Cell, Vol. 68, 479-489,

February

7, 1992. Copyright

0 1992 by Cell Press

The Drosophila Learning and Memory Gene rutabaga Encodes a Ca2+/CalmoduIin-Responsive Adenylyl Cyclase Lonny R. Levin,’ Pyung-Lim Han,t Paul M. Hwang,’ Paul G. Feinstein,” Ronald L. Davis,** and Randall R. Reed,’ *Howard Hughes Medical Institute and Department of Molecular Biology and Genetics The Johns Hopkins School of Medicine Baltimore, Maryland 21205 tDivision of Neuroscience *Department of Cell Biology Baylor College of Medicine Houston, Texas 77030

Summary Four putative adenylyl cyclase genes from Drosophila melanogaster were identified by virtue of their extensive sequence homology with mammalian cyclases. One corresponds to the learning and memory gene rutabaga and is most similar to the mammalian brain Ca*+/calmodulin (CaM)-responsive cyclase. In a mammalian expression system, rutabaga cyclase activity was stimulated approximately S-fold by the presence of Ca2+/CaM. A point mutation, identified at this locus in rut7 mutant flies, resulted in loss of detectable adenylyl cyclase activity. New P element insertion-induced rutabaga mutations mapped to within 200 nucleotides of the 5’end of the rutabaga cDNA. These data confirm the identity of the rutabaga locus as the structural gene for the Ca2+/CaM-responsive adenylyl cyclase and show that the inactivation of this cyclase leads to a learning and memory defect. Introduction Adenylyl cyclase generates cyclic AMP (CAMP) in response to a variety of extracellular signals via the actions of a well characterized membrane-associated cascade. A transmembrane receptor, when activated by hormone or neurotransmitter ligand, modulates the activity of a guanine nucleotide-binding regulatory protein (G protein). The G protein interacts with adenylyl cyclase to alter the rate of CAMP synthesis. The ability of a ligand to elicit complex responses is at least in part due to multiple forms of each of the protein components (Reed, 1990; Simon et al., 1991). Differentially regulated forms of adenylyl cyclase were originally suggested by biochemical (Brostrom et al., 1977; Westcott et al., 1979; Pfeuffer et al., 1989), immunological (Rosenberg and Storm, 1987; Mollner and Pfeuffer, 1988; Pfeuffer et al., 1989), and genetic studies (Livingstone et al., 1984). Mammalian brain contains two distinct adenylyl cyclase activities, one stimulated by the addition of calciumlcalmodulin (Ca*+/CaM) (Brostrom et al., 1975; Cheung et al., 1975; Smigel, 1986) and theother unresponsive to CaM (Brostrom et al., 1977; Westcott et al., 1979). Biochemical and genetic studies in the fruit fly,

Drosophila melanogaster, also suggested the existence of Ca*+/CaM-stimulated and -insensitive forms of adenylyl cyclase (Livingstone et al., 1984; and see below). Recently, cDNA clones encoding mammalian enzymes with each of these biochemical properties have been isolated (Krupinski et al., 1989; Feinstein et al., 1991; Gao and Gilman, 1991). The forms of mammalian adenylyl cyclase characterized at the molecular level, types I, II, Ill, and IV, haveeach been expressed to high levels in eukaryotic expression systems (Bakalyar and Reed, 1990; Feinstein et al., 1991; Gao and Gilman, 1991; Krupinski et al., 1989; Tang et al., 1991). Their enzymatic activity can be modulated by receptors and G proteins, yet each exhibits distinct modes of regulation. The type I adenylyl cyclase, expressed to high levels in brain, is stimulated by C3’1CaM (Krupinski et al., 1989; Tang et al., 1991). The activity of the type II enzyme appears to be insensitive to CaM and is expressed in brain as well as several peripheral tissues (Feinstein et al., 1991). The third type of adenylyl cyclase, type Ill, is expressed in the olfactory epithelium, and its low intrinsic basal activity may be an important adaptation for mediating olfactory signal transduction (Bakalyar and Reed, 1990). The type IV isozyme is widely expressed and biochemically resembles the type II enzyme (Gao and Gilman, 1991). Structurally, these enzymes share a similar predicted structure with two clusters of transmembrane segments separating two homologous cytoplasmic domains (Krupinski et al., 1989). Interestingly, sequences within each cytoplasmic domain display significant homology with catalytic portions of guanylyl cyclases, suggesting their involvement in cyclase activity. The generation of memory in mammals through the D, dopamine receptor (Sawaguchi and Goldman-Rakic, 1991) and the sensitization of the gill-withdrawal reflex in Aplysia through the serotonin receptor (Goelet et al., 1986) are thought to require stimulation of adenylyl cyclase and regulation of CAMP levels. In Aplysia, coordination of the conditioned stimulus with the unconditioned stimulus may involve Ca*+/CaM activation of adenylyl cyclase (Abrams and Kandel, 1988). One type of long-term potentiation, a possible cellular model for learning and memory, is thought to be mediated by Ca2+ flux into the postsynaptic nerve terminal via N-methyl-D-aspartate receptors (Brown et al., 1988; Nicoll et al., 1988). Receptor-mediated activation of the phosphoinositide-specific phospholipase C and the subsequent generation of inositol 1,4,5-trisphosphate also serve to raise intracellular Ca*+ levels and may be responsible for non-N-methyl-o-aspartate receptor-type long-term potentiation (Nicoll et al., 1988). The ability of Drosophila melanogaster to learn and remember simple associative tasks has enabled the genetic identification of mutations that affect the acquisition and/ or storage of information (Dudai, 1988; Dauwalder and Davis, 1991). Flies can be effectively trained to avoid a particular odor by coupling exposure to that odor with a

