Proc. Nail. Acad. Sci. USA Vol. 88, pp. 10178-10182, November 1991 Biochemistry
Cloning and expression of a widely distributed (type IV) adenylyl cyclase (cyclic AMP/guanine nudeotide-binding protein/signal transduction)
BONING GAO AND ALFRED G. GILMAN Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235
Contributed by Alfred G. Gilman, August 14, 1991
We have cloned and expressed a cDNA that ABSTRACT encodes a widely distributed form of mammalian adenylyl cyclase (EC 4.6.1.1). Although those adenylyl cyclases described previously have a rather narrow tissue distribution, this enzyme (type IV) is apparently synthesized in a variety of peripheral tissues and in the central nervous system. The protein resembles the other adenylyl cyclases in its proposed structure. It most resembles the type II adenylyl cyclase described in the preceding paper [Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Nall. Acad. Sci. USA 88, 10173-10177] in its amino acid sequence, lack of response to calmodulin, and synergistic activation by a combination of the Gs a subunit and the G-protein By subunit complex.
Intracellular concentrations of cAMP are regulated by membrane-bound, multiprotein complexes of hormone receptors, heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), and adenylyl cyclases (1, 2). Hormones that stimulate cAMP accumulation bind to appropriate receptors in the plasma membrane and activate a G protein, Gs, by catalyzing exchange of GDP for GTP on the G-protein a subunit; Gs, in turn, activates adenylyl cyclase. Both calmodulin-sensitive and calmodulin-insensitive forms of adenylyl cyclase have been identified in brain and other tissues (3-6), and a soluble, calmodulin-sensitive (but G-proteininsensitive) adenylyl cyclase has been found in sperm (7). Molecular cloning has permitted characterization of a brainspecific, calmodulin-activated adenylyl cyclase (type I) (8) and a calmodulin-insensitive enzyme (type II) that is found largely in brain but is also expressed in lung (9). An olfactoryspecific, calmodulin-activated adenylyl cyclase (type III) has been identified as well (10). We describe herein the cloning and characterization of a type IV adenylyl cyclase.* It is insensitive to calmodulin and appears to be distributed more widely than the enzymes described previously. All of the mammalian adenylyl cyclases that have been cloned share unusual structural features, including two large hydrophobic domains, each with multiple putative transmembrane helices, and two roughly 40-kDa cytoplasmic domains. Portions ofthe cytoplasmic domains are homologous to each other and are conserved within the members of the adenylyl cyclase and guanylyl cyclase families.
cyclase (8). Hybridization was performed at 500C in S x SSPE (0.9 M NaCl/50 mM sodium phosphate, pH 7.4/5 mM EDTA)/5x Denhardt's solution/0.1% SDS containing calf thymus DNA (100 jig/ml) (11). Filters were washed with 5 x SSC (0.75 M NaCl/75 mM sodium citrate, pH 7)/0.1% SDS at 500C. Positive inserts were released from the vector by digestion with EcoRI and were subcloned into phage vector M13mpl9. Dideoxynucleotide sequencing was performed with a modified T7 DNA polymerase (Sequenase, United States Biochemical). Northern Blot Analysis. Total RNA was purified and poly(A)+ RNA was selected using an mRNA separator as suggested by the supplier (Clontech). Poly(A)+ RNA was electrophoresed through formaldehyde/agarose gels (11) and transferred to nylon membranes. A full-length type IV adenylyl cyclase cDNA was inserted into the EcoRI site of plasmid pBluescript SK(-) (Stratagene), and the plasmid was linearized at the Hind III site 286 base pairs from the 5' end of the insert. T3 RNA polymerase was then