performed by RC3 in the postsynaptic neuron. Acknowledgments-We gratefully acknowledge Dr. David Millar and. Dr. Pat JeMings for helpful discussion and ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Ine.
Vol. 269,No.35,Issue of September 2, pp. 22420-22426,
1994
Printed in U.S.A.
Mutational and Biophysical Studies Suggest RC3Neurogranin Regulates Calmodulin Availability* (Received for publication, April 15, 1994)
Dan D. Gerendasy,Steven R. Herron, Joseph B. Watson$, and J. Gregor SutcliffeP From the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 and the @Department of Psychiatry and Biobehavioral Sciences, Mental Retardation Research Center and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024
RC3heurogranin is a forebrain-enriched, postnatalin dendritic spinedevelopment and plasticity (Morreale de Esonset, thyroidhormone-dependent,protein kinase C cobar et al., 19831, and these deficits may correlate with RC3 substrate of dendritic spines that interacts with calmod-deficiencies. ulin. These characteristics suggest aprominentrole The primary amino acid sequence of RCS/neurogranin conwithin the Caz+-mediated second messenger cascades as- tains a stretch of 15/19 residues that areidentical to a region sociated with neonatal synaptogenesis and adult neural near the amino terminus of GAP143/neuromodulin that conplasticity. To understand the molecular interactionsbe- tains its site of phosphorylation by protein kinase C (PKC) tween RC3 and calmodulin, we characterized recombi- and its calmodulin-binding domain (Fig. 1). Neurogranin was -+ Ala, nant RC3 andfoursequencevariants:Ser-36 identified independently by Baudier and colleagues (Baudier Ser-36 + Asp, Ser-36 + Lys, and Phe-37 4 Trp. Interacet al., 1989) by virtue of its ability to act in crude brain homotions between CaM and variant Phe-37 + Trp can be monitored by fluorescence spectroscopy, allowingus to genates and in hippocampal slices as a Ca2+-dependent phosdetermine, by competitiveassays, the relative affinities phorylation substrate for PKC that was soluble in perchloric acid. Unphosphorylated neurogranin and GAP-43 both bind to of the wild-type and variant proteins for calmodulin. calmodulin-Sepharose (CaM-Sepharose) inthe absence but The effects of salt and Ca2+on the rank order of these not the presence of Ca2+(Baudier et al., 1989, 1991; Coggins et affinities permitpartial dissection ofhydrophobic, of the RC3-CaM inter- al., 1991; Ape1 et al., 1990; Coggins and Zwiers, 1989; Alexionic, and structural components action and suggest that it is bimodal. We demonstrate ander et al., 19881, but neither molecule binds when phosphoGAP-43/neuromodulin that RC3 binds preferentially to CaM when Ca2+is ab- rylated. Thus,RC3heurograninand sent and that the addition of a negative chargeto resi- share several biochemical properties. Nevertheless, these two due 36 is sufficient to disrupt all detectable RC3-CaM proteins are distinct in their anatomicalregions of expression, interactions. We propose a model wherein a Ca2+-"sensi- subcellular distributions, and time of expression during neutive," bimodal interaction betweenRC3 and CaM regu- ronal development. lates the transduction of postsynaptic Ca2+fluxes into The timing of RC3 expression, its enrichment in dendritic physiological responses through the modulation of Ca2+/ spines of forebrain neurons and deficit in hypothyroidism, and CaM availability. its biochemical properties related to PKC, Ca'', and CaM suggest a role for RC3 in the chemistryof neonatal synaptogenesis and adult neural plasticity, perhaps as a postsynaptic component of the long term potentiation (LTP) cascade. The latter RCSIneurogranin is a78-amino acid, postnatal-onset propossibility is supportedby the observation that immunoprecipitein, originally identified in a subtractive hybridization study tation of RC3 from hippocampal slices in which LTP has been (Watson et al., 1990), that is highly enriched in rat forebrain induced yields significantly more of the phosphorylated form areas but absentfrom cerebellum and peripheral tissues.RC3 than do control slices (Chen et al.,1993). Additionally, Cohen et and itsbovine homologue neurograninBICKS (bovine D-50 imal. (1993) have proposed that phospho-RC3 enhances Ca2+momunoreactive C-kinase substrate) (Baudier et a l . , 1991; Cogbilization, and this could be one mechanism by which RC3 gins et at., 1991) are neuron-specific, as demonstrated by both participates in the initiationof LTP. in situ hybridization and immunohistochemistry (Watson et al., RC3 is known to interact with two proteins, PKC and CaM, 1990; Represa et al., 1990). In the neostriatum of adult rats, and its putativePKC recognitiodCaM-binding domain constiimmunoelectron microscopy has demonstrated that accumuit tutes fully one-third of the entire protein. Thus, a close relalates principally in dendritic spines, where it is observed in tionship must exist betweenthe structureof this region and the association with postsynaptic structures (Watson et al., 1992). function of RC3. We recently described the expression and puThis ultrastructural assignmentis intriguing because the conrification of RC3 and three variants containing different amino centration of RC3 is severely reduced selectively and reversibly acids at position 36, normally a serine residue that is the puin theforebrain regionsof hypothyroid rats (Munoz et al.,1991; tativesite of PKC phosphorylation.' Additionally, weconIniguez et al.,1992). Hypothyroidism is associated with deficits structed andpurified a variant form containing a tryptophan in place of phenylalanine 37, the only aromatic residue in native * This work was supported in part by National Institutes of Health RC3. The elution profiles generated by these variants during Grants NS2211 and NS22347. The costs of publication of this article were defrayed in part by the payment ofpage charges. ThisarticIe must ' The abbreviations usedare: GAP, 43-kDa growth-associated protein; therefore be herebymarked "aduertisement"in accordance with 18 PKC, protein kinase C; CaM, calmodulin; LTP, long term potentiation; U.S.C. Section 1734 solely to indicate this fact. 0 To whom correspondence should be addressed: Dept. of Molecular NMDA, N-methyh-aspartate. D.D. Gerendasy, S. R. Herron, K. K. Wong, J. B. Watson, and J. G. Biology, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Sutcliffe, manuscript in preparation. Jolla, CA 92037. Tel.: 619-554-8064; Fax: 619-554-6112.