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B Drosophila AC-62D Drosophila AC-76E Drosophila AC-l 2F

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rat brain G Cyclase Figure

1. Summary

of Cloned

GIHTEPVCAGVVGLKMPRYCLFGDTV~TAS~~ESN~ Drosophila

Adenylyl

Cyclase

Homologs

(A) Approximate sizes of EcoRl fragments from Drosophila genomic phage isolated using mammalian adenylyl cyclases as probes. Fragments hybridizing to the mammalian adenylyl cyclase cDNA probes indicated in the last column are shaded. Chromosomal location was determined by in situ hybridization to polytene chromsomes. (B) The predicted amino acid sequences of a portion of three of the four putative adenylyl cyclases are aligned with mammalian type I adenylyl cyclase (Krupinski et al., 1989) and rat brain guanylyl cyclase (Chinkers et al., 1989). Identities with mammalian type I cyclase are shaded.

mild electric shock (Quinn et al., 1974). These flies display a high degree of learning for 1 hr after training and show some retention for at least 24 hr (Tuliy and Quinn, 1985). Genetic screens have identified mutants that fail to avoid the trained odor (Dudai et al., 1976; Livingstone et al., 1984). Of the several mutants isolated, the molecular and resultant biochemical nature of the defect is known for only one. The dunce mutation mapped to the same locus as a gene for CAMP phosphodiesterase, and biochemical studies showed that dunce flies were deficient in this enzyme activity(Byers et al., 1981; Davis and Kiger, 1981). Subsequent cloning and expression of the duncegeneconfirmed that it encodes a Drosophila CAMP phosphodiesterase (Chen et al., 1986; Qiu et al., 1991). The identification of the biochemical activity of dunce prompted a survey of other learning and memory mutants to determine whether they might encode components of the CAMP pathway. rutabaga flies had lower levels of adenylyl cyclase activity than wild-type flies and specifically lacked a Ca’+/CaM-stimulated cyciase activity (Livingstone et al., 1984). Because these flies had normal levels of CaM, it was postulated that rutabaga encoded a Ca2+I CaM-responsive adenylyl cyclase, similar to the mammalian type I enzyme, or a subunit conferring Cd+lCaM responsiveness to a catalytic subunit of adenylyl cyciase. Alleles of rutabaga partially suppress a female sterile phenotype caused by the phosphodiesterase deficiency in dunce flies (Beilen et al., 1987; Livingstone et al., 1984).

These dunce-, fur flies have near wild-type levels of CAMP (Livingstone et al., 1984), suggesting that mutations at rutabaga compensate for elevated CAMP. Interestingly, these doubly mutant flies are still unable to learn, suggesting that memory requires complex spatial and temporal regulation of CAMP as opposed to absolute levels of CAMP. Elucidating the functional significance of the different forms of adenylyi cyclase has proven difficult in mammals. individual isoforms have distinct biochemical properties and tissue distribution; however, the in vivo relevance of these unique properties remains unclear. A genetic approach is perhaps the simplest and most convincing method of understanding the roles of the many adenyiyl cyclase isozymes. Characterization of Drosophila adenylyl cyclases may provide insight into the role of the homologous mammalian enzymes. Results fdentification of Four Adenyiyi Cyciase Genes in Drosophila meianogaster DNA fragments from bovine type I cyciase (Krupinski et al., 1989) and rat type II cyclase (Feinstein et al., 1991) were independently hybridized at low stringency to a Drosophila melanogaster Canton S genomic library (5 x lo5 recombinant phage) (Maniatis et al., 1978). A total of 30 independent, positive recombinant phage were isolated

Biochemical 481

and Genetic

Characterization

of rutabaga

suggests that these three Drosophila genes encode distinct forms of adenylyl cyclase. The analogous region of the fourth Drosophila gene was not isolated; however, sequences displaying significant homology with other regions of mammalian adenylyl cyclases suggested that it also encodes a Drosophila adenylyl cyclase (data not shown). Chromosomal Mapping of Drosophila Adenylyl Cyclase Loci DNAfragments from each locus that hybridized to a single band on a genomic Southern DNA blot were biotinylated and used for in situ hybridization to polytene salivary gland chromosomes. Threeof the loci mapped to the autosomes of Drosophila (Figure 1A) and were not pursued further. The fourth gene mapped to the X chromosome at bands 12F5-13Al (data not shown). The rutabaga locus has been mapped by deficiency complementation analysis to X chromosome bands 12F5-7 (Livingstone, 1965; Livingstone et al., 1964).