utilized to make a full-length, type IV adenylyl cyclase RNA probe. Hybridization was performed at 650C with 50%o formamide/5x SSC/0.1 M sodium phosphate, pH 7/5x Denhardt's solution/0.1% SDS containing calf thymus DNA (100 ,ug/ml). Filters were washed at 650C with 0.1x SSC/0.1% SDS. Calf liver rRNA (Pharmacia) and a 0.24- to 9.5-kilobase RNA "ladder" (Bethesda Research Laboratories) were used as RNA molecular size standards. Calculated molecular sizes were the average of three experiments. Polymerase Chain Reaction. A GeneAmp RNA polymerase chain reaction kit (Perkin-Elmer/Cetus) was used for all reactions. Two DNA primers specific for type IV adenylyl cyclase were utilized-BG45 (5 '-GCTTGGGTCTGAGGTCA; corresponding to amino acid residues 55-60) and BG35 (5'-AGCCAGCCTACCCAGGTT; nucleotides -86 to -103). The thermal profile used was the same as that for the positive control in the kit, except that the annealing temperature was 70'C instead of 600C. A type IV adenylyl cyclasespecific probe corresponding to amino acid residues 1-15 was used to identify the product (5'-GATCTTCGCTGGGAG-
GAGGCCGGGGACTGAAGAGGCGGGCCATGGATCCA). Hybridization was at 600C in 5 x SSPE/5 x Denhardt's solution/0.2% SDS containing calf thymus DNA
CAGCTCATCAAAGTCAGCAATGATCTCATT-3') (see Fig. 1), which encodes amino acids 901-916 oftype I adenylyl
(100 Ag/ml). Antibodies and Western Blot Analysis. Two peptides, ACIV-1 and ACIV-2, with sequence specific for type IV adenylyl cyclase were synthesized for generation of antibodies. The ACIV-1 sequence is located in the second large cytoplasmic domain (residues 927-940; see Fig. 1) and that for ACIV-2 is at the carboxyl terminus of the protein (residues 1047-1064). Peptides were coupled to the purified protein derivative of tuberculin (PPD), and rabbits were immunized with the coupled peptide as described (12). Prior
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: GTP[yS], guanosine 5'-[y-thio]triphosphate. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M80633).
MATERIALS AND METHODS cDNA Cloning and Sequencing. An oligo(dT)-primed rat testis Agtll cDNA library (Clontech) was screened with oligonucleotide 31C (5'-TTGTAGAAGTCCTTGTCCAT-
10178
Biochemistry: Gao and Gilman
Proc. Natl. Acad. Sci. USA 88 (1991)
cggggcagccagcctacccaggttccctcctggagcctagctttgctac gzigacic tec: .ggagcttaacacaaagcaggcaggggctccgggctggggcgggggatatc
MARLFSPRPPPSEDLFYETYYSLSQOYPLLILLLVIVLCAIVALPAVAWASGRELTSDPS
60
FLITTVLCALGGFSLLLGLASREOQLQRWTRPLSGL;IWAALLALGYGFLFTGGWSAWQV 120 SFFLFI IFTVYAMLPLGMRDAAGOVSSLSHLLVLGLYLGWRPESORDLLPOLAANAVL 180 FLCGNVVGAYHKALMERALRATFREALSSLHSRRRLDTEKKHOEHLLLSI LPAYLAREMK AE IMARLOAGQSSRPENTNNFHSLYVKRHQGVSVLYADIVGFTRLASECSPKELVLMLNE FGKFDOIAKEHECMRIKILGDCYYCVSGLPLSLPDHAINCVRMGLDMCRAIRKLRVATG AYAVERADMEHRDPYLRELGEPTYLVIDPWAEEEDEKGTERGLLSSLEGHTMRPSLLMTR .L:SWAA; F.F AHt SHVDSPASTSTPLPEKAFSPOWSLDRSRTPRGL HDELDTGDAKFFO
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600
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660 720 780 840
VALGTATILLVFTMAVVSLLFLPVSSDCPFLAFGVSSVAF®DTSWELPASLPL1SIPYSMH CCVLGFLSCSLFLHMSFELKLLLLLLWLVASCSLFLHSHAWLSDCLIARLYQGSLGSRPG LKL K LMtiA IYF FIlFF FTt LLLARQNE YYCRLDF LWJKKK L RQE REE TETMENVLPAHVA rCL GO PRNREDI.YH jSYECVCVL FAS IPDFKEFY SE SN IN HEOGLECLRL LLE II AD FF| E3)KFSG 0VEKIKTIGSTYMAATGLNATPGODTQQDAERSCSHLGTMVEFAVALGSKLG J'IN KiiSFNN4F'RLRVGLNHu'PVVAGVIGAQKPQYIW0GNTVNVASRMESTGVLGKIQVTEE IA.S T Y SR V I KVKGK LCTY FLNT LTRTGSPSAS tagacacc t gagc t c:c r-t' tr.t i'ntr t caa taaaatgtctccaggcatctg
nucleotide was removed. Activated Ga was incubated with Sf9 cell membranes for 10 min prior to assay. Calmodulin, Ca2+, EGTA, forskolin, or G-protein fry subunit complex was added immediately prior to assay.