22420
Interaction of RC3 INeurogranin with Calmodulin
22421
RESULTS
F37W-Calmodulin Interactions Mon.itored by Fluorescence Spectroscopy-The F37W variant contains a single tryptophan located within itsCaM-binding domain; thus,we expected that interactions with CaM would alter its fluorescence emission spectrum aswas previously found for GAP-43/neuromoduP*********** linCaM interactions. To test this, we titrated a known concentration of F37W with CaM. Tryptophan fluorescence was measured by excitation a t 295 nm and subsequent collection of the emission spectra. Addition of saturating concentrationsof CaM FIG.1.Alignment of RC3 (residues1-51) with similar region of to F37W caused a blue shift in the emission maximum from 351 GAF"43 (GAP; residues 1-55). The overlapping sites of calmodulin nm to 334 nm both in the presence and absence of Ca" (Fig. binding ( ' h ) and PKC phosphorylation ( p ) for GAP-43 are indicated. Gaps were introduced by eye to optimize alignment. 2 A ) , probably caused by an increase in the hydrophobicity of the tryptophan residue's environment. The emission intensity was greater in the absence of Ca2+. This was not due to affinityCaM-Sepharose chromatography suggested that interactions between RC3 and CaM were influenced by these amino acid dependent differences in complex formation, as the spectra substitutions. Here, we verify and extend this observation by were collected after maximal saturationof F37W with CaM, as fluorescence spectroscopy using the tryptophanyl variant as a determined from data obtained from titration of F37W with reporter protein againstwhich to measure relative affinitiesof CaM in the presence and absence of Cat+and/or 200 mar NaCl wild-type RC3 and serine 36 variants for CaM. We also inves- and plotting the fractional increase influorescence versus the tigate the behavior of RC3 and variantswhen applied to CaM- total concentration of CaM a t each titrationpoint (Fig. 2B ). The Sepharose in the presence of Ca". Together, these approaches linear stoichiometric increasein fluorescence that occurred when CaM was added to 4.5 PM F37W in the absence of Ca'+ reveal ionic and hydrophobiccomponents to the interaction between RC3 and CaM and, by analogy, between GAP-43 and indicates that the concentrations used in this experiment were CaM. We propose that RC3 interacts with CaM through two well above the K,,. When 100 nnl F37W was titrated with CaM fundamentally different modes in the absenceof Ca2+ and that in 10 nM increments, a linear dependence was also observed phosphorylation of RC3 a t serine 36 abolishes both of these (data not shown),placing an upper limit on the K,, of 100 na1. binding interactions. One of these modes may also be utilized in F37W bound to CaM less tightly, but only slightly so, when Ca" the presence of CaZ+ under certain circumstances. Ourobser- was present, as indicated by the increased hyperbolic character vations lead toa hypothesis inwhich RC3 serves asa biochemi- of its titration curve (Fig. 2). cal "capacitor" in that it transduces local Ca2+fluxes into kiWe examined the stoichiometry of these interactionsby plotnetic parametersaffecting the availabilityof Ca2+/CaMand the ting the increase in fluorescence intensity versus the ratio of rapidity with which it is madeaccessible to other proteins. [CaM] to [F37W] (Fig. 2B, inset). The bindingcurves generated by titrating F37W with CaM in the absence ofCa", with or EXPERIMENTAL PROCEDURES without NaCl, plateaued when this ratio reached a value of 1, Determination ofProtein Concentration-Colorimetric protein assays indicating a stoichiometryof 1to 1. Titrationscarried out in the tend to underestimate RC3 concentrationswhen traditional standards presence of Ca2+ and NaCl resulted inhighly variable titration such as bovine serum albumin are used. We synthesized a 78-amino curves, possibly caused by denaturation and/or the formation of acid peptide based on the predicted sequence ofRC3. The results of higher multimers. amino acid content analyses performed on this peptide and on pure Relative CaM-binding Affinities of RC3 Variants-The relapreparations of recombinant RC3 indicated that theBCA protein assay kit (Pierce)yielded results thatwere consistently off by 20% when the tive affinities of three Ser-36 variants were assessed by comparing their abilities to compete with F37W for CaM, using synthetic peptide was used as a standard. This was probably due to chromogenic contaminants within the synthetic peptide.All of the profluorescence emissionspectroscopy as an assay for binding. The tein concentrations assayed in the experiments described below were binding curves that resulted when F37W was titrated with performed by amino acid content analysis. CaM in the presence of RC3 WT (20 p ~ ) S36K , (20 phl), S36A SpectroJluorimetry-All spectrofluorimetry was performed with an or S36D (20 p ~ are ) displayed in Fig. 3 (A-E). The SPF-5OOC spectrofluorimeter a t 20 "C using a path length of 1 cm. The (19 p ~ ) , presence of competing ligand displaced the bindingcurve tothe excitation beam (295 nm) was passed through a 2-nm slit, and the emitted spectrum (334 and 360 nm) was collected in 1-nm increments right and the degree to which it did so is indicative of the through a 7.5-nm slit.Eachdatapointwassampled32timesand relative affinity of the competitor. A concentration of 4.5 u31 averaged. Titrations of F37W with CaM were all performed in 50 mhf F37W was used when Ca" was absent, while 8.5 pa1 was used Tris, pH 7.5, 2 mM EGTA or CaCl,, and, where indicated,200 mM NaCl. in its presence to compensate for lower signal strength. In the Calmodulin was added