9.5 kb e 7.5 kb e

Figure 2. Northern Message

Blot Analysis

Message was detected cDNA as orobe.

by using

of Drosophila

Adenylyl

random-primer-labeled

Cyclase full-length

and purified. Individual EcoRl restriction fragments hybridizing to either of the cyclase probes were subcloned from each of the phage and back hybridized at high stringency to the other clones. The recombinants segregated into four nonoverlapping classes (Figure 1A). A restriction map was generated for each of the putative adenylyl cyclase loci by digesting the recombinant phage with enzymes other than EcoRl and DNA hybridization analysis or by the alignment of overlapping phage where available. Hybridization with the type I adenylyl cyclase identified phage belonging to only three of these classes, while type II exclusively recognized recombinant phage belonging to the fourth. This suggested the possible existence of functional homologs for type I and type II mammalian cyclases in Drosophila. Small DNA fragments that hybridized strongly with the mammalian cyclase cDNA probes were cloned and sequenced. Three of the four putative adenylyl cyclase fragments contained an open reading frame that exhibited greater than 67% identity with a conserved region within the second catalytic domain of bovine type I cyclase (Figure 1 B). Specific sequences in this region are absolutely conserved among metazoan adenylyl cyclases, but not among guanylyl cyclases (Krupinski et al., 1969). These sequences can therefore be used to differentiate between members of the cyclase superfamily, and their presence

Identification of cDNA Clones Encoding the Adenylyl Cyclase That Maps to the rutabaga Locus Genomic fragments from the adenylyl cyclase mapping in the vicinity of the rutabaga locus were used to screen a library derived from Drosophila head RNA (Papazian et al., 1987). Several overlapping cDNA clones were isolated, and nucleotide sequence analysis revealed an open reading frame that extended through an EcoRl site at the 3’ end of the longest of these clones (approximately 3.7 kb). This open reading frame was homologous to a major portion of the mammalian adenylyl cyclases (see below). Northern blot analysis of RNA isolated from Canton-S flies indicated two messages of approximately 7.5 kb and 9.5 kb (Figure 2). The discrepancy between the sizes of the cDNA clones and the messages, the lack of a termination codon, as well as an inferred incomplete amino acid sequence implied that these cDNAs resulted from insufficient methylase protection of an internal EcoRl site. Subsequent analysis revealed the existence of an EcoRl site at this position in genomic phage DNA. Nucleotide sequencing of the adjacent region extended the cyclase homology beyond this EcoRl site. Additional cDNA clones were isolated using this EcoRl fragment contained within the original genomic phage (Figure 3). These cDNA clones continued the open reading frame and extended the homology with mammalian adenylyl cyclase. The complete cDNA was reconstructed from three fragments and spanned 7545 bp. Nucleotide sequence analysis revealed an open reading frame predicted to encode a protein of 2249 amino acids (Figure 4A). The amino-terminal half of the predicted protein product appears to share the same overall structure as the previously isolated mammalian adenylyl cyclases (Bakalyar and Reed, 1990; Feinstein et al., 1991; Gao and Gilman, 1991; Krupinski et al., 1989). The hydropathy profile of the N-terminal half of the Drosophila cyclase is very similar to that of the mammalian type I enzyme and suggests a similar arrangement of twelve membrane-spanning segments

Cell 402

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r

I

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4.7

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(1.1 0.9 0.9)

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t

I

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1951 769 1084

~ Figure

3. Genomic

17612080

Organization

of rutabaga

Adenylyl

Cyclase

and Localization

of P Element

Insertions

Genomic phage spanning the rutabaga locus are indicated on top. pGE and pGRF are plasmids containing regions not isolated in any phage. The EcoRl restriction map of the rutabaga locus is shown in the middle. site described in the text. Exons corresponding to the rutabaga cDNA are indicated as boxes below the gene. open reading frame and open boxes are predicted noncoding sequences. Sites of P element insertion are direction of f3galactosidase transcription.

(Figure 48). These structurally conserved hydrophobic domains exhibit almost no amino acid sequence identity with any of the members of the adenylyl cyclase family (Figure 4A; Gao and Gilman, 1991). Two potential N-linked glycosylation sites exist in the predicted extracellular loop between membrane spans 9 and 10 at amino acid positions 800 and 807. Sites for N-linked glycosylation also exist between predicted membrane spanning regions 9 and 10 of the mammalian types I, II, III, and IV adenylyl cyclase. Krupinski et al (1989) proposed that portions of the two large cytoplasmic domains (C,, and C,J, which exhibit amino acid similarity with each other as well as with the catalytic domains of guanylyl cyclases, are responsible for catalytic activity. The primary sequence of these proposed catalytic domains is conserved between Drosophila and the mammalian adenylyl cyclases. Among the known adenylyl cyclase genes, the putative cytoplasmic C, and Cna domains of the Drosophila cyclase are most similar to the bovine type I cyclase (75% and 57% identity, respectively) (Figure 4A). Moreover, the Ca2+/CaM-stimulated adenylyl cyclase from mammalian brain (type I) shares greater homology with this Drosophila cyclase than with other characterized mammalian cyclases (Gao and Gilman, 1991). The carboxy-terminal half of the protein (starting immediately after the second putative catalytic domain) displays no obvious similarity with any region of the mammalian adenylyl cyclases nor to any sequence in the Genbank and NBRF databases. This region contains several polyglutamine stretches indicative of the OPA repeat, found within many Drosophila genes (Wharton et al., 1985). The significance of these sequence motifs is not known. Biochemical Characterization of the Adenylyl Cyclase That Maps to the rutabaga Locus Three of the mammalian adenylyl cyclases (Krupinski et al., 1989; Bakalyar and Reed, 1990; Feinstein et al., 1991)