240 300 360
420 480 540
VD INMRVGVHSGSVLCGVIGLQKWQYDVWSHDVTLANHMEAGGVPGRVHITGATLALLAG
900 96,0 1020
4n064
FIG. 1. Deduced amino acid sequence of type IV adenylyl cyclase. The sequence corresponding to probe 31C is boxed. Peptides ACIV-1 and ACIV-2, which were used for raising antibodies, are underlined. The two circled asparagine residues are likely glycosylation sites. Shaded sequences indicate potential transmembrane spans. Nucleotide sequences upstream and downstream of the coding region are given in lowercase letters.
to electrophoresis, membranes were treated as described by Tang et al. (13); the Western blotting procedure was as described (14). Production of Recombinant Baculovirus. The full length type IV adenylyl cyclase cDNA was released from the pBluescript SK(-) plasmid and was inserted into the Bgl II and Sma I sites of baculovirus vector pVL1393 (15). Spodoptera frugiperda (Sf9) cells (2 x 106) were cotransfected (using calcium phosphate) with 1 ,ug of wild-type viral DNA and 18 ,ug of baculovirus vector containing type IV adenylyl cyclase cDNA (15). Screening and purification of recombinant virus were as described (13). Sf9 Cell Membrane Preparation and Adenylyl Cyclase Assays. Sf9 cells were infected with recombinant baculovirus at a multiplicity of infection of 1 plaque-forming unit per cell. Cells were usually harvested 48 hr after infection and were lysed by nitrogen cavitation at 550 psi (1 psi = 6.89 kPa) for 30 min in 20 mM Na Hepes, pH 8/1 mM EDTA/1 mM EGTA/150 mM NaCl/2 mM dithiothreitol. Membranes were prepared as described by Tang et al. (13). Adenylyl cyclase assays were performed in the presence of 10 mM MgCl2 for 20 min (4). Recombinant (Escherichia coli-derived) G. was purified as described (16); the protein was activated with guanosine 5'-[y-thio]triphosphate (GTP[yS]) and unbound
RESULTS cDNA Cloning and Protein Structure. Screening of -300,000 clones from a rat testis cDNA library with oligonucleotide 31C yielded 24 positives; 5 of these clones were sequenced and 1 was homologous to type I adenylyl cyclase. The deduced amino acid sequence of this clone, designated type IV adenylyl cyclase, is shown in Fig. 1. The first potential translation initiation codon is preceded by 109 nucleotides, including an in-frame stop codon, and is followed by an open reading frame encoding 1064 amino acid residues. The nucleotides surrounding the first ATG codon are characteristic of those found in eukaryotic translation initiation sequences (17). The hydrophobicity profile of type IV adenylyl cyclase resembles those of types 1 (8), 11 (9), and III (10), suggesting that all of these proteins contain two large hydrophobic regions, each consisting of six putative transmembrane helices. The rest of the sequence is relatively hydrophilic and is presumed to be cytoplasmic. The entire sequence of the type IV protein was divided into seven regions for more detailed comparisons. As shown in Table 1, regions C1a and C2a, comprising the bulk of each of the two large cytoplasmic domains, are the most conserved portions of the four adenylyl cyclases; the similarities between type II and type IV adenylyl cyclase are particularly high in these regions (76% and 79% amino acid identity). These two domains are also similar to each other and to portions of the catalytic domains of several guanylyl cyclases (18), and they have limited homology with the adenylyl cyclase from