in0.25-1.00 phl increments toa cuvette containing 1ml of 2.33.4.45, or 8.90 p~ F37W. The titrations displayed in Figs.absence of Ca2+,S36A displaced the binding curve furthest to 2 and 3 were performed in the presence of 4.45 or 8.90 p F37W as the right (Fig. 3A). RC3 WT and S36K shifted the binding they less tightly to indicated. Competition experiments were performed in the same man- curves to lesser extents, indicating that bind ner described except20 V M competitor was included in the cuvette prior CaM than S36A. S36D did not compete with F37W for CaM to the titration. (Fig. 3E). The relative order of affinities was different when CaM-SepharoseColumnChromatography-Pure or partially pure Ca2+ was present (Fig. 3B). In thiscase, the RC3 WT competed (perchloric acid-soluble, trichloroacetic acid-insoluble) recombinant proteins, obtained from 1 liter of induced cells as described previously,* less strongly than S36A and S36K, which displayed similar were applied toa CaM-Sepharose column (1cm x 40 cm) containing 15 affinities, while S36D exhibited no affinity (Fig.3E ). The same rank order of affinities was observed with low and high Ca2+ ml of CaM-Sepharose 4B (PharmaciaBiotech Inc.) a t 4 "C. The column was washed and the protein eluted in the indicated buffers. Fractions when 2.34 p~ F37W was titrated with CaM in the presence of (10 ml) were collected and assayed by resolution of aliquots on 1 2 8 4-fold excess of the RC3 Ser-36 variants (data not shown). SDS-polyacrylamide gels that were silver-stained. The same column We examined the effect of increasing ionic strength on the was used throughout, allowing direct comparison of elution profiles. Variantsexaminedwerewild-type(RC3 WT), serine36 alanine interactions between CaM and each of the variants (Fig. 3, C (S36A), serine 36 -> lysine (S36K), serine 36 -> aspartate (S36D), and and D ) . F37W alone or with each of the competitors was titrated with CaM in the presence or absence of 200 mv NaCI. phenylalanine 37 -, tryptophan (F37W).
0
-+
Interaction of RC3 INeurograninCalmodulin with
22422
B
A 61
I
1.O
0.8 0.6
/ \
P F37W+EGTA
0.4 0.2
0.0 1.0
0.5
1.5 2.5
2.0
[CaM]/[F37W]
300
340 380 460 420 Wavelength (nm)
0.0 0.0
5.0
10.0
FIG.2. Fluorescence emission spectroscopy of F37W.CaM interactions.A, emission spectra of 4.5 p~ F37W in 2 mM EGTA or 2 m~ CaCl, obtained in the presence or absence of 10 p~ CaM. Emissions were scanned between 300 and 460 n~ in 1 n~ intervals while exciting at 295 nM. B, titration of4.5 or 8.5 VM F37W with CaM (0.25-1.00 JIM increments) in the presence or absence of Ca2+or salt. The fractional change in fluorescence emission at 334 nM (F,,, - FJF,,,, - Fo) was plotted against the total concentration ofCaM added ([CaM],,,) where F334 is the fluorescence observedat each titration point, Fo is the initial fluorescence observedprior to the addition of any CaM, and F,, is the maximum fluorescence observed.All measurements were corrected forRaman fluorescence. CaM was determined to have no intrinsic fluorescence (data not shown). Inset to B , the stoichiometry of binding in the presence or absence of Ca2+was determined by plotting the relative fluorescence (F334/Fo) as a function of added CaM. 0, F37W + EGTA; A, F37W + 2 nm Ca2+;0, F37W + 0.2 M NaCl.
NaCl had no significant effect on the interactionbetween F37W and CaM or on the ability of RC3 WT t o compete for CaM (Fig. 3C). Variants S36K and S36A, on the other hand, exhibited significant although opposing salt effects (Fig. 30). Theaffinity of mutant S36K for CaM was attenuated by 200 m~ NaCl below that of RC3 WT. The affinity of S36A for CaM appeared to increase in thepresence of salt. Effects of Salt and ea2+ on Interactions between RC3 and CaM-Sepharose-We previously demonstrated that recombinant RC3, S36A, and S36K were retained on CaM-Sepharose columns developed in EGTA at 200 mM NaC1, but elutedrapidly when Ca2+ was added to the column buffer. When F37W was applied to a CaM-Sepharose column in thepresence of 100 mM NaCl and washed with 200 mM NaCl, it bled from the column in a prolonged fashion that was hastened only slightly by the 4A).Variant F37W, therefore, appeared to addition of Ca2+ (Fig. interact with CaM-Sepharose more strongly than RC3 WT or any of the variants when both salt and Ca2+ werepresent. To investigate salt effects more closely, partially purified F37W was applied to the column in thepresence of Ca2+ andabsence of NaC1, washed with buffer containing Ca2+and 100 mM NaCl, then with buffer containing 200 mM NaCl, and finally with buffer containing 200 m~ NaCl and EGTA. F37W did not appear in theflow-through, but began to eluteas the washbuffer containing 100 mM NaCl entered the column, and continued to bleed off as the salt concentration increased to 200 mM NaC1. No further protein eluted after addition of buffer containing EGTA (Fig. 4B). For comparison, in similar experiments with S36A (Fig. 4C) or RC3 WT (Fig. 4D), some of the protein loaded in thepresence of Ca2' emerged in theflow-through and the restbled from the column during the subsequent salt washes. They eluted prior to, or were apparently unaffected by, the application of buffer containing EGTA. When RC3 WT was applied to thecolumn in loading buffer containing Ca2+and 200 mM NaCI, little if any significant bindingwas observed (Fig. 4E). Thus, variantF37W appeared to interact with CaM in the absence of Ca2' more strongly than RC3 WT but less strongly than variant S36A. In the presence of Ca2+, it interacted more strongly than anyof the others, especially at low ionic strength. In thepresence of salt, RC3 WT bound to CaM preferentially when Ca2+ was absent.