EcoRl genomic fragments that cover The star indicates the internal EcoRl Black boxes represent the predicted indicated, with arrows indicating the

have been successfully characterized by high level expression under the control of the cytomegalovirus promoter in human kidney cell line 293 (Gorman et al., 1990). The similarity between the Drosophila adenylyl cyclase and the three mammalian cyclases suggested that it could be expressed in this system. Adenylyl cyclase activity in crude membrane preparations from cells transfected with the full-length cDNA was approximately forty-fold higher than control cells transfected with expression vector alone (Figure 5A). Forskolin, which directly activates mammalian adenylyl cyclases, stimulates this basal activity approximately 40-fold. The cyclase activity in Drosophila cyclasetransfected 293 cells was stimulated in the presence of GTPyS or AIF,- , but not GDPf3S (Figure 5B), indicating that this Drosophila adenylyl cyclase is capable of coupling to the endogenous human G, protein. Assays of crude fly homogenates detected a specific loss of the CaM-responsive cyclase activity in rut7 flies (Livingstone et al., 1984). EGTA decreased the basal cyclase activity seen in Figure 5A and Figure 58, indicating a contribution of Ca2+ to the observed activity (Figure 5C). The addition of CaM in the presence of 30 gM Ca’+ to Drosophila cyclase-transfected 293 cell membranes resulted in a 5-fold increase in activity over that seen in the presence of EGTA (Figure 5C). The adenylyl cyclase activity displayed the characteristic biphasic Ca*+ dependence (Brostrom et al., 1975). The enzyme was activated over a broad range of low Ca*+ concentrations (maximal activation between 1 and 30 nM Ca*+) but was inhibited by higher concentrations. The CaM stimulation seen in fly homogenates by Livingstone et al. (1984) was only approximately 2.5-fold and occurred between 0.1 and 1 PM. Their experiments included the contribution to basal activity from at least one other cyclase that was not CaM responsive. The difference in Ca*+ optimum may reflect differences due to the heterologous components of this expression System.

A 74

RUT bAc1

100

RUT bAC1

CGLFAWLVLLQCSVIKDHHLPTLCYGILLFTASI----CWSM--PTLGSVFP~TKE~EG~QI~WF~Y~PLQIWEAVAFGIALPS~ISL . . . ... . . .. . . . .. .. . .. . CVLFLALLWTNVRSLQVPQLQQVGQLALLFSLTFALLCCPFALGGPAGAHAGAAAVPATADQGWJQLLLLVTFVSYALLP~SLLAIGFGLWAASHLLV

..

RUT

??ryKIFTDALRYLEYNQLIANIVIFIGVNVAGLVVNIMMERAQRRTFLDTRNCIASRLEIQDENEKLERLLLSVLPQHVAMQMKNDILSPVAGQFHRIYI . . . . . . .. .. . . . ... .. .. .. .. .. . .. .. . .. . . . . .. . .. . . TA--TLVPAKRPRLWRTLGRNALLFLGVMrYGIFVRILAEEDFLKPPERIFHKIYI

. .

bAC1

168

.

200

..

.. .

268 298

QKHENVSILFADIVGF~SSQCSAQELVRLLNELFGRFDQLAHDNHCLRIKILGDCYYCVSGLPEPRKDHAKCAVEMGLDMIDAIA?lrVEATDVILNMR ..% . . . . . . . . . . . . . . . . . .. .. ... . . . . ... .. .. ... .. . . ... . ... . ... .... . . .. QRHDNVSILFADIVGFTGLASQCTAQELVKLLNELFGKFDELATENHCRRIKILGDCYYCVSGLn2~KT;r;HCCVEMGLDMIDTITS;A~~:rE;D~~

368

468

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VGIHTGRVLCGVLGLRKWQFDVWSNDVTLAMIMESGGEPGRVHVTRATLDSLSGEYEVEAGHGDERSSYLRDHGVDTFFIVPPPHRRKPLML~LGVRSA . . . .. .. ... . ... ... . ... . .. ... .. .. . .. . . . VGLHTGRVLCGVLGLRKWQYDWISNDVTLANVMEAAGLPG~I~T~~AC~N~D~~~~~~~H~~~F~KT~IE~~~~~~SHR~K---IFPG~-ILBD

RUT bAC1

IGSRRKLSFRNVSMiVMQLLHTIK-FSEPVPFSNIATGSFPSAASALGGGGVSV~GGGGGVALQKILHATPPP ;KPAKRMK~KT;ICyLLV~~~CR~~~EI~~~~--------------------------~-----~~----~----DDDK~~L~---.-------

568

RUT bAC1 RUT

398

494

542

RUT bAC1

667

RUT

NARSVDCDKSEHWRLTLRFRQSDMEREYHKDFDLGFTTAMCSLLLLILGAALQVTALPRTLILLLLFLFAFIWVSAILMLLLAVRLKWIIWDISESFS

761

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672

bAC1

575

958

RUT bAC1

RUT bAC1

866

LQEINSHSYNNFMLRVGINIGPWAGVIGARKPQYDIWGNTVNVASRS~VPGYSQVTQEVVDSLVGSHFEFRCRGTIKVKGKGDMVTYFLCDSGNKS .. . . . .. .. . ... ... ... .. .. ... ... .... ... ... ... . .. . . . . .. . LDEINYQSYNDFVLRVGINVGPWAGVIGARRPQYDIWGN'DINVASRMDSTGVQGRIQVTEEVHRL~RRGSYR~VST;~KVS;~d~~EMLT;~~EGRTDGN

