James J. CaliS, John C. ZwaagstraSP, Nicole Monsnll, Dermot M. F. Cooperll, and ...... Largent, B. L., Jones, D. T., Reed, R. R., Pearson, C. A,, and Snyder, S. H.
THEJOURNAL OF BIOL~CICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269,No. 16, Issue ofApril 22, pp. 12190-12195, 1994 Printed in U.S.A.
Type VI11 Adenylyl Cyclase A Ca2+/CALMODULIN-STIMULATED ENZYME EXPRESSED IN DISCRETE REGIONS OF RAT BRAIN* (Received for publication, December 8, 1993, and in revised form, January 26, 1994)
James J. CaliS, John C. ZwaagstraSP, Nicole Monsnll, Dermot M. F. Cooperll, and John -pinski*** From the $Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2610, the lUaboratoire de Neurocytochimie fonctionnelle, Unite‘ de Recherche associe‘e, Centre National de la Recherche Scientifique 339, Universite de Bordeaux I, 33405 Talence Cedex, France, and the IlUniversity of Colorado Health Sciences Center, Department of Pharmacology, Denver, Colorado 80262
A cDNA that encodes type VI11 adenylyl cyclase has dance, and modes of regulation (3-12). In a recent attempt to been isolated from two rat brain libraries. The open explore the diversity in the adenylyl cyclase family, messages reading frame encodes a 1248-amino acid protein pre- for eight distinct forms, designated types I-VIII, were identidicted to have twosets of six transmembrane spans and fied in various tissuesby the polymerase chain reaction (PCR)’ two putative nucleotide binding domainsas is charac- (11). Full-length cDNAs have been reported for types I-VI (3teristic of other mammalian adenylyl cyclases. T w o type 12), and cDNA fragments from at least two other adenylyl VI11 messages are detected in rat brain with estimated cyclases have been isolated (11, 13, 14). sizes of 5.5 and 4.4kilobases. In situ hybridization indiThough some tissues appear to express only subsets of the cates that the type VI11 messages are most abundantly eight adenylyl cyclases identified thus far, all are detected in expressed in the granule cells of the dentate gyrus, the brain (11). The importance of CAMP i n the brain is underscored pyramidal cells of hippocampalfields CA1-CA3, the enby this finding and by the identification of specific neuronal torhinal cortex, andthe piriform cortex. Hybridization is also detected in the neocortex, the amygdaloid com- functions that clearly require the modulationof CAMP concentrations. Studies of classical conditioning in the marine molplex, and regions of the thalamus and hypothalamus. Stable expression of the type VI11 cDNA in human em- lusc, Aplysia (15); the Drosophila learning mutant, rutabaga in the mammalian hipbryonal kidney cells leads to the appearance of a novel (16, 17); and long-term potentiation 165-kDa glycoprotein in the membrane fraction. Stimu- pocampus (18, 19) all provide examples in which CAMP synthelation of these cells with agents that increase intracel- sis by adenylyl cyclases has been proposed to play a role in lular Ca2+results in up to 43-fold increases in CAMPac- aspects of memory formation and learning.A full understandcumulation over that of control cells transfected with ing of CAMP-dependent processes suchas these requires identhe expression vector. Addition of isoproterenol alone tification and characterizationof the distinct adenylyl cyclases does not leadto type VIII-specificeffects in intact cells. involved. Here we report the isolation of the rat type VI11 Adenylyl cyclase activity in membranes prepared from adenylyl cyclase cDNA, the localization of its expression to type VIII-transformed cells is stimulated up to 40-foldby discrete regions inrat brain, and the preliminary characterizathe addition of Ca2+/calmodulin(EC, = 53 n~ calmodu- tion of the enzymatic properties observed upon expression of lin). The addition of activated recombinant a subunit of type VI11 in human embryonal kidney cells. Possible functional G, synergistically increases the Ca2+/calmodulin-stimu- roles for this enzymeare discussed. lated activity.A possible role for typeVI11 adenylyl cyclase in long-term potentiation is discussed. EXPERIMENTALPROCEDURES cDNA Cloning-A 172-bp cDNA fragment unique to the type VI11 adenylyl cyclase message was previouslyidentified by the polymerase The synthesis of cyclic AMP (CAMP) by mammalian, mem- chain reaction (11).The PCR product was labeledby random priming to brane-bound adenylyl cyclasesis modulated by hormones and a