direct immunohistochemical representsthe PKC phosphorylation site and the predicted cal- studies revealed differences in the distribution and develop- modulin-binding domain, suggestinga possibleevolutionary link mental expressionofthe 3 PKC isozymesin the brain, suggesting between p17 and neuromodulin (LaBate and Skene, 1989; J. that the different isozymesof PKC have distinct functions (Brandt Baudier, J.C. Deloulme, H. Matthes, and M. Sensenbrenner, et al., 1987; Huang et al., 1987; Hashimoto et al., 1988; Yoshida unpublished observations). et al., 1988). In the present study, we report the tissue distribution and expressionof pl7 in the rat brain. Becausepreliminary electron Received Apr. 2, 1990; revised June 14, 1990; accepted July 12, 1990. microscopic studieshave shown that p 17 is often found in gran- We would like to thank Dr. T. Janet for his help in immunizing rabbits. We ulelike structures in pyramidal cells of the hippocampus and are also indebted to Miss Boudoin and Miss S. Guidasci for their technical and photographic assistance. We wish to thank Dr. K. Langley for carefully reading cortex, we will hereafter refer to the pl7 protein asneurogranin. the manuscript. This work was supported by the French Association pour la Recherche sur le Cancer Grant ARC 645 1 and by INSERM. Materials and Methods Correspondence should be addressed to Dr. J. Baudier, Centre de Neurochimie du CNRS, INSERM Unite 44, S Rue Blaise Pascal, 67084, Strasbourg, France. PuriJicationof proteins.Bovine brain neurogranin(p 17)was purified as J.C.D. is a recipient of a fellowship from the Association Franqaise Contre les previouslydescribed (Baudier et al., 1989).For rabbit immunization, Myopathies. a final purificationstep was included, consisting of reverse-phasechro- Copyright 0 1990 Society for Neuroscience 0270-6474/90/123782-l 1%03.00/O matographyon a Pro RPC column.Neurogranin was eluted at 0.5 ml/ The Journal of Neuroscience, December 1990, fO(12) 3783 min with a 50-ml linear gradient of O-50% acetonitril with 0.1% tri- fluoroacetic acid. Neurogranin eluted at about 20% acetonitril. The protein was then extensivelv dialvzed aaainst 20 mM Tris-HCl CDH. 7.5) plus 10 mM dithiothreitol (DTT)and 1 mu EDTA and stored a; -2OT: Bovine brain neuromodulin and 7 proteins were prepared as previously described (Baudier et al., 1987, 1989). Production of antibodies. Antibodies against purified neurogranin, neuromodulin, and 7 proteins were prepared in New Zealand white rabbits. The proteins (300-500 pg) were emulsified in complete Freund’s adjuvant and injected intradermally at multiple dorsal sites. At 2-3- week intervals, the rabbits were given 2 other booster injections of 150- 250 pg proteins in incomplete Freund’s adjuvant. The rabbits were bled repeatedly, and the antibody titers were determined by Western blotting. Antisera that showed significant activity at 1:lOOO dilution were col- lected. A second type of rabbit antisernm against neurogranin was ob- tained by injecting the neurogranin monomer, which was cut directly from the SDS-polyactylamide (0.1%: 12.5%) gels and emulsified in in- Figure 1. Distribution of neurogranin in adult rat tissues. Perchloric complete Freund’s adjuvant. This antiserum gave similar immuno- acid-treated extracts of adult rat tissues were resolved by SDS-PAGE staining patterns on rat-brain sections to the first antiserum (see Results). and processed for immunoblotting as described in Materials and Meth- Purified antibodies against neurogranin were obtained by affinity chro- ods. N, purified bovine-brain neurogranin monomer; Cr. cerebrum; Cb, matography of purified serum IgG on neurogranin coupled to a glutar- cerebellum; He, heart; Lu, lung; Sp, spleen; Li, liver; Ad, adrenal gland, aldehyde-activated Trisacryl column. Ki, kidney; Te, testis. The immunoreactive bands correspond to the Tissue and cell extracts. Tissues were freshly excised from rats killed neurogranin monomer. In each lane, the proteins loaded correspond to by decapitation. The tissues (0.5 gm/ml) were homogenized in buffer approximately 20 mg tissue. A 1100 mM Tris-HCl CDH. 7.4). 2 mM EDTA. 2 mM DTT. 100 mM NaCl and 1% Triton k-lb01 using a Dounce’ homogenizer, and the reproducible. The use of affinity-purified IgG against neurogranin gave extract was clarified by centrifugation. Perchloric acid was added to the a similar staining pattern to that of the crude antisera. supematant (final concentration 2.5%), which was immediately centri- fuged for 15 min at 15000 x g. The supematant was then dialyzed Results overnight against 40 mM Tris-HCl (pH, 6.9), 50 mM NaCl, and 2 mM DTT. Cortical neuronal cell cultures were obtained from fetal rats, as Tissue distribution and developmental expressionof previously described (Gensburger et al., 1986). The cultures were rinsed neurogranin 3 times with PBS. The cells were disrupted by brief sonication in buffer The specificity of the polyclonal antibodies raised against pure A and processed in the same way as the tissue extract. Zmmunoblotting. Tissue and cell extracts were electrophoresed in 0.1%: neurogranin can be seen from transblot analysis of total and 12.5% SDS-polyacrylamide gels, then transferred electrophoretically to partially purified rat-brain extracts (Figs. 1, 2). The major band an Immobilon blotting membrane (Millipore). After transfer,
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