1064 1258

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1158

1134

ANGGLPNIRENGNGHNGEHQQQQQQQQHQQQQQHQQQQQHGGFMVVAVPLQPQHHQLQFQHPHQHPLPSAVSVPVQHQILLHHQLQLQHQPV PSVMLREFNIIENPTSGGRHQQMEQLPPHHGSLDLSGMGMGVGAGVGGDCFMMPRRDRERTYVPPLNQHGHHPPHHLHSNLNLNQSQHPPSFTSLGYGQ CRESEPLLHASSVAPVAKIMPMQHRPKYEPPRYTSPHTMLSQQHQQQQQHQHQQPQSQSQSAQDQQTHPAQDPHPLQRQYAMYSQQPQLPPKPVLRTYMK PLPKLPTDLEESRDMSSTDDLSSRPHSPSMSSSDESYSKTTEGEGEGDEDSPRMVNGGHLHHRNGYHLPAGGLVNPLQWLYPCDIQVDPTSPVMMAHLH DFELSSTTESQGHHTNSNTTSNTQQKGDSCNSFDFQKATVGTRAGAAIATKSPFERELQRLLNESSRARCLATATTTAGAISTTDQTASNGSRELSYSLS NGKLSSANGHGVGGSGSGSGSGSGSAVGNGSGGSGSSNGNIKEITR NKNPSESSQMQTSDTESCEILHENRNQMHVLAML EMHTAKELNGSHAHHGQHHHQQPQRTHRQRPRSKELQYSHESLDGLDGAVQSQSQQRNQRYHHHHH HQQRQQQQQRYNHVQEQEERDDTEDNLADEEFEDDEEFEDD~GRD~QKRLQKSELNHKRSEVATEAGNHHDD~EEEDDDDDEEEDHRNGGREAAFL,TNGSMRG LERNVINDELKYGATHLNHQSMDSNPLESQSEWSDDDCRE~~GAES~YITDEPGLENISLLNEAGLTDAEGALSDVNSLYNAPDVDDTSVSSRASSSSR LLSLDSLSGLYDCDLDSKHELAIVNASHKISSKFGQPLSPAQQQHQQQQQQQQQQQQQHQQQQLQQNPQHTQAQSHLAPVQFQSAEELRE

1358 1458 1558 1658 1758 1858 1958 2058 2158 2248

B RUTABAGA

TYPE I Cyclase

Figure

4. Amino

Acid Sequence

of rutabaga

Adenylyl

Cyclase

and Homology

with Mammalian

Type

I Cyclase

(A) The predicted amino acid sequence is shown aligned with mammalian type I adenylyl cyclase. Identities are indicated by dots, The cytoplasmic C,, and Czs domains, as described in Tang and Gilman (1991) are overlined. The alignment was produced by the ALIGN program in the GCG software package. No attempt was made to align residues following amino acid 1152 of rutabaga with the carboxyl terminus of the type I cyclase. (E) The hydrophobicity profile of rutabaga and mammalian type I cyclases. Hydrophobicity was determined by the method of Kyte and Doolittle (1952) by the Strider DNA analysis program with a window size of 11 amino acids. Putative membrane spans are shown in black. The approximate locations of cytoplasmic C,. and Cz. domains are indicated.

Cell 484

rutabaga

vector

LUU

vector

rutabaga

-&RUT.4SAGA+ cm - + - RUTASAGA cd4 VECTOR+ ‘AM -*-VECTOR-Cd

0-I

a

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a-109

IO”

lo4

1O‘3

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[Ca’+] Figure

5. Adenylyl

Cyclase

Activity

of rutabaga

(A) Adenylyl cyclase activity was assayed in membranes of 293 cells transfected with vector alone (vector) or with the rutabaga cDNA under the control of the cytomegalovirus promoter (rutabaga). Activity was measured either in the absence (black bars) or presence (striped bars) of 100 pM forskolin. Activity is given as picomoles of CAMP formed per min per mg of total protein. Values represent averages of triplicate determinations from at least two pooled transfection plates. Standard deviations around the means are indicated. (B) Cyclase activity was measured in the presence of the G protein activators, AIF4- (20 $4 AICI, + 10 mM NaF) (thin striped bars) or 100 uM GTPTS (striped bars), or a G protein inhibitor, 100 uM GDP5S (dark striped bars). (C) Membranes prepared from vector transfected 293 cells (circles) and rutabaga transfected 293 cells (triangles) were assayed in the presence of varying amounts of Cap+ with (open symbols) or without (closed symbols) the addition of 50 kg/ml bovine CaM.