Saccharomyces cerevisiae (19). There is little or no sequence similarity among adenylyl cyclases in the regions designated N1, Clb, and C2b. The length of the amino-terminal portions of the proteins (N1 and N2) is quite variable (e.g., 28 residues for type IV, 77 residues for type III). The carboxyl-terminal region designated C2b is present only in the type I and type III proteins. In general, sequence homologies are also modest among the hydrophobic domains, although the type II and type IV proteins again show the greatest similarities. There are four consensus sequences for N-linked glycosylation within the type IV sequence. Two ofthese sites (N595 and N927) are presumed to be intracellular; the other two (N694 and N701; circled in Fig. 1) are located between transmembrane spans 9 and 10. There is one such site in types I and III adenylyl cyclase and two in the type II protein in the same
Table 1. Comparisons of different adenylyl cyclases
Types compared
M1 M2
% identity
N2
Ml
Cia
Clb
10179
M2
C2a
C2b
28 56 17 58 35 28 43 79 31 76 52 56 IV/Il N 2 NH 51 32 54 28 33 IV/III 52 NH NH 58 33 18 II/I 25 56 27 54 35 19 II/Ill NH NH 54 NH 54 NH 35 III/I Ct1a C1b C2a The structural model of mammalian adenylyl cyclases is shown at right. N denotes the amino-terminal region of the proteins; the N1 domain is largely absent from type IV adenylyl cyclase. M1 and M2 are the first and second membrane-spanning domains. C1 and C2 are the predicted cytoplasmic domains. C2b is present only in types I and III adenylyl cyclase. The tabulated numbers are the percentage of identical amino acid residues in each region for the indicated pairs of adenylyl cyclase. Gaps were introduced to obtain the best alignment. NH (nonhomologous) indicates that the percentage identity score is less than or equal to 15. The residues in each region are as follows: type I, N2 = 46-63, M1 = 64-237, Cla = 238-432, Clb = 433-588, M2 = 589-806, C2a = 807-1070, C2b = 1071-1134; type II, N2 = 28-44, Ml = 45-211, Cl. = 212-441, Clb = 442-595, M2 = 596-821, C2a = 822-1088; type III, N2 = 61-77, M1 = 78-247, Cia = 248-471, Clb = 472-624, M2 = 625-858, C2a = 859-1122, C2b = 1123-1144; type IV, N2 = 11-28; M1 = 29-195; Cia = 1%-425; Clb = 426-577; M2 = 578-805; C2a = 806-1064.
IV/I
t~~~Cb
10180
Proc. Natl. Acad Sci. USA 88 (1991)
Biochemistry: Gao and Gilman
A
B
k7.
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28 S ....--
S.0
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4
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FIG. 2. (A) Distribution of mRNA for type IV adenylyl cyclase. Nine micrograms of poly(A)+ RNA from brain, kidney, and testis and 10 j&g of 28S and 18S ribosomal RNA were electrophoresed in a formaldehyde/0.9% agarose gel. The blot was hybridized with an RNA probe corresponding to type IV adenylyl cyclase. Each preparation of poly(A)+ RNA contained approximately the same amount of actin mRNA (data not shown). (B) One microgram of poly(A)+ RNA from each tissue was used for polymerase chain reaction as described in Materials and Methods. The integrity of each preparation of poly(A)+ RNA was tested with actin-specific primers (data not shown). There is an intervening sequence of -200 base pairs (bp) in the portion of the type IV adenylyl cyclase gene that is flanked by the two primers used for the polymerase chain reaction. Thus, the RNA preparations were not contaminated with genomic DNA.