DISCUSSION
Variant Phe-37 + Trp associated withCaM in both the presence and absence of Ca2+.This interaction was monitored by fluorescence emission spectroscopy providing an assayfor comparing relativeaffinities of the otherRC3 variants for CaM. In the absence of Ca2+, the order of affinities was: F37W > S36A >> S36K 2 RC3 WT >> S36D. Salt decreased S36K affinity, increased S36A affinity, and had minimal effects on RC3 WT and F37W, resulting in the following order: S36A > F37W >> RC3 WT > S36K >> S36D. The relative affinities of F37W and S36A for CaM in the presence and absence of salt is supportedby the stoichiometry of binding when both are present(Fig. 3F), which plateaued a t approximately 1.0 in the absence of salt but at 0.8 when salt was present, indicating that the affinity of S36A for CaM is greater than or equal to that of F37W. Similar treatment of fluorescence data obtained in thepresence of Ca2+ impliesthat F37W binds more tightly than any of the others (data not shown). F37W clearly had a higher affinity than S36A in the absence of salt and Ca2+.When Ca2+was present, theaffinities were generally reduced, but still measurable: F37W >> S36A = S36K > RC3 WT >> S36D. M n i t y orders could not be studied in thecompetition assay when Ca2+ and salt were simultaneously present because of effects on the reporter F37W, however, from the chromatography studies, a partial order of affinities based on the rate at which each eluted after salt was added, could be determined: F37W > S36A = RC3 WT. Although RC3 WT and S36A interacted withCaM-Sepharose in a salt-resistant manner when first applied to thecolumn in the absence of salt (Fig. 4, C and D ) , little interaction was observed in the case of RC3 WT when salt was present initially. Thus, while the relative order of binding in thepresence of Ca2+ and absence of salt may be informative, it is not necessarily indicative of that which would occur under physiological conditions. Nevertheless, the nature of the amino acid replacements and the effects of ionic conditions on the relative CaM affinities of the RC3 variants suggests both hydrophobic and ionic components to these interactions and, by extension, to the RC3 WTCaM interaction.
22423
Interaction of RC3 INeurogranin with Calmodulin
A
FIG.3. Relative affinities of RC3 WT and variants determined by fluorescenceemissionspectroscopy. F37W was titrated with CaM (0.25-1.00 p increments) in the presence of 20 p~ competitor as indicated. 4.5 p F37W was used in the absenceof Ca", while 8.5 was used in its presence. In panels A-E, the fractional change in the difference between the fluorescenceobserved at 351 and 334 nm was plotted against the log of the total concentration of CaM, where FdiR is the difference (F,,, - F351) observed at each titration point, Fo is the difference observed prior to the additionof CaM, and F,, is the maximum differenceobserved. In panel F , the fractional change in fluorescence measured at 334 nm (as in Fig. 4) was plotted against the ratio of the concentration of CaM added to the total con+ centration of ligandpresent(F37W S36A).A, competition between F37W and RC3 WT, S36K, or S36A in the absence of Ca2+ and salt; B , competitionbetween F37W and RC3 WT, S36K, or S36A in the presence of Ca2+and absence of salt; C , effect of 200 mM NaCl on F37W-CaM interaction and the ability of RC3 WT to compete in the absence of Ca"; D , effect of 200 mM NaCl on the ability of S36K and S36Ato compete with F37W in the absence of Ca"; E, competitionbetween F37W and S36D in the absence (EGTA)or presence (Ca2+) of Ca"; F, competition between F37W and S36A in the presence or absence of 200 mM NaCl when Ca2+is absent. All spectra were corrected for Raman fluorescence.