specific activity of approximately 10’ c p d p g (20) and used to screen 6.5 x lo5 plaques from a rat brain cDNA library in A GTlO (Clontech; that coupleagonist neurotransmittersactingviareceptors oligo(dT)and random priming). Final washes were performed at 62 “C binding to regulationof the enzyme through direct activation in 1%sodium dodecyl sulfate, 1mM disodium EDTA, and 40 mM sodium by G proteins and cross-talkwith other signaling pathways (1, phosphate, pH 7.2 (20).A single type VI11 clone, encoding aminoacids 2). While adenylyl cyclase activityis ubiquitous in mammalian 344-1160 in Fig. 1, was isolated. Two fragments (240-bp 5’, and 200-bp and 3’) derived from the ends of the partial rat cDNA were used as probes tissues, it has beendemonstratedbothbiochemically through molecular cloning that various forms of the enzyme to screen 4 x lo5 plaques from a randomly primed bovine brain cDNA library in theA GTll vector (Clontech),and six overlappingbovine type exist that differ widely with regard to tissue distribution, abunVI11 clones were isolated. Two cDNA fragments encoding the bovine equivalent of amino acids 144-317 and 865-1205 were used to screen * This work was supported by National Institutes of Health Grants 3.5 x lo5 plaques from a rat brain cDNA library in the A ZAP I1 vector GM46395 (to J. K.) and GM32483 (to D. M. F. C.). The costs of publica- (Stratagene; oligo(dT) and random priming). Five overlapping clones tion of this article weredefrayed in part by the payment of page were selected from 3.5 x lo6 plaques screened. The cDNA inserts were S ’ (Stratagene) for sequencing using dyecharges. This article must therefore be hereby marked “advertisement” subcloned into Bluescript K labeled chain terminator chemistry and an automated sequenator (Apin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide seguence(s) reported in this paper has been submitted plied Biosystems).The amino acid sequencepresented in Fig. 1is based to the GenBankTMIEMBL Data Bank with accession number(s)L26986. on the nucleotide sequence determined on both strands of at least two 8 Present address: National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada. ** To whom correspondence should be addressed: Weis Center for The abbreviations used are: PCR, polymerase chain reaction; D m , Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822- dithiothreitol; HME, 20 m~ NaHepes, pH 8.0,2.0 mM MgCI,, and 1.0 m~ EDTA rGG,,,recombinant OL subunit of G,; bp, base pairb). 2610. Tel.: 717-271-8256;Fax:717-271-6701. ~
~
~~
12190
Type VIII Adenylyl Cyclase
12191
293 Cell Membrane Preparation-Membranes were prepared from stable, polyclonal293cell populations transfected withthe pCMV5-neo 56 GGGGGGSRKASNPAGSGPNHHAPQLSSDSVLPLYSLGSGE~HNTGGT~FPERS vector (control), type VIII, type I (23), or type VI (11)adenylyl cyclase expression constructs. Confluent monolayers of cells on 150-mm culture 111 GSGSASGSGGGGDLGFLHLDCAPSNSDFFLNGGYSYRGVIFPTLRNSFKSRDLER dishes were washed twice in 137 m~ NaCl, 2.7 mM KC1, 10.14 m~ 166 LYQRYFLGQRRKSEVVMNVLDVLTKLTLLVLHLSLASAPMDPLKGILLGFFTGIE Na,HPO,, 1.76 mM KH,PO, and harvestedby scraping inice-cold 40 m~ Tris-HC1,l mM EDTA, 150 m~ NaCl. All subsequent steps werea t 4 "c. 221 W I C A L V W R K D T T S H T Y L Q Y S G P G D G I G Y Y L F T L Cells were collected by centrifugation for 10 min a t 250 x g and resus276 FATYSMLPLPLTWAILAGLGTSLLOVTLOVLIPRLAVFSINQVLAOWLFMCMNT pended in 20 mM NaHepes, pH 7.5,5 m~ EDTA, 1m~ EGTA, 2 mM D?T, 200 nm sucrose, and protease inhibitors A (22 mgfliter each of L-1331 AGIFISYLSDRAQRQAFLETRRCVEARLRLRLETENQRQERLVLSVLPRFWLEMIN tosylamido-2-phenylethylchloromethyl ketone, 1-chloro-3-tosylamido386 DMTNVEDEHLQHQFHRIYIHRYENVSILFADVKGFTNLSTTLSAQELVRMLNELF 7-amino-2-heptanone, and phenylmethylsulfonyl fluoride, 3.2 mgfliter of leupeptin and lima bean trypsin inhibitor, and 0.5 mgfliter 441 A R F D R L A H E H H C L R I K I L G D C Y Y C V S G L P E P R Q R ~ H C ~ G W ~ ~ X R Feach W aprotinin). Cells were lysed by cavitation following equilibration for 20 496 R T I M D V D B R f O f R S G S V Q F D V W S W D V D I A N K L E S G G I P G R I H I S K min in 350 p.s.i. nitrogen.Nuclei and unbroken cells wereremoved by centrifugation for 7 min at 500 x g. The membranes werecollected by 551 ATLDCLSGDYNVEEGHGKERNEFLRKHNIETYLIKQPEESLLSLPEDIVKESVSC centrifugation for 30 min at 110,000 x g. The membrane pellet was 606 S D R