Identification of the rut1 Mutation Understanding the role of the rutabaga adenylyl cyclase in learning and memory requires knowledge of the genetic defect which leads to the rut7 phenotype. The rut7 mutation was isolated following ethyl methane sulfonate mutagenesis (Livingstone et al., 1984), which primarily induces single base changes. Southern blot analysis of genomic

DNA from homozygous rut7 and wild-type Canton-S flies did not reveal any DNA rearrangements in the rutabaga gene (data not shown). In addition, Northern blot analysis cDNA probe detected a similar with a full-length rutabaga pattern of hybridization to RNA isolated from homozygous rut7 and wild-type flies (data not shown). To identify the presumed single base change responsi-

Biochemical 485

and Genetic

Characterization

of

rutabaga

R t KE KTVGSTYMAVVGLI P TYPEI(bovine) K I KT I GSTY M AAVGLAP TYPE II (rat) KIKTIGSTYMAATGLSA TYPE III (rat) UKTIGSTYMAASGVTP RUTABAGA

Adenylyl cyclase region Cea

RIKI

RUTABAGA

Adenylyl cyclase region Cla

TYPEI(bovine) TYPE

II (rat)

TYPE

Ill (rat)

membrane-bound

guanylyl

cyclase

(rat brain)

membrane-bound

guanylyl

cyclase

(S. purpuratus)

soluble Yeast Figure

guanylyl adenylyl

cyclase cyclase

6. ldenitification

(bovine

lung)

(Saccharomyces

cerevisiae)

LGDCYYCVSGLPE RI K I LGDCY Y CVSGLTQ RIKI LEDCYYCVSGLPI RIKI LGDCYYCICGLPD KV ET I GDAYMVVSGLPV KV ET I GDAY N LVSGlP KVETVGDKYMTVSGWE EVKTEGDAFMV..AFPT

L

of the rut1 Mutation

Amino acid sequences of both proposed catalytic (Feinstein et al., 1991) rat type Ill adenylyl cyclase soluble bovine lung guanylyl cyclase (Koesling et the region surrounding GIY”‘~~ rn rutabaga adenylyl Shading indicates residues conserved in at least

domains of the bovine type I adenylyl cyclase (Krupinski et al., 1989) rat type II adenylyl cyclase (Bakalyar and Reed, 1990) as well as of the rat brain guanylyl cyclase (Chinkers et al., 1989). al., 1988) and Saccharomyces cerevisiae adenylyl cyclase (Kataoka et al., 1985) are aligned in cyclase. The arrow indicates the Gly 1026+Arg substitution found in the genomic DNA of rut1flies. seven of the twelve sequences.

ble for the rut1 phenotype, the 0.5 kb, 4.5 kb, and 1.5 kb fragments (see Figure 3) were isolated EcoRl genomic from homozygous rut7 flies as well as from the Canton-S parent strain from which the rut7 flies were derived. Comparison of the nucleotide sequences identified a single point mutation (adenine for the guanine at position 3459 in the wild-type cDNA), which would result in an arginine substituted for the glycine at amino acid 1026 (Figure 6). GIY ‘Oz6is conserved in both cytoplasmic domains of the rutabaga adenylyl cyclase as well as in the known mammalian adenylyl cyclases and guanylyl cyclases (Figure 6). The adenylyl cyclase from Saccharomyces cerevisiae, which displays very limited homology with the higher eukaryotic enzymes, encodes a conservative alanine substitution at the analogous position. The Gly’“*6 -Arg mutation was introduced into the wildtype cDNA clone by oligonucleotide-directed mutagenesis, and adenylyl cyclase activity associated with the resulting mutant cDNA was assayed in the mammalian expression system. Adenylyl cyclase activity in Gly1026-Arg transfected cells was indistinguishable from cells transfected with expression vector alone, indicating that the mutation abolished catalytic activity (Figure 7A). To confirm that no other mutations were introduced in the course of mutagenesis, the mutant gene was reverted back to the wild-type sequence by a subsequent round of oligonucleotide-directed mutagenesis. Expression of the reverted Arg 1026-+Gly gene led to a restoration of catalytic activity (Figure 7A). Western blot analysis of transfected cells, using polyclonal antisera generated against a poropen reading frame, confirmed that tion of the rutabaga

similar levels of each of the proteins were expressed (Figure 78). These data indicate that the Gly1026+Arg mutation is sufficient to cause the complete loss of cyclase activity in vitro and result in the biochemical and phenotypic defects seen in vivo (Livingstone et al., 1984). P Element insertions into the rutabaga Locus The learning and memory gene dunce is preferentially expressed in mushroom bodies, a region of the insect brain thought to be responsible for integrating and storing sensory information (Nighorn et al., 1991). In an “enhancer detector” experiment @‘Kane and Gehring, 1987), a collection of P element insertions that resulted in P-galactosidase expression in mushroom bodies was isolated (P.-L. H. and Ft. L. D., unpublished data). Cytological mapping identified the site of several of these P element insertions as the 12F region of the X chromosome, suggesting locus. Five of the they might be inserted at the rutabaga lines(178,2080,1084,769, and 1951)were tested for their ability to learn in an odor avoidance paradigm (Tully and Quinn, 1985) and for behavioral complementation with the rut7 mutation. These data shall be presented in detail elsewhere, but in summary, all five showed significantly reduced learning, and at least three (178, 2080, and 1084) failed to complement the rut7 allele (P.-L. H., L. Ft. L., Ft. R. Ft., and R. L. D., unpublished data). The inability of these P element insertion mutants to complement the rut7 allele suggests that the transposable elements disrupted the rutabaga gene. The genomic DNA sequences surrounding the P element insertion sites in the enhancer detector lines were

Cell 466

A

vector wild type mutant (Glyl026-Arg) revertant (Argl026-Gly)

basal

forskolin-stimulated

5.4 f 0.4 201.5 f 31.3 4.7 + 0.2

180.7 + 6.9 7121.6 + 904.0 205.1 + 16.6

181.7 f

5723.4 + 328.5

15.5

6

s

2?a r#+

3b&S{83> >3EO!