B
-7.5 -7
-6.5 -6
-5.5 -5 -4.5 -4 Log[CaMl,,,
-7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4 Log[CaMlm, OF37W (no Competitor) OS36K
C
Iw S36A
D
1.o
0.8 0.6 0.4
0.2 0.0 -7
-6.5
-6
-5.5
-5
-4.5
Log[CaMl,,l V A
E
F
-7
-6
-51-7
-6
-5
Log[CaMl,,,, 0 F37W (no Competitor)
Phosphorylation Is Required for Complete Abrogation of RC3.CuMAfinity-Of the variants examined,all except S36D had measurableaffinity for CaM, evenwhen Ca2+was present. The behavior of S36D is consistent with that of an analogous GAP-43/neuromodulin Ser-41 + Asp mutant that was notretained on a CaM-Sepharosecolumn in the absence of Ca2+ (Chapman et al., 1991). These observations suggest that the inability of phosphorylated RC3 to bind CaM is due to the negative charge contributedby the phosphatemoiety and that phosphorylation of RC3 is required to abolish completely its association with CaM in dendritic spines. Interestingly, the S36K variant,which contains a positively charged residuein place of serine 36, was promotedin the rank affinity order when Ca" was present, suggesting that charge repulsion of phosphorylated RC3 or S36D is more important after Ca2+ has conferred a structural change on CaM, RC3, or, possibly, both. If RC3 does interact, albeit weakly, with CaM when Ca2+ levelsare highin uiuo, its phosphorylation would be expected to disrupt this interaction as well. The CaM-binding Domain of Variant S36K Resembles That of GAP-43-While GAP-43/neuromodulin binds more tightly to CaM in theabsence of Ca2+,its affinity is sensitive tosalt and, at physiological salt concentrations, binding becomes essentially Ca'+-independent. RC3, on the other hand, maintainsits
V S36K+0.2M NaCl A S36A+0.2M NaCl
S36K S36A
1.o
0.8 1 .o [CaMl I [F37W + S36AI
0.6
A S36D
higher affinity for CaM in theabsence of Ca2+,even a t 200 nm NaCl. Fluorescence spectroscopy and CaM-Sepharose chromatography indicated that the S36K variant exhibited greater affinity for CaM when no salt was present, whereas no difference was observed for RC3WT. Examination of the aminoacid sequence of GAP-43 (Fig. l),whose affinity for CaM is also decreased by salt, revealsthat it contains more basic residues to the NH,-terminal side of its PKC-targeted serine thandoes RC3. The increase in the net positive charge when Ser-36 is replaced with lysine may, therefore, lower CaM affinity in the presence of salt by subjecting this variant tosome of the forces and/or constraintsthat act upon GAP-43. Sequences NH,-terminal to the siteof PKC phosphorylation may be implicated in the unusual ability of RC3 and, underlow ionic conditions, GAP-43 to bind CaM more tightly when Ca2+ levels are low; a 17-mer GAP-43 peptide, all but 2 residues of which are COOH-terminal to the serine residue phosphorylated by PKC (Ser-41) and containinga Phe + Trp substitution, bound to CaM with an affinity similar to that of GAP-43 when Ca2+was absent and witha much greater afflnity when it was present (Alexander et al., 1988; Chapman et al., 1991). This behavior is markedly different from that of F37W, which contains the analogous substitution but which binds CaM less tightly in thepresence of Ca2+ than in absence. its Additionally,
22424
Interaction of RC3 INeurograninCalmodulin with A. Phe37-rTrp (EGTA) 2W mM
Proteln 100 mM NaCl
NaCl
.
FIG.4. Effects of salt and Ca2+on CaM-Sepharose column elution profiles of RC3 W T and variants. Pure (panel B )or partially pure (panels A and C-E) proteins obtained from 1 liter of induced cells was applied to a CaM-Sepharose column, washed, and eluted in the indicated buffers. Arrows on the top of each stripmarkthe fractionemerging from the bottom of the column as buffer containing the additional component was added to the top. Faint bands that appear in the first lanes of gels A , B, and E are caused by leakage of markerproteins from the left. Fractions (10ml) were collected and assayed by resolution of aliquots on 12% SDS-polyacrylamidegels that were silver-stained. Arrows on the left side of each gel indicate which band is RC3 or variant thereof. The samecolumn wasusedthroughout,allowingdirect comparison of elution profiles. F37W was loaded in Ca2+-freebuffer containing 100 mM NaCI, washed with 200 mM NaCI, and EGTA eluted with wash buffer containing Ca2+ ( A , Phe-37 -,T r p ) or was loaded in salt free buffer containing Ca", washed with 100 mM NaCI, then 200 mM NaCI, and finally 200 mM NaCl containing EGTA(B, Phe-37 + Trp); S36A ( C , Ser-36 -, Ala) and RC3 WT ( D ,RC3 W T ) were treated as in panel B. RC3 WT was also loaded in buffer containing 200 mM NaCl and Ca" (E, RC3 WT).
CeZ'
t
1"I
,
-;
~
"
B. Phe37+Trp (Ca2+) Proteln No NaCl
t ,
- .
1WmM NaCl
-
2 W d
Ne1
t
EGTA
.
r""""" I I
C. Ser36+Ala (Ca2+) . , 1WmM NaCi 4
Protein NO NaCi 4
2WmY Necl
EGTA
! I
2
1
D. RC3 WT (Ca2+) Proteln No NaClNaCl
ZOOrnM
l 0 0 d NeCl
1
L E. RC3 WT (Ca2+) Proteln 2M) mM N s c l t
EGTA ".
-1
-b
..