R N S G A T F T E G S W S P E L P F D N I V G K Q N T L A A L T R N S l N Q A L H V Q S G resuspended with a motor-drivenTeflon homogenizer (10 slow strokes) in a wash buffer (20mM NaHepes, pH 7.5,2 r m EDTA, 2 mM DIT, 200 661 PEEINKRIEHTIDLRSGDKLRREHIKPFSLMFKDSSLEHKYSQMRDEVFKSNLVC nm sucrose, and protease inhibitors A), diluted 8-fold in the same buffer, 716 ~ R L M P M T I O F S I L I M L H S A L V L I T T A E D Y K C L P L I L R K and once again collected by centrifugation. The final membrane pellet was resuspended by homogenizationinaminimal volume of wash 771 TCCWINETYLARNVIIFASILINFLGAVINILWCDFDKSIPLKWLTFWSSAVFTD buffer to give a final protein concentrationof approximately 10 mg/ml 826 ICSYPEYFVFTGVLAMVTCAVFLRLNSVLKLAVLLIMIAIYALLTETIYAGLFLS a s determined by dye binding usingbovine serum albumin as standard (24). 881 YDNLWHSGEDFLGTKEASLLLMAMFLLAVFYHGQQLEYTARLDFLWRVQAKEEIN Immunochemical Analysis-A peptide with the amino acid sequence 936 EMKDLREHNENMLRNILPGHVARHFLEKDRDNEELYSQSYDAVGVMFASIPGFAD TPSGPEPGAQAEGTDKSDLP (corresponding to residues1229-1248 of type VI11 in Fig. 1)was synthesized on a n Applied Biosystems model 991 FYSQTEMNNQGVECLRLLNEIIADFDELLGEDRFQDIEKIKTIGSTY~VSGLSP 431A peptide synthesizer andcoupled to the tuberculin-purified protein 1046 EKQQCEDKWGHLCALADFSLALTESIQEINKHSFNNFELRIGISHGSWAGVIGA derivative (Ref. 23, Statens Seruminstitut, Copenhagen).New Zealand White rabbits were injected on day 0 with 0.2 mg of peptide-carrier 1101 KKPQYDIWGKTVNLASRMDSTGVSGRIQVPEETYLILKDQGFAFDYRGEIYVKGI complex in anemulsion of Freund's complete adjuvant.Boosts of 0.1 mg 1156 SEQEGKIKTYFLLGRVQPNPFILPPRRLPGQYSLAAWLGLVQSLNRQRQKQLLN of peptide-carrier in Freund's incomplete adjuvant weregiven on days 14 and 28. Rabbits were bled biweekly thereafter, and serum from the 1211 ENSNSGIIKSHYNRRTLLTPSGPEPGAQAEGTDKSDLP third bleed of rabbit D-88 was used for the blot (see Fig. 4). FIG.1. Translation of type VI11 adenylyl cyclase cDNA seSDS and D I T were added to samples (2 pg of 293 cell membrane quence. The type VI11 adenylyl cyclase protein sequence shown was protein in HME (20 mM NaHepes, pH 8.0, 2.0 m~ MgCl,, and 1.0 nm deduced from a series of overlapping cDNAs that were isolated as de- EDTA)) to final concentrations of 2% and 0.2 m ~ respectively, , before scribed under"ExperimentalProcedures."TheGenBank accession heating for 5 min at 56 "C. N-Ethylmaleimide was added t o a 50 mM number for the nucleotide sequence is L26986. The shadedsequence is final concentration before a 10-min incubation a t room temperature. encoded by the PCR product used as the initial probe in the cloning For deglycosylation of membrane proteins from type VIII-expressing strategy. The underlined sequences are predicted to form membraneassociated helices (46). Asterisks indicate the positions of potential N- cells, samples were diluted 10-fold t o a final concentration of 20 m~ linked glycosylation sites that areproposed to be on the extracellular NaPO,, pH 7.2, 1 mM EDTA, 0.2 m~ DIT, and 0.6% Nonidet P-40 and heated for 5 min at 56 "C. After addition of 0.6 units of N-glycosidase F side of the membrane. (Boehringer Mannheim) and protease inhibitorsA, these samples were incubated for 1 h a t 37 "C. The deglycosylated samples were precipitated with 10% trichloroacetic acid, washed in ice-cold acetone, dried, independent clones. A full-length type VI11 cDNA was assembled in and resuspended in HME, 2% SDS, and 0.2m~ DIT. A glycerol-loading Bluescript by ligation of convenient restriction fragments of the first partial ratcDNA and two of the final ratclones. dye was added to all samplesbefore electrophoresis on 7.5% polyacrylNorthern Blotting-Total RNA waspreparedfromadultmale amide gels. Contents of gels were electrophoretically transferred to Sprague-Dawley rat brains andpoly(A)+-enrichedusing oligo(dT) cellunitrocellulose membranes with a Bio-Rad semi-dry blotting apparatus lose (Pharmacia LKB Biotechnology Inc.) as described (20).5 pg of the (45 min, 15 V). A 50,000-fold dilution of the D88 antiserum and enRNA was size fractionated on a 0.8% agarose/formaldehyde gel and hanced chemiluminescence (Amersham Corp.) were utilized for detectransferred to a Duralon W nylon membrane (Stratagene) by capillary tion of immune complexes (manufacturer's protocol). action (20).An antisense riboprobe (Promega System11) to nucleotides Cyclic AMP Accumulation-293 cells stably expressing