presumed extracellular loop. There is evidence for glycosylation of types I and III adenylyl cyclase (10, 13); glycosylation of the type I protein appears to occur in the carboxylterminal half of the molecule (13). The hydrophobic domains of type IV adenylyl cyclase contain some curious amino acid sequences, all involving leucine residues. There is a leucine-rich region in the adenylyl cyclase of S. cerevisiae (19). This region is composed of tandem leucine-rich repeats of 23 amino acids that span about 600 amino acid residues. There are 7 hydrophobic amino acids in the repeat, including 4 leucines. This structure, which is highly amphipathic, is also found in the leucine-rich
A__ MO _'__
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FIG. 3. Western blot analysis ofaccumulation of type IV adenylyl cyclase after infection of Sf9 cells with recombinant baculovirus. Membrane proteins (2.7 Ag) were electrophoresed through a 9O acrylamide gel. The antibody was against peptide ACIV-1. Adenylyl cyclase specific activities (assayed with 5 mM MnCl2 and 100 ,uM forskolin) are shown at the bottom. For lane 48*, cells were infected with wild-type (nonrecombinant) baculovirus. Molecular masses (kDa) are shown at left.
Table 2. Adenylyl cyclase activities of Sf9 cell membranes containing the type IV or type I enzyme % maximal activity Type I Type IV Assay condition 5.2 1.7 Mg2+ 14 4.4 Mn2+ 68 20 Mn2 , forskolin 18 5.1 Mn2+, GTP[yS] 46 39 G,,-GTP[yS] 74 39 Mn2+, G. GTP[yS] 100 100 Mn2+, G.,GTP[yS], forskolin 85 24 GsGTP[yS], forskolin 30 1.7 Ca , CaM 9 1.7 Ca CaM, EGTA In each case, the maximal activity was observed in the presence of Mn2 , G~.-GTP[yS], and forskolin: type IV, 6 nmol min- mg 1; type I, 16 nmol min-l mg-1. The concentrations of effectors were as follows: Mn2+, 5 mM; forskolin, 100 ALM; GTP[yS], 10 tiM; G.-GTP[yS], 50 nM; EGTA, 100 ,M; Ca2+, 50 nM; and calmodulin (CaM), 100 nM.
a2-glycoprotein of human serum (20). A similar sequence is found in a presumed intracellular loop between membrane spans 8 and 9 in type IV adenylyl cyclase (residues 640-662). Membrane spans 10 and 11 each contain the 7-amino acid sequence SCSLFLH. Sixteen amino acid residues, including a stretch of 6 leucines, separate this repeat. There is a periodic repeat of leucine residues at every seventh position (residues 55, 62, 69, 76) in the region encompassed by membrane span 2. Tissue Distribution of Type IV Adenylyl Cyclase. An RNA probe for type IV adenylyl cyclase was used to detect the mRNA that encodes the protein. A single band of -3.5 kilobases was detected in poly(A)+ RNA from brain and kidney (Fig. 2A). Since this band was difficult to detect, the polymerase chain reaction was utilized to determine the distribution of mRNA in other tissues. Positive signals were detected in brain, heart, intestine (minor), kidney, liver, and lu.ng, but not testis (Fig. 2B). The failure to detect mRNA in testis is curious in view of the fact that the clone was isolated from a commercial testis cDNA library. Type IV adenylyl cyclase appears to be widely distributed. Expression and Characterization of Type IV Adenylyl Cyclase in Sf9 Cells. The type IV adenylyl cyclase cDNA was inserted into the baculovirus genome under the control of the strong polyhedron promoter, and Sf9 cells were infected with the recombinant virus. There was a notable increase in adenylyl cyclase activity 2 days after viral infection. The maximal specific activity achieved was 1.2 nmol min-'-mg(assayed in the presence of Mn2' and forskolin) (Fig. 3). Enzymatic activities in uninfected cells or in cells infected with wild-type virus were lower by a factor of -10. Antiserum from a rabbit immunized with peptide ACIV-1 was used to detect the expressed protein. A diffuse band with an apparent molecular mass of 110 kDa was present in cells infected with the recombinant baculovirus (Fig. 3). We presume that the diffuseness of the band was due to varying degrees of glycosylation or other modifications, including proteolysis. An antiserum raised against the ACIV-2 peptide recognized the same band (data not shown). The intensity of the signal on immunoblots suggested that the level of expression of type IV adenylyl cyclase was considerably greater than that seen previously for the type I enzyme (13). This was verified by examination of Coomassie blue-stained gels, where a distinct protein band at 110 kDa was evident 2 days after infection. Since the specific activity of the type-IV adenylyl cyclase in Sf9 cell membranes was less than that seen with type I, we assume that a considerable amount of the protein was denatured and/or aggregated. Adenylyl cyclase