r
U"
a 9-amino acid GAP-43 peptide (residues 43-51) bound to CaM- these dataimply that two modes of binding occur under low salt Sepharose in the presence, as well as the absence, of Ca2+. conditions depending on ambient Ca2+levels. RC3 Has High and Low Affinity Forms-The F37WCaM While these observations do not allow one to infer that NH,terminal sequences contribute tobinding in theabsence of Ca2+, binding curves in Ca2+with or without competing ligands all they do suggest that the NH, terminus attenuatesCaM binding had similarcontours, while the curves generated in the absence when Ca2+ is present. of Ca2+appear to fall into two classes. Those for F37W alone Ca2+ AlterstheQuality of RC3CaM Znteractions-Trypand with S36A as competitor were very similar toeach other in tophanyl fluorescence emitted by F37W when binding saturat- shape andwere differentfrom those obtainedwith WT RC3 and ing quantitiesof CaM was dramatically quenched by Ca2+,in- S36K, which appeared tobe less ideal competitors than S36A. dicating that the tryptophan residueexperiences different en- Their no Ca2+curves appear to be a hybrid of the curves they vironments depending upon Ca2+ concentrations.Calcium also generated in thepresence of Ca2+and theno Ca2+S36A curve. had differential effects upon the affinities of the RC3 variants This is consistent with the notion that RC3 WT and variants for CaM, suggesting that binding in thetwo conditions utilizes cycle between low and high affinity states when Ca2+is absent distinct mixes of hydrophobic and ionic components. Evidence and that the tightest binders, F37W and S36A, spend more for differential ionic contributions, for example, can beseen in time in thehigh affinity state than do RC3 WT and S36K. In the behavior of S36K, which displays a similar affinity to RC3 the presence of Ca2+,only the low affinity mode is observed. Model for a Bimodal Interaction between RC3 and CaM-We WT in the absence of Ca2+ but binds more strongly in its presence. Since phosphorylation or substitution of ser-36 with as- have found recently that an a-helix involving residues 25-47 partate abolishes affinity, i t is possible that thenegative dipole within RC3 is stabilized by CaM under physiological salt con.~ thetwo modes of associated with thehydroxyl moiety of serine actually detracts centrations only when Ca2+is a b ~ e n tThus, from CaM affinity when Ca2+is present. binding inferred from the data presented here appear to have Differential hydrophobic contributions to the interaction can conformational equivalents. Replacement of residue 36 with be inferred from the behavior of the F37W, which suggests that alanine may increase a-helical tendencies, accounting for the increasing the hydrophobic bulk of residue 37 increases the higher CaM affinity. Hence, the biochemical phenotype exhibaffinity of RC3 for CaM regardless of ambient Ca2+,but espe- ited by each of the variants could depend upon the ease with cially when it is present. Thehigh affinity of S36A for CaM in which an a-helix can be realized. This notion can be reprethe absence of Ca2+ may also indicate hydrophobic interactions, sented formally as shown by Reaction 1, with the portion of although recently obtained data from circular dichroism spectroscopy suggest that the major contribution of alanine 36 is D. D. Gerendasy, S. R. Herron, P. Jennings, and J . G. Sutcliffe, probably structuralratherthan hydrophobic. Collectively, manuscript in preparation.
Interaction of INeurogranin RC3 molecules occupyingeach affinity statedetermined by equilibrium constants K,,, through Keq4.
Calmodulin with PIP-PIP
J
DAG
1
Kql
[RC3] + [CaM] e [RC3CaM]
22425
'I IP,
J
NMDA-R
w CaM
[RC3*] + [CaM] e [RC3*CaMI 1 REACTION
RC3, unstructured CaM-bindingdomain; RC3*, a-helical CaMbinding domain; RC3CaM, low affinity complex; RC3*CaM, high affinity complex. Equilibrium constant K,,, governs the portion of molecules containing an a-helical CaM binding domain and is greatest in the case of variant S36A. Those factors that influence Keqswould also be expected to influence K,,,. Equilibrium constants K,,, and Keql govern the conversion of free RC3 and CaM to low and high affinity complexes, respectively. When Ca2+is absent, RC3 appears to cycle between low and high affinity states; however, it only binds to CaM through the low affinity mode when Ca2+is present. Therefore, K,,, can be examined in isolation by including Ca2+.When RC3 WT was applied to a CaM-Sepharose column in buffer containing Ca2+ and 200 mM NaC1, little if any interaction was observed. When it was applied in the absence of salt, RC3 interacted with the matrix and continued t o do so even after NaCl was increased from 0 to 100 m ~ and , then to 200 mM. These observations imply that K,,, can be dissected into its component rate constants, KO,, and Kom by varying NaCl concentrations in the presence of Ca". Salt decreases K,, while leaving KoErelatively unaffected. After KO,,,was diminished by salt, thelength of time required for RC3 WT t o elute indicates that Kom is relatively slow (on the order of seconds). Thus, under physiological conditions, we would predict that RC3 binds t o CaM slowly when Ca2+levels are high and dissociates slowly regardless. When salt and Ca2+were initially present, only F37W interacted strongly with CaM-Sepharose, suggesting that an increase in the hydrophobic component of the interaction resulted in a K,,, that was relatively insensitive to salt and that the biochemical phenotype of this variantinvolves stronger low affinity binding. Variant S36A, whichbinds CaM very tightly in the absence of Ca2+,interacts with CaM-Sepharosein much the same way as thewild-type whenCa2+is present, indicating that its phenotype is mediated by a stronger high affinity state. However, fluorescence spectroscopy suggests that both F37W and S36A spend more time in the high affinity mode than RC3 WT or S36K. The concentration of low affinity complex is determined by K,,,; thus, an increase in this value, such as that caused by the substitution of Phe-37 with Trp, results primarily in a greaterconcentration of the high affinity form in reactions 1and 2 of the threereactions describing possible relationships between the two affinity forms.
&,I
[RC3] + [CaM] =s[RC3.CaMl 1
k
1L K w z
3
[RC3*] + [CaM] [RC3*.CaM] 2 REACTION [RC3] + [CaM]
11
Keq1
=== [RC3CaM]
Keq4
[RC3*] + [CaM] e [RC3*CaM] REACTION 3
FIG.5 . Hypothesis that places RC3 in the post-synaptic second messenger cascade.Two separate but intertwined processes are proposed toresult in the disassociation of the RC3.calmodulin complexand subsequent phosphorylation of RC3 by PKC. Phosphorylation of phosphatidylinositol4-monophosphate(PIP)results inphosphatidylinositol 4,5-bisphosphate (PIP,), which is hydrolyzed following stimulation of some receptors into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IPJ.The mobilization of internal stores of Ca2+by inositol 1,4,5trisphosphate along with the possible influx of extracellular Ca2+ through the NMDA receptor may cause the disassociation of RC3 and CaM. The final concentrations of free CaM and RC3 are dependent on the Ca2'-"sensitive" rate constants K,,, and Kq2, which are, in turn, influencedby the conformationalstate or preference of the two proteins. The rapid disassociation of RC3 proposed to occur when CaM undergoes a large Ca2+-inducedconformational change is illustrated. PKC, which is activated by diacylglycerol in the presence of Ca2+,phosphorylates numerous substrates, including RC3. Other Ca2'-dependent enzymes are also activated, most of which require CaM, such as Ca2+/CaMdependent kinase 11. Phosphorylated RC3 (RC3-P),whichno longer interacts with CaM, may have other, as yet unknown, biochemical activities.