the cDNA encoding amino acids 572-1151 (Fig. 1)was transcribed by T3 polym- constructs were preparedfor measurement of CAMPaccumulation and erase from a linearized typeVI11 cDNA construct in Bluescript. Prehy- analyzed a s described (11). Final incubations were for 7 min a t 37 "C bridization, hybridization, washing,and message size estimations were after additionof the indicated agents (Table I). To examine the response as described (11). to ATP in theabsence of extracellular Ca", cells were washed twice in In Situ Hybridization-Corona1 sections (30 pm) of male SpragueCa2+-free medium (0.25 PM Fe(NO,),, 5.36 m~ KCl, 0.81 nm MgSO,, 109 Dawley rat brains were prepared for in situ hybridization analysisas nm NaCl, 0.78mM Na,HPO,, 25 m~ D-glucose, and 25 m~ NaHepes, pH described (21). The probe ( T G A C G I T G C G G G C C A G T C T C G T - 7.4) and then incubated for 20 min a t 37 "C before additions were made TAATCCAACAACAGG'ITITGCGG) is anantisense oligonucleotide (Table I). In this case, cells were also incubated in the same medium with a sequence complementary to the cDNA encoding amino acids supplemented with 1.8 mM CaCl, for comparison. Reported values are 768-785 of the type VI11 protein (Fig. 1).Following hybridization (21), mean 2 S.D. ( n = 6). sections were washed sequentially in 4 x SSC (1 x SSC: 0.15 M NaC1, Assay of Adenylyl Cyclase-The adenylyl cyclase assay mixture was 0.015 M sodium citrate; 15 min, room temperature), 1 x SSC (15 min, as previously described except that 10 nm MgCl, was substituted for room temperature), 1 x SSC (1 h, 37 "C), and 1 x SSC (1 h, room MnC1, and [a-32P]ATPwas omitted (25). Assays were performed for 10 temperature). Sections were mounted (21) exposed and t o 8-max hyper- min at 30 "C using 5 pg of membrane protein before reactions were film (Amersham Corp.)for 2 days at room temperature. stopped by adding a 10-fold excess of 0.1 N HCl and 1.1m~ EDTA. Total Expression of n p e VIII Adenylyl Cyclase-The full-length typeVI11 CAMP was measured by an automated radioimmunoassay (26), and cDNA was subcloned into thepCMV5-neo vector (11)for expression in synthesis was linear with respect to time and amount of membrane 293 human embryonal kidney cells (ATCC CRL 1573). Thevector alone protein added. Assays with indicated concentrations of calmodulin (Calwas used asa control. 3 pg of the test construct and 7 pgof Bluescript biochem) were performed in the presence of 100 p~ EGTA, plus or plasmid carrier DNA were used to transfect the cells by calcium phos- minus 250 p~ CaCl,, to give 17 p~ free Ca" in thereaction mixture as phate co-precipitation (22). Tissue culture and selection of antibiotic determined with the program, EQCAL (Biosoft). Purified r G s a was a (geneticid-resistant, polyclonal populations of cells were performedas generous gift fromDr. Michael P. Graziano (Merck, Sharp, and Dohme described (11). Research). The protein had been purified to apparent homogeneity after 1 MELSDVHCLSGSEELYTIHPTPPMDGGSGSRPQRLLWQTAV~ITEQRFIHGHR
Type VIII Adenylyl Cyclase
12192
TABLEI CAMPaccumulation in 293 cell populations stably expressingtype VIII adenylyl cyclase a 7-min incubation CAMPaccumulation, given as the mean S.D. ( n= 6), was determined a s described under “Experimental Procedures,” after with the indicated additions. Ro 20-1724 a t a final concentration of 100 lyrl was always included. Other additions applied individually or in a medium combinations wereas follows: 50 p~ ATP, 1 p~isoproterenol (INE), 10 p~ A23187. ATP minus CaCI, values were determined in defined that lacked CaCI, (see “Experimental Procedures”). When CaCI, was addedback to this medium and ATP-stimulated accumulation was measured, values were 1.1k 0.4 nmoVmg protein for type VIIU293 cells, and 0.02 k 0.002 nmoVmg protein for vector-transformed controls. Expression vector
CAMP Basal
ATP
ATP minus CaCI,
Vector
0.01 -c 0.002
0.03 2 0.006
0.02 2 0.006
Type VI11
0.23 2 0.12
2.0 2 0.3
0.05 = 0.03
A23187
INE
INE and ATP
INE and A23187
0.110 -c 0.03
0.1 -c 0.03
nrnollrng protein
0.04
f
0.004
10 f 1.6
0.11 0.005 0.3 -c 0.1
2.4 -c 0.5
expression in Escherichia coli (27). Assays including rG,, substituted MgSO, for MgCI,. G r,was activated with GTPyS as described (23) , prior to addition to the assay mixtures. The fraction of active a,was approximately 60%a s assessed by the stoichiometry of GTPyS binding measured in each experiment (28).