-200kD

4

Figure

7. Adenylyl

Cyclase

Activity

of the Gly’U6-Arg

Mutant

- 97.4 kD

Protein

(A) Membranes from 293 cells transfected with either vector alone, wild-type rutabaga, the rutabaga cDNA containing the Gly’nZB-+Arg mutation, or the mutant cDNA with the point mutation reverted to the wild-type sequence by site-directed mutagenesis were assayed in the absence or presence of 100 uM forskolin. Activity is given as picomoles of CAMP formed per min per mg of total protein. Values represent averages of triplicate determinations from at least two pooled transfection plates. Standard deviations around the means are indicated. (B) Extracts from each assay were Western blotted using rabbit ployclonal sera raised against a carboxy-terminal portion of rutabaga. Detection was by enhanced chemiluminescence.

isolated and used to determine their position relative to the cloned adenylyl cyclase. The P elements in the independently isolated lines 178 and 2080 inserted at exactly the same site, 155 nucleotides upstream from the Y-most nucleotide of the cloned adenylyl cyclase cDNA (see Figure 3). The P elements in strains 1084 and 769 inserted 5 nucleotides and 20 nucleotides, respectively, further upstream of the 178/2080 insertion site. These observations are consistent with the notion that this group of insertions is within the promoter region for the adenylyl cyclase and disrupts its expression. The observation that mutations resulting from these P element insertions are allelic to rut7 provides strong evidence that the Drosophila adenylyl cyclase described here is rutabaga. Discussion Type I and type II adenylyl cyclases are biochemically and genetically distinct enzymes that catalyze the production of the second messenger CAMP in mammalian brain. These genes were used to isolate four Drosophila adenylyl cyclase homologs. Several criteria establish that the Drosophila cyclase mapping to 12F and most similar to the type I mammalian isozyme corresponds to the learning and memory gene rutabaga. This Drosophila adenylyl cyclase cytologically maps to the rutabaga locus. Biochemical analysis of rut7 flies implicated a deficiency in a CaMactivated form of adenylyl cyclase as being responsible for

the learning defect (Livingstone et al., 1984). The activity of this cyclase is responsive to CaM, and a point mutation in this gene isolated from rut7 flies abolishes adenylyl cyclase activity. Lastly, P element insertions, characterized as new alleles of rutabaga, map to the 5’ upstream region of this cyclase. The mammalian type I adenylyl cyclase is activated by G proteins as well as by Ca2+/CaM (Tang et al., 1991) and is expressed in the regions of the brain thought to be involved in learning and memory (Xia et al., 1991; C. Glatt and S. Snyder, personal communication). The rutabaga cyclase is most closely related to the mammalian type I enzyme (Krupinski et al., 1989). Heterologous expression of rutabaga in mammalian cells indicates its activity is also responsive to activation by both G proteins and Ca2+/CaM. Additionally, P element insertions in rutabaga, as well as in situ hybridization and immunological localization, show that the rutabaga gene is expressed in the mushroom bodies of the fly brain ( P.-L. H., L. R. L., R. R. Pi., and R. L. D., unpublished data). The ability of these type I adenylyl cyclases to be activated by two pathways provides a potentially important site for the integration of second messenger systems. Previous models for learning and memory (Livingstone, 1985) that proposed distinct adenylyl cyclases, one modulated by monoamines acting through a G protein, and a second, the Ca2+/CaM-stimulated rutabaga adenylyl cyclase, appear incorrect. Current models for learning and memory

Biochemical 487

and Genetic

Characterization

of rutabaga

have posited at least one molecular component in the system to be sensitive to the spatial and temporal responses of distinct neurotransmitter pathways (Abrams and Kandel, 1988 ). The biochemical properties of the rutabaga cyclase and reduction of memory in rutabaga mutants may indicate that the rutabaga adenylyl cyclase performs this integrative function. The rutabaga adenylyl cyclase was active in a human embryonic kidney cell line and was capable of coupling to the endogenous G protein. In addition to being structurally homologous, the rutabaga cyclase is functionally conserved with its mammalian counterparts. Quan et al. (1991) have demonstrated a similar conservation of function in which Drosophila G, was capable of receptorindependent activation of the endogenous mammalian adenylyl cyclase in a mouse lymphoma cell line. The ability to interchange mammalian and Drosophila counterparts is not universal; in the same system, a mammalian receptor did not activate the Drosophila G protein. The dunce and rutabaga mutations result in qpposite changes in intracellular CAMP levels, yet each results in similar types and degrees of memory loss. Similarly, overexpression of the catalytic subunit of CAMP-dependent protein kinase or constitutive inactivation of protein kinase led to learning deficiency (Drain et al., 1991). Flies carrying the double mutation, dunce- rufabaga-, while exhibiting approximately normal levels of CAMP, still fail to learn (Livingstone et al., 1984), emphasizing the significance of precise regulation of CAMP levels. The isolation of mutations disrupting learning in Drosophila, biochemical dissection of associative learning and sensitization in Aplysia, and electrophysiological studies on higher mammals implicate CAMP and Ca2+ as important components in acquisition and memory. Experimental