In the third case, increasing K,,, increases low affinity complex concentrations at the expense of high affinity complex concentrations. Amodel that emphasizes K,,, and Keq2to the exclusion of Keq3and Keq4would imply that thelow affinity binding mode must be traversed to gain access t o the high affinity state. This simplifies any hypothetical mechanisms by which a negative charge aEliated with residue 36 could effectively abolish interactions between RC3 and CaM; phosphorylation of RC3 or the substitutionof Ser-36 with aspartate need only prevent the formation of the low affinity complex in order to prevent any interaction. The second equation implies that KO,,, increases when Ca2+ is withdrawn and that the rate of binding can be facilitated by a coinciding increase in K,,,. When RC3 is purified on CaM-Sepharose in the presence of EGTA, addition of Ca2+causes rapid elution, suggesting that slow dissociation prescribedby Kom can be circumvented. Possibly, Ca2+-dependentconformational changes within CaM actively drive the low affinity complex apart at the same time that K,,, is effectively decreased to 0, while a low KO,,,prevents reassociation. Since K,,, is small even when salt is absent and because Konlis strongly attenuated, if not completely abolished, by 200 mM NaCl unless Ca2+levels are low, all of the RC3 in dendritic spines probably disassociates rapidly from CaM when Ca2+levels rise and a significant proportion likelyremains disassociated for the duration. Should a change in Ca2+levels not be sufficient to cause the conformational rearrangements in CaM required for completeand rapid disassociation, one might imagine that an equilibrium concentration of free and bound CaM would beattained at a relatively slow rate determined by Kom. A Physiological Model for RC3 Interactions-The hypothesis depicted in Fig. 5 places RC3 in the dendritic spine in the context ofPKC and CaM, the two proteins with which it is known to interact. There, acascade is initiated by an influx of Ca2+through the NMDA receptor, which is essential for the induction of Hebbian LTP in the dentate gyrus and the CA1
22426
Interaction of RC3lNeurogranin with Calmodulin
region of the hippocampus (Brownet al., 1990; Madisonet al., 1991).Two of the NMDA subunits, NMDAR2A and NMDARZB, are expressed in many of the same locations as RC3 (Nakanishi, 1992; lnsel et al., 1990). While the NMDA receptor may play a role in physiologicalphenomena relevant to RC3, it isnot essential, as any event leading to increased Ca2+concentrations within a dendritic spine would cause RC3 to relax its grip on CaM and stimulate its phosphorylation by PKC. A transient increase in Ca2+is also sufficient for the induction ofLTP (Madison et al., 1991; Lynch et al., 1983; Malenka et al., 1988). When Ca" levels within a spine are very low, equilibrium constants K,,, and K,,, ensure that little free CaM exists. A sudden rise in Ca2+would alter these equilibria, causing the immediate disassociation of RC3 and CaM and subsequent activation of Ca2+lCaM-dependent proteins such as CaM kinase I1 and calcineurin. Reassociation would depend on the reestablishment of K,,, and K,, which are very low when Ca2+conand K,, may centrations are high. Intermediate values of be possible when fewer than four of CaMs Ca2+-binding sites are occupied. Such values would be possible if the rateconstant KO,,, and/or the average amount of time spent in thehigh affinity mode were dependent upon the number of occupied Ca2+binding sites. Additionally, the rate of disassociation could be regulated by the size of a Ca2+flux and base-line Ca2+levels prior to its induction. Together, these two variables could determine whether CaM undergoes a sufficiently dramatic conformational change to cause rapid disassociation of the RC3CaM complex orwhether the rateof disassociation would be limited to Kom.In short, RC3 may serve as a biochemical "capacitor," in that iteither releases Ca2+/CaMgradually or in a rapid pulse, depending on the size and durationof a Ca2+flux. The validity of this model requires that RC3 not be limiting with respect to CaM. When RC3 is purified from bovine brain, it is obtained in yields that suggest its abundance is equal to, or greater than, thatof CaM, and in thisrespect it isanalogous to GAP-43 (Martzen and Slemmon, 1994; Alexanderet al., 1987). An important characteristic of the bimodal interaction described here is that rates of association andlor disassociation may be regulated independently of, but concurrently with, the final steady-state concentrations of free CaM and RC3. Amechanism that allowed the modulation of equilibrium concentrations based on Ca2+levels is attractive inthat itwould provide a means by which Ca2+levels and free CaM concentrations could be coordinated. This type of mechanism could also be used to alter therelative weighting of activities contributed by different classes of Ca2+lCaM-dependent enzymes depending on their abilities to compete for CaM.Consistent with this notion, GAP-43 and RC3 have recently been demonstrated, in vitro,to increase the Ca2+requirement of CaM-dependent nitric oxide synthetase activation within the physiologically relevant Ca2+ concentrations of 0-10 p ~ (Slemmon . and Martzen, 1994; Martzen and Slemmon. 