-9.5 Kb -7.5 Kb
RESULTS
Cloning of Type VZZZ Adenylyl Cyclase-A 172-bp fragment unique to the type VI11 adenylyl cyclase message was previously identified by PCR using guinea pig brain cDNA as template (11).The type VI11 PCR fragment was the initial probe used in a cloning strategy that resulted in theselection of six overlapping rat cDNAs covering a total of 4601 bp, and encoding a protein of 1248 amino acids (Fig. 1).The shaded amino acid sequence in Fig. 1 is identical to that encoded by the guinea pig PCR probe, and thecorresponding cDNA sequences were 88% identical (data not shown), consistent with a high degree of conservationacross species. The first methionine codon in the open reading frame is in a context that is in reasonable agreement with the consensus for a eukaryotic ribosome binding site (29), and each reading frame contains a t least three stop codons upstream of this putative initiatorATG (data not shown). The type VI11 adenylyl cyclase protein conforms well to the topography typical of the cloned, membrane-bound, mammalian adenylyl cyclases (3-12), including the arrangement of potential transmembrane spans (underlined inFig. 1)and the putative nucleotide binding domains (3,30). The amino-terminal cytoplasmic domain of type VI11 is most similar tothose of types V and VI in that they share short stretches of amino acid homology and they are long (179 amino acids for type VIII) compared with those of other mammalian adenylyl cyclases. Amino acids 814, 818, and 885 are predicted to be potential N-linked glycosylation sites located on the extracellularside of the membrane. The carboxyl-terminal cytoplasmic domain of type VI11 has a tail that extendsapproximately 60 amino acids beyond the region of homology shared with the guanylyl cyclases, making it most like type I in this regard (3). Painvise comparison of the complete amino acid sequences of the published mammalian adenylyl cyclase sequences indicates that the type VI11 protein is most closely related to eithertype V or VI (Ref. 31, about 42% identical), with most of the sequence similarity clustered in the putative nucleotide binding regions. Expression of Type VZZZ Message-Using a PCR approach, expression of the type VI11 message was detected in rat and guinea pig brain but not in heart, kidney, liver, testes, or skeletal muscle (11).In Fig. 2, poly(A)+-enrichedrat brain RNA was analyzed by high stringency blot hybridization using an antisense type VI11 riboprobe. Two distinct type VI11 messages of 5.5 and 4.4 kilobases are observed (Fig. 2). Consistent with the PCR results (111, no specific signals could be detected in rat heart, kidney, or liver poly(A)+ RNA,which were also examined by blot hybridization (data notshown). While the 4601-bp cDNA is exceeded in length by the 5.5-kilobase message, the
11-c 1
-4.4 Kb
-2.4 Kb
- 1.4 Kb
FIG.2. Blot hybridization analysis of type VIJJ adenylyl cyclase mRNA. 5 pg of poly(A)f-enriched RNA from rat brain was size fractionated by agarose gel electrophoresis and transferred to a nylon membrane. A 1741-nucleotide32P-labeled riboprobe, complementary to the cDNA sequence encoding amino acids 572-1151 of the type VI11 protein, was used to screen the membrane as described under “Experimental Procedures.” The membrane was exposed to Kodak XAR-5 film for 8 h. The relative migrationof RNA size standards isindicated.
cDNA could be incomplete at either end,since no attempt was made to find longer clones once the open reading frame was complete. A poly(A) addition sequence (AATAAA) is present, but not utilized, in the 3“untranslated region of the cDNAs cloned (data not shown), suggesting thepossibility of multiple transcripts. Though shorter than the reported cDNA, the 4.4kilobase mRNA is still of sufficient length to encode the 1248amino acid type VI11 protein. Type VIII-specific antisense oligonucleotides were hybridized to sections of rat brain to furtherlocalize the expression of this message (Fig. 3). Type VI11 adenylyl cyclase, like type I(21, 321, is expressed in several regionsof the brainassociated with learning and memory. The strongest hybridization signals are detected in thepyramidal cell layers of the CA2 and CA3 fields of the hippocampal formationand the granule cell layers of the dentate gyms.Somewhat less intense signals are also observed in the pyramidal cells of the CA1 field, while no signals are detected in the molecular layer. Moderate levels of type VI11 mRNA are found in the neocortex, with no significant differences in the distribution pattern throughout layers 11-VI. The ventral part of the neocortex, including the entorhinal and
Cyclase
Type VIII Ade nylyl
12193 NGaseF:
U
U
Membranes:
c
I VVVI I1I111V1 I
U
U
-
+
-
“
I
-205 koa
-117 koa
-80 koa
FIG.4. Immunoblotting of membranes from 293 cells expressing type VI11 adenylyl cyclase. Antiserum D88 was raised against a
FIG.3. Expression of type VI11 adenylyl cyclase message within the rat brain. Coronal sections were hybridized to a “‘S-labeled oligonucleotide probe complementary to the cDNA encoding type V I 1 1 amino acids 768-785 a s described under “Experimental Procedures.” The autoradiogram shows type VI11 adenylyl cyclase mRNA expression in pyramidal cells of the CA1-CAS fields of the hippocampus (CA); granule cells of the dentate gyrus ( D G ) ;regions of the neocortex (Cr)including the entorhinal cortex (En), amygdaloid complex ( A m ) , and piriform cortex (Pir);the thalamus (2‘); hypothalamus ( H y p ) ;and zona inserta (Zi).