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Gao and Gilman
2 CD
0
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10181
of Ca2+ and calmodulin. This is true even when membranes have been washed extensively with EGTA and then assayed in the presence of calmodulin and Ca2+ at concentrations ranging from 50 nM to 500 AM (data not shown). The effects of G. and forskolin on type IV adenylyl cyclase activity are interactive and are dependent on divalent cations. In the presence of Mn2+, the effects of G, and forskolin are greater than additive (Table 2; Fig. 4). This is not true of type I adenylyl cyclase (Table 2; ref. 13). In the absence of Mn2 , forskolin actually inhibits activity in the presence of maximal concentrations of G.", while the diterpene enhances activity synergistically with low concentrations of G, and increases the apparent affinity of adenylyl cyclase for the G-protein a subunit (Fig. 4). G-protein jBy subunit complex inhibits the activity of stimulated (G,, or calmodulin) type I adenylyl cyclase (13, 21, 22). The pfy subunit complex has the opposite effect on type II adenylyl cyclase, stimulating the enzyme markedly if G~a is also present (22). Type IV adenylyl cyclase behaves much like the type II protein (Fig. 5). The f3y subunit complex has no effect on type IV adenylyl cyclase when tested alone. However, in the presence of activating concentrations of G.", enzymatic activity is increased roughly 5-fold by nanomolar concentrations of fry.
DISCUSSION
Gs., nM FIG. 4. Activation of type IV adenylyl cyclase by forskolin and G,,GTP[,yS]. Adenylyl cyclase activity of membranes from Sf9 cells infected with recombinant baculovirus was assayed in the presence (A) or absence (B) of 5 mM Mn2+. The forskolin concentration was 0 (0), 1 ,uM (A), 10 AuM (o), or 100 AM (K).
activity in Sf9 cell membranes decreased after 2 days of infection, although the amount of immunoreactive material was relatively constant for an additional 1-2 days. This, too, was presumably a result of denaturation or aggregation. At these times immunoreactive material was seen at the top of the gel. Type IV adenylyl cyclase activity in Sf9 cell membranes can be activated by Mn2 , forskolin, or Ga (Table 2). However, in contrast to the type I enzyme, there is no effect
The family of membrane-bound adenylyl cyclases appears to be a relatively large one. cDNAs for four of these proteins have now been cloned, and additional members of the group have been detected with strategies based on the polymerase chain reaction (J. Krupinski, personal communication). A certain number of common features are obvious, and it is beginning to be possible to correlate different structural features with different functional properties. All of the adenylyl cyclases cloned to date share the same general topology with regard to the membrane and contain conserved, duplicated cytoplasmic domains. Preliminary evidence suggests that interactions between these domains may be necessary for catalysis (13). It is thus particularly interesting that the soluble guanylyl cyclase is a heterodimer, with each subunit containing nonidentical domains homologous to
3i
.0
2
a) E C
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1
Gsa(xnM)
py(nM)
0 20 20 20 20 20 20 0 20 20*1 00 1 00*
0 100100 100 100100 1 00 0 1 00 1 00* 300300*
FIG. 5. Effect of G-protein fry subunit complex on activation of type IV adenylyl cyclase by Gsa GTP[-yS]. Sf9 cell membranes containing type IV adenylyl cyclase were incubated with 20 nM (A) or 100 nM (B) GoGTP[yS]. The indicated concentration of 8ry or boiled fry (*) was added at the start of the assay.