1994). The rate at which these equilibria are established may also be important for the proper interpretation of cyclical or intermittent Ca2+fluxes of varying magnitudes and degrees of overlap. The ability to modulate these rates and final steady-state concentrations in opposite directions may be advantageous and made possible by two binding modes linked in series. We have demonstrated that S36D, a mimic of phosphorylated RC3,does not display a detectable affinity forCaM in the presence or absence of Ca2+or salt, implying that activation of
&,
PKC and its consequent phosphorylation of RC3 abrogates all interactions between CaM and RC3 within the dendritic spine. This may explain why PKC activation within the postsynaptic neuron is required for the induction ofLTP (Madison et al., 1991; Malinow et al., 1989; Silva et al., 1992). Another, not necessarily exclusive,possibility is that the phosphorylated form of RC3 has an unidentified biological activity of its own, facilitating neuroplastic events such as LTP. Cohen et al. (1993) have suggested that thephosphorylated form of RC3 may serve to mobilize internal post-synaptic stores of Ca". Additionally, numerous functions have been attributed to GAP-431 neuromodulin, phosphorylated and unphosphorylated, which may perform a presynaptic function partially analogous to that performed by RC3 in the postsynaptic neuron. Acknowledgments-We gratefully acknowledge Dr. David Millar and Dr. Pat JeMings for helpful discussion and advice concerningthe generation and interpretation of fluorescence emission spectroscopy data and for critically reading this manuscript. We are also grateful to Jeff Burns for dedicated technical assistance. REFERENCES Alexander, K. A,, Watkim, B. T., Doyle,G. S.,Walsh, K. A. & Storm, D. R. (1988) J. B i d . Chem. 263, 7544-7549 Apel, E. D., Byford, M. F.,Au, D., Walsh, K. A. & Storm, D. R. (1990) Biochemistry 29,2330-2335 Baudier, J., Bronner, C., Kligman, D. & Cole,R.D. (1989) J . Biol. Chem. 264, 1824-1828 Baudier, J.,Deloulme, J. C., Van Dorsselaer, A,, Black, D. & Matthes, H. W. (1991) J. Bid. Chem. 266, 229-237 Brown, T. H., Kairiss, E. W. & Keenan,C.L. (1990) Annu. Reu. Neurosci. 13, 475-511 Chapman, E. R., Au, D., Alexander, K. A,, Nicolson, T.A. & Storm, D. R. (1991) J. Bid. Chem. 266,207-213 Chen, S.J., Klann, E. & Sweatt, J. D.(1993) Proceedings of the 23rd Annual Meeting of the Society for Neuroscience in Washington D. C., November 7-12, 1993, Abstr. 703.5 Coggins, P. J. & Zwiers, H. (1989) J. Neurochem. 63,1895-1901 Coggins, P. J., Stanisz, J., Nagy, A. & Zwiers, H. (1991)Neurosci. Res. Commun. 8, 49-56 Cohen, R.W, Margulies, J. E., Coulter, P. M., I1 & Watson, J. B. (1993) Brain Res. 627, 147-152 Iniguez, M. A,, Rodriguez-Pena,A,, Ibarrola, N., Morreale de Escobar,G. 81 Bernal, J. (1992) J. Clin. Invest. 90, 554-558 Insel, T. R., Miller, L. P. & Gelhard, R. E. (1990) Neuroscience 36, 3 1 4 3 Lynch, G., Larson, J., Kelso, S., Banionuevo, G. & Schottler, F. (1983) Nature SOB, 719-721 Madison, D. V, Malenka, R. C. & Nicoll, R. A. (1991) Annu. Reu. Neurosci. 14, 379-397 Malenka, R. C., Kauer, J. A,, Zucker, R.J. & Nieoll, R. A. (1988) Science 2 4 2 , 8 1 4 4 Malinow, R., Sehulman, H. & Tsien, R. W. (1989) Science 246,862-866 Mcrtzen, M. R. & Slemmon. R. J. (1994) J. Neurochem., in press McLeod, M., Stein, M. & Beach, D. (1987) EMBO J. 6,729-736 Morreale de Escobar,G . ,Escobar delb y , F.& Ruiz-Marcos,A.(1983) in Congenital Hypothyroidism (Dussault, J. H. & Walker, P., eds) pp. 85-126, Marcel Dekker, New York Munoz, A,, Rodriguez-Pena, A,, Perez-Castillo,A,, Ferreiro, B., Sutcliffe, J. G. & Bernal, J. (1991) Mol. Endocrinol. 6,273-280 Nakanishi, S. (1992) Science 268,597403 Represa, A,, Deloulme, J. C., Sensenbrenner, M., Ben-Ari,Y. & Baudier, J. (1990) J. Neurosci. 10, 37823792 Saiki, R. It, Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, IC B. & Erlich, H. A. (1988) Science 239, 487491 Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.5.A. 74, 5463-5467 Silva, A. J., Paylor, R., Wehner, J. M. & lbnegawa, S. (1992) Science 267,206-211 Slemmon, R. J. & Martzen, M. R. (1994) Biochemistry 33,5653-5660 Studier, W. F.,Rosenberg, A. H., Dunn, J. J. & Dubendor6 J. W. (1990) Methods Enzymol. 186,6&88 Towbin, H.,Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S. A. 76, 435w354 Watson, J. B., Battenberg, E. F., Wong, K. K., Bloom, F. E. & Sutcliffe, J. 0. (1990) J. Neurosci. Res. 26, 397408 Watson, J. B., Sutcliffe, J. G. & Fisher, R. S. (1992) Proc. Natl. Acad. Sci. U.5.A. 89,858144585 Wojtkowiak, Z., Briggs, R. C. & Hnilica, L. S. (1983)AnaZ.Biochem. 129,486