peptide with the sequence of the last 20 amino acids of rat type VI11 adenylyl cyclase. Plasmid constructs stably expressed in the cell populations analyzed were the pCMV5-neo vector ( C ) ,type I adenylyl cyclase ( I , Ref. 23), type VI adenylyl cyclase (VI, Ref. ll),and type V I 1 1 adenylyl cyclase (VZII). 2 pg of membranes from cells expressing type V I 1 1 were subjected to an additional denaturation protocol and a I-h incubation at 37 “C with (+) or without (-) N-glycosidase F prior to precipitation and preparation for electrophoresis (see “Experimental Procedures”). Untreated membranes ( U )did not receive any of the additional manipulations required for treatment with N-glycosidase F. Relative migration of molecular weight standards is indicated.
cells with 50 p~ ATP indicates that peak intracellular Ca2+ concentrations are approximately 400 nM, and theseeffects are piriform cortex and the amygdaloid complex, give strong hy- insensitive to adenosine deaminase (indicating no role for P, bridization signals (Fig. 3). Positive hybridization is also ob- receptors in the elevation of intracellular Ca2+concentrations served in several parts of the thalamus and hypothalamus. Type (data not shown)).Stimulation of type VIIV293 cells with 50pv VI11 expression inthe cerebellum is low and can only be detected ATP results ina 9-fold increase in CAMPover the basal level. If at the level of the granulecell layer after prolonged exposure, the extracellular medium is prepared without Ca2+,cAMP acand no signal above background is seen in themolecular layer cumulation in thepresence ofATPis reduced to approximately (Table I). Addi(additional data not shown).The probe used in Fig. 3 is a 50-base 5% of the value obtained when Ca2+ is present oligonucleotide antisense to the region encoding amino acids tion of 10 VM A23187, a calcium ionophore, increases CAMP 768-785, but an indistinguishable labeling pattern was ob- accumulation 43-fold over basal in the cells expressing type tained with a completely independent 50-mer using sequence VI11 (Table I). The measurements with ATP and A23187 are antisense to the cDNA encoding amino acids 717-733 (data not consistent with the interpretation that aninflux of extracellushown). Consistent signals from two independent oligonucle- lar Ca2+ canstimulate type VI11 adenylyl cyclase, analagous to otide probes provide strong, positive evidence that the signals the Ca2+-dependentstimulation of type I (33). cAMP accumulation in type VIIV293 cells is not stimulated significantly by observed are specific to the typeVI11 message. Expression of the Type VIII Adenylyl Cyclase cDNA in 293 the p-adrenergic agonist, isoproterenol, relative to what isobCells-The type VI11 adenylyl cyclase cDNA was placed under served for the sum of the basal activity of the type VI11 cells the control of the humancytomegalovirus promoter and trans- plus the isoproterenol-stimulated activity of the vector-transfected into 293 human embryonal kidney cells to examine the formed cells (Table I). Isoproterenol (1p ~ does ) enhance the properties of the expressed enzyme. Polyclonal populations of effects of either ATP or A23187 on CAMPaccumulation in type 293 cells, which had stably incorporated the expression con- VIIV293 cells even though it has no additional effect on the struct into their genomes, were selected with the antibiotic control population, suggesting Ca2+may potentiate activation geneticin. A diffuse 165-kDa band is observed in immunoblots of type VI11 by G,. However, measurements of fura-2 fluoresof membranes preparedfrom the cells transfected with the type cence indicate that intracellular Ca2+ concentrations are inVI11 construct but not in those preparedfrom cells transfected creased to approximately 600 nM when isoproterenol is added with the expression vector alone or with typeI or VI expression with ATP (data not shown), implying that enhanced Ca2+-deconstructs (Fig. 4). The apparentmolecular mass of the immu- pendent stimulation could also account for this effect. Adenylyl cyclase activity in membranes isolated from type noreactive proteinis reduced to 125kDa following treatment of the membranes with N-glycosidase F indicating a t least one VIIV293 cells is increased in a concentration-dependent manpotential N-linked glycosylation site isutilized in an intactcell ner by the addition of exogenous Ca2+/calmodulin;membranes (Fig. 4). prepared from vector-transformed cells are insensitive to this Cyclic AMP accumulation in response to hormonal stimuli treatment (Fig. 5A). Fitting the type VI11 data gives a calcuhas been measured in the stable polyclonal293 cell populations lated EC,, value of 53 nM for calmodulin and a maximal activity overexpressing the typeVI11 adenylyl cyclase cDNA. Inhibition of just over 4 nmol/min/mg of protein. The latter value is apof cAMP phosphodiesterases with Ro 20-1724 results in a 23- proximately 600-fold higher than the activity of the membranes fold increase in basal cAMP accumulation in type VIIV293 cells prepared from the vector-transformed cells. Chelation of free relative to the vectod293 control cells, indicating that typeVI11 calcium with EGTAcompletely eliminates theeffects of calmodadenylyl cyclase has significant basal activity (Table I). TheP2 ulin on the membranes expressingtype VI11 protein (Fig. 5A, purinergic agonist, ATP, induces a biphasic increase in intra- open squares). The addition of 1 PM exogenous calmodulin incellular Ca2+ concentrationsin 293 cells with a fast component creases the type VI11 activity 10-fold relative to the activity that isunaffected by EGTA and a slow component that iselimi- measured without exogenous calmodulin, although this maxinated by chelation of extracellular Ca2+. Calibration of the mal stimulation is apparently increased to 40-fold if it is calchanges in fura-2 fluorescence following stimulation of 293 culated relative to the constant type VI11 activity measured
12194
Type VIII Adenylyl Cyclase DISCUSSION
A
30 n
.