Procedures

DNA Methods DNA manipulations were carried out according to Sambrook et al. (1989). Nucleotide sequencing was performed using synthetic oligonucleotide primers and the Sequenase kit (US Biochemical@ according to manufacturers instructions. The Drosophila libraries used were a randomly sheared genomic library in the cloning vector Charon 4A (Maniatis et al., 1978) and a Drosophila head cDNA library in ZAP (Papazian et al., 1987). The genomic library was screened using bovine type I adenylyl cyclase clone l-6 (Krupinski et al., 1989) or rat type II adenylyl cyclase clone 28 (Feinstein et al., 1991). Hybridizations were performed under standard conditions (Jones and Reed, 1989). Low stringency washes were at 55OC with 2x SSC. High stringency washes were performed in 0.5 x SSC at 65%. Mutagenesis was performed as described (Kuret et al., 1988). In Situ Hybridization to Polytene Chromosomes In situ hybridization of polytene salivary gland chromosomes ments from each locus was performed as previously (Langer-Safer et al., 1982).

with fragdescribed

Northern Blot Analysis RNA was isolated from Canton-S flies by guanidinium isothiocyanate extraction followed by cesium chloride ultracentrifugation (Chirgwin et al., 1979). Poly(A)+ mRNA was selected over Poly(A) Quick columns (Stratagene). Twenty micrograms of poly(A)’ RNA was size fractionated in 1% agaroselformaldehyde gels, transferred to Nytran filters, and hybridized following standard protocols (Sambrook et al., 1989).

Heterologous Expression and Adenylyl Cyclase Assay The human embryonic kidney cell line 293 was transfected with 5 ng of expression vector (pCIS) by the calcium-phosphate method (DhalIan et al., 1990; Gorman et al., 1990). Cells were collected in phospatebuffered saline and EDTA three days after transfection, sonicated for I min in lysis buffer (50 mM Tris [pH 7.5],1 mM EDTA, 1 mM dithiothreitol, 0.1 mglml Leupeptin, I mM phenylmethylsulfonyl fluoride), and pelleted at 70,000 rpm for ten min. Membranes were resuspended in lysis buffer by passing repeatedly through a 22 gauge needle. An amount equivalent to approximately 1120 of a 10 cm plate was assayed for adenylyl cyclase activity as in Salomon (1979) under the conditions described in Bakalyar and Reed (1990). CAMP production was measured after 30 min at 31%. Protein concentrations were determined by Bio-Rad protein assay kit using bovine serum albumin as standard. Ca2+/CaM responsiveness was measured in the presence or absence of 50 ug/ml CaM (Calbiochem). Ca2+ concentrationsof 1 nM and below were achieved by buffering with 1 mM EGTA (Caldwell, 1970). Identification of the rut1 Mutation Genomic DNA from homozygous rut7 flies and parental Canton-S flies (W. Quinn, personal communication) was isolated, restricted with EcoRI, cloned into ZAP (Stratagene), and packaged. Recombinant phage containing genomic fragments corresponding to the rutabaga gene were isolated by screening at high stringency with the reconstructed full-length cDNA. Cloned fragments were rescued and sequenced. Western Blot Analysis Polyclonal sera were generated against a fusion protein bearing amino acids 1131 through 16460f the rutabaga adenylyl cyclase. Gel-purified fusion protein was expressed using the GEMEX system (Promega) and injected into rabbits according to Harlow and Lane (1988). Fusion protein was induced by addition of isopropyl 5-D-thiogalactopyranoside in strain JM109[DE3]. Induced cells were lysed in T7 detergent lysis buffer (50 mM Tris [pH 8.0). 2 mM EDTA, 100 mM NaCI, 1% deoxycholate, and 1% NP-40) by sonication and insoluble protein was electrophoresed on a 6.5% SDS-polyacrylamide gel. The gel was lightly Coomassie stained and the band corresponding to 110 kd fusion protein was excised. The gel slice was pulverized, mixed with Freunds adjuvant, and injected into New Zealand white rabbits. Membrane protein (1 pg) from transfected cells was submitted to 6.5% SDS-polyacrylamide gel electrophoresis and Western blot analysis as described (Bakalyar and Reed, 1990). rutabaga protein was detected using 1:200dilutionof sera byenhancedchemiluminescence (Amersham). P Element Rescue Sequences flanking the inserted P elements were rescued (Wilson et al., 1989) by digesting total genomic DNA with either Xhol or Hindlll, circularizing the resulting fragments, and transforming bacteria to isolate the pBLUESCRlPT vectors. The sites of P element insertion were determined by direct nucleotide sequencing as well as by determination of distances between sequence-specific oligonucleotides by polymerase chain reaction. Acknowledgments We thank Chip Quinn for supplying fly stocks and Phil Beachy for assistance in maintaining them. We also thank Celeste Berg and Alan Spradling for assisting with the in situ localization. This work was supported by a grant from the National Institutes of Mental Health to R. R. R. and by a grant from the National Institutes of Health to R. L. D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

3, 1991;

revised

November

13, 1991,

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Accession

The accession M81887.

number

Number for the sequence

reported

in this

paper

is