B
a 'G 25c.
.9
15 -
0
10
100
[Calmodulin] (nM) FIG.5. Effects of calmodulin (A) and calmodulin plus GTPyS.rG,, ( B ) on type VI11 adenylyl cyclase in 293 cell membranes. Membranes (5 pg of protein) prepared from 293 cells stably transformed with the pCMV5-neo vector (0and A) or the type VI11 adenylyl cyclase expression construct (W and 0) were assayed for 10 min as described under "Experimental Procedures."A , increasing amounts of calmodulin were added to samples in the presence (W and 0)or absence (0and A) of 17 p~ free Ca2+.E , assays were performed with varying amounts of calmodulin in the presence of 17 p~ free Ca2+plus (W and 0)or minus (0and A) 40 n~ GTPyS.rG O1. The GTPyS.rG,, was approximately 60% active as determined by [3'SlGTPyS binding (28). Values are means (n = 4) with standard deviations indicated by the error bars except where the error is smaller than the size of the points.
when Ca2+is chelated with EGTA (Fig. 5 A ) . This discrepancy can be explained if endogenous calmodulinfrom the 293 cells is not completely removed during the preparation of the membranes, and low nanomolar concentrations of the endogenous regulator can stimulate typeVI11 when free Ca2+is present. Addition of both activated rG,, and Ca2+/calmodulinto membranes expressing type VI11 results in synergistic increases in adenylyl cyclase activity (Fig. 5B).A subsaturating dose of 40 nM rG,;GTPyS alone increases type VI11 adenylyl cyclase activity &fold to approximately 1.1 f 0.1 nmol/min/mg of protein, while 90 I"calmodulin alone stimulates the activity to 1.7 2 0.1 nmol/min/mg of protein. When both regulatory molecules are added together to the membranes, theobserved activity is 24.3 1.3 nmol/min/mg of protein or 8.7-fold higher than the sum of the activities observed when the activators are added separately (Fig. 5B 1. The calculated EC,, for activation by calmodulin (44 nM, under these conditions) is not affected significantly by the presence of rGs,.
The enzymatic properties of type VI11 adenylyl cyclase are most closely related tothose of type I, despite the fact that the two proteins areonly 38%identical at the aminoacid level. The most striking characteristicof both of these adenylyl cyclases is their ability to be stimulated by Ca2+/calmodulin(Ref. 23, Fig. 5). The EC,, for activation by calmodulin is about 20 I"for type I and 50 I"for type VIII, implying that theenzymes have the potential to be fully activated at the micromolar concentrations of calmodulin that exist in neurons(34). The purinergic receptor-mediated increases in CAMP accumulation in type VIIIexpressing cells (Table I) indicate that intracellular Ca2+concentrations of about 400 I"can cause a robust stimulation of the enzyme. Therefore, type VI11 should be potently activated by 1 p~ free Ca2+concentrations that can be achieved in CA1 neurons following agonist-dependent stimulation of Ca2+ influx gated by the N-methy1-D-aspartate receptor (35). The inability of type VIIU293 cells to respond to isoproterenol with increases inCAMPaccumulation is notable given that the additionof recombinant G,, clearly stimulates theadenylyl cyclase activity in membrane preparations (Fig. 5B).Similar paradoxical observations have been made for type I (5,8, 23, 36). However, this does not appear to be an artifact of the expression system, since other adenylyl cyclases can be stimulated significantly through G, activation when they are expressed in 293 cells (5, 8, 11, 36). The results of membrane assays imply that type VI11 can functionally couple to G,, and that stimulationby both Ca2+/calmodulinand G,, is synergistic (Fig. 5B).Further analysisof subcellular localization and regulation by G protein subunits will be required to resolve these discrepancies. It seems very likely that the typeVI11 adenylyl cyclase described here is the full-length rat homolog of a partial human form which has been cloned (131, since the proteinsencoded by these cDNAs are 98% identical over the 675 amino acids they have in common. Approximately 1000 amino acids of bovine 95%identical to the type VI11 (data notshown) are greater than corresponding rat sequence indicating that the typeVI11 protein is highly conserved across these species, presumably to maintain an importantfunction. In contrast, type VI11 is only about 42% identical to the most closely related rat adenylyl cyclases, types V and VI, implying that the sequence differences among the members of this enzyme family have been maintained to preserve distinct functions. The pattern of expression for the type VI11 message within the rat brain (Fig. 3) is distinct from that of any one adenylyl cyclase previously examined (12,21,32).For example, type I is not detected in the hypothalamus (21,32)and theexpression of type VI11 message there may explain the fact that hypothalamic plasma membranes display a robust Ca2+/calmodulinstimulated adenylyl cyclase activity (data notshown). The cerebellum represents a region in which little expression of the type VI11 message could be detected with two independent rat antisense oligonucleotides, while type I and I1 messages are abundant (12, 21, 32, and data not shown). This is in contrast to a previous report inwhich a 2-kilobase riboprobe transcribed from a partial human typeVI11 cDNA was utilized to examine message distribution inrat brain, and strong hybridization was detected in the cerebellum (37). Cross-hybridization may have occurred in those experiments because of the reduced stringency conditions utilized to compensate for species differences. Type VI11 expression, like that of types I and 11, is most abundant in thehippocampus, a region functionally associated with the formation of new memories. Type VIII message is detected in allpyramidal cells of CA1-CA3 fields and the granule cells of the dentategyrus, whereas typeI is most abundant
Type Cyclase VIII Adenylyl in the dentate gyrus and the CA2 fields (12, 21, 32). The distinct pattern of type I and VI11 messages in the CA fields suggests that these two adenylyl cyclases may have specific, but complementary, physiological functions in the hippocampus. Type I message is more abundant overall than thatof type VIII; however, there is no simple method to correlate message to protein abundance when comparing across types. For example G,, message is the most abundant G protein message observed in brain (381, but at the protein level Go, is at least 10-fold more abundant comprising approximately 1%of brain membrane protein (39). Quantitative immunohistochemistry will be required to determine the relative protein levels and subcellular localization of these proteins within neurons. A physiological role for Ca'Ycalmodulin-sensitive adenylyl cyclases has been proposed in a variety of animal models of learning and memory. One example is classical conditioning in the marine mollusc, Aplysia, in which a Ca2+/calmodulin-sensitive adenylyl cyclase has been proposed as the point of convergence forthe inputsfrom both conditioned (Ca''-dependent) and unconditioned (G,-dependent) stimuli (15).Unfortunately the relative sequence homology between these adenylyl cyclases cannot be reported since a cDNA clone encoding the Aplysia enzyme has not yet been described. The study of the Drosophila mutant, rutabaga, provides a second example. This mutant demonstrates impaired learning as assessed by an olfactory discrimination test and aselective loss of Ca2+/calmodulin-stimulated adenylyl cyclase activity in enzymatic assays (16). The product of the rutabaga gene is a protein of 2249 amino acids, and the first 1151 amino acids are predicted to have the topographical hallmarks of a mammalian adenylyl cyclase (17). Pairwise comparison of amino acid sequences indicates that thefirst half of the rutabaga gene product is 47% identical (67% similar) to bovine type I and 43% identical (64% similar) to rat type VI11 (31). Thus, the Drosophila protein is slightly more closely related to both the mammalian types I and VI11 than the latter two proteins are t o each other. The best studied synaptic model of memory in mammals is thatof longterm potentiation in the hippocampus (40, 41). Within field CA1, this phenomenon is dependent on the class of glutamate receptors that can also be activated by N-methyl-D-aspartate (42). The channel gated by this receptor is permeable to Ca2+ (43, 441, and activation of the receptor by either glutamate or N-methyla-aspartate results in the stimulation of a Ca'+/calmodulin-dependent adenylyl cyclase activity and increases in CAMPwithin field CA1(18,45). Both type VI11 and type I (21, 23, 32, 33) could contribute to this step in the development of long-term potentiation in the hippocampus, although the sequence differencesbetween the two proteins suggest that crosstalk with other signaling pathways may define more specific roles for each of these enzymes. The increases in CAMP following Ca2+influx would be expected to lead to activation of CAMPdependent protein kinase and subsequent phosphorylation of transcription factors that would modify gene expression and result inlong-term synaptic changes (19). Both the localization of type VI11 expression and the functional properties of the enzyme imply that it iswell suited to contribute to the development of long-term potentiation in themammalian hippocampus, although genetic knockout experiments will berequired t o determine the precise role of adenylyl cyclases in this process.
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