10182
Biochemistry: Gao and Gilman
the C1a and Cu domains of adenylyl cyclase. Coexpression of both subunits is necessary for catalytic activity (23). Membrane-bound guanylyl cyclases have a single subunit that contains one such domain. Perhaps this protein functions as a dimer. The possibility that adenylyl cyclase can function with two identical Cia or Cu domains should be tested. If interactions between the cytosolic domains is crucial, it seems reasonable to suggest that the primary role of the two sets of transmembrane spans and of activators of the enzyme is to promote interaction between the two cytosolic portions of the protein. The most obvious functional differences noted to date among the cloned adenylyl cyclases are their sensitivity to stimulatory effects of calmodulin (types I and III) and their capacity to be inhibited (type I), unaffected (type III), or stimulated (in the presence of G..) (types II and IV) by the G-protein f3ry subunit complex. Type II and type IV adenylyl cyclase also show potentiative interactions between G. and forskolin, whereas type I does not. Types II and IV adenylyl cyclase are the most similar in terms of sequence, and, based on this general criterion, it is perhaps not surprising that their regulatory properties are also similar. They are also similar structurally in lacking the C2b domain at the carboxyl terminus. The general structure of mammalian adenylyl cyclases resembles those of membrane channels or transporters, although there is no amino acid sequence homology between the cyclases and their topographical homologs. This structural similarity originally led to the speculation that adenylyl cyclases might have dual roles, as both enzymes and transporters. This hypothesis now seems less likely in view of the poor conservation of amino acid sequence in the transmembrane spans of the various adenylyl cyclases. Although it is possible that each cyclase might have a distinct role as a transporter, there are no obvious hypotheses as to what group of molecules might serve as substrates for these proteins. It is difficult to fathom the evolutionary pattern of regulated adenylyl cyclase systems. Adenylyl cyclases in both S. cerevisiae and higher organisms are activated by G proteins. In Saccharomyces, however, the G protein is RAS and the cyclase bears little structural resemblance to the mammalian enzymes (19). This is the case despite the fact that yeast contain heterotrimeric G proteins that mediate the response to mating hormones. Thus, the basic regulatory relationship between GTP and adenylyl cyclase has been retained from yeast, but the molecules that transduce the information are totally different. The functions of the G proteins have changed, and structurally distinct adenylyl cyclase molecules have evolved.
Proc. NatL Acad. Sci. USA 88 (1991) We thank Marian Stanzel and Lisa Ortlepp for superb technical assistance, Dr. Wei-Jen Tang for membranes containing type I adenylyl cyclase and for help with the baculovirus expression system, Dr. Barbara Barylko for bovine brain calmodulin, Dr. Maurine Linder for recombinant G,~,, and Ethan Lee for the bovine brain (3'y subunit complex. This work was supported by United States Public Health Service Grant GM34497, American Cancer Society Grant BE-30M, The Raymond and Ellen Willie Chair of Molecular Neuropharmacology, The Lucille P. Markey Charitable Trust, and the Perot Family Foundation.
1. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649. 2. Simon, M. I., Strathmann, M. P. & Gautam, N. (1991) Science 252, 802-808. 3. Yeager, R. E., Heideman, W., Rosenberg, G. B. & Storm, D. R. (1985) Biochemistry 24, 3776-3783. 4. Smigel, M. D. (1986) J. Biol. Chem. 261, 1976-1982. 5. Pfeuffer, E., Drehev, R.-M., Metzger, H. & Pfeuffer, T. (1985) Proc. Natl. Acad. Sci. USA 82, 3086-3090. 6. Pfeuffer, E., Mollner, S. & Pfeuffer, T. (1985) EMBO J. 4, 3675-3679. 7. Gross, M. K., Toscano, D. G. & Toscano, W. A., Jr. (1987) J. Biol. Chem. 262, 8672-8676. 8. Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R. & Gilman, A. G. (1989) Science 244, 1558-1564. 9. Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Nat!. Acad. Sci. USA 88, 10173-10177. 10. Bakalyar, H. A. & Reed, R. R. (1990) Science 250, 1403-1406. 11. Maniatis, T., Fritsch, E. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 12. Lachmann, P. J., Strangeways, L., Vyakarnam, A. & Evan, G. (1986) CIBA Found. Symp. 119, 25-57. 13. Tang, W.-J., Krupinski, J. & Gilman, A. G. (1991) J. Biol. Chem. 266, 8595-8603. 14. Mumby, S. M., Kahn, R. A., Manning, D. R. & Gilman, A. G. (1986) Proc. Nat!. Acad. Sci. USA 83, 265-269. 15. Summers, M. & Smith, G. E. (1987) Bulletin 1555 (Tex. Agric. Exp. Stn., College Station, TX), pp. 1-56. 16. Graziano, M. P., Freissmuth, M. & Gilman, A. G. (1989) J. Biol. Chem. 264, 409-418. 17. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. 18. Chinkers, M. & Garbers, D. L. (1991) Annu. Rev. Biochem. 60, 553-576. 19. Kataoka, T., Broek, D. & Wigler, M. (1985) Cell 43, 493-505. 20. Takahashi, N., Takahashi, Y. & Putnam, F. W. (1985) Proc. Nat!. Acad. Sci. USA 82, 1906-1910. 21. Katada, T., Kusakabe, K., Oinuma, M. & Ui, M. (1987) J. Biol. Chem. 262, 11897-11900. 22. Tang, W.-J. & Gilman, A. G. (1991) Science, in press. 23. Nakane, M., Arai, K., Saheki, S., Kuno, T., Buechler, W. & Murad, F. (1990) J. Biol. Chem. 265, 16841-16845.
Cloning and expression of a widely distributed (type IV) adenylyl cyclase (cyclic AMP/guanine nudeotide-binding protein/signal transduction)
BONING GAO AND ALFRED G. GILMAN Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235
Contributed by Alfred G. Gilman, August 14, 1991
We have cloned and expressed a cDNA that ABSTRACT encodes a widely distributed form of mammalian adenylyl cyclase (EC 4.6.1.1). Although those adenylyl cyclases described previously have a rather narrow tissue distribution, this enzyme (type IV) is apparently synthesized in a variety of peripheral tissues and in the central nervous system. The protein resembles the other adenylyl cyclases in its proposed structure. It most resembles the type II adenylyl cyclase described in the preceding paper [Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Nall. Acad. Sci. USA 88, 10173-10177] in its amino acid sequence, lack of response to calmodulin, and synergistic activation by a combination of the Gs a subunit and the G-protein By subunit complex.
Intracellular concentrations of cAMP are regulated by membrane-bound, multiprotein complexes of hormone receptors, heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), and adenylyl cyclases (1, 2). Hormones that stimulate cAMP accumulation bind to appropriate receptors in the plasma membrane and activate a G protein, Gs, by catalyzing exchange of GDP for GTP on the G-protein a subunit; Gs, in turn, activates adenylyl cyclase. Both calmodulin-sensitive and calmodulin-insensitive forms of adenylyl cyclase have been identified in brain and other tissues (3-6), and a soluble, calmodulin-sensitive (but G-proteininsensitive) adenylyl cyclase has been found in sperm (7). Molecular cloning has permitted characterization of a brainspecific, calmodulin-activated adenylyl cyclase (type I) (8) and a calmodulin-insensitive enzyme (type II) that is found largely in brain but is also expressed in lung (9). An olfactoryspecific, calmodulin-activated adenylyl cyclase (type III) has been identified as well (10). We describe herein the cloning and characterization of a type IV adenylyl cyclase.* It is insensitive to calmodulin and appears to be distributed more widely than the enzymes described previously. All of the mammalian adenylyl cyclases that have been cloned share unusual structural features, including two large hydrophobic domains, each with multiple putative transmembrane helices, and two roughly 40-kDa cytoplasmic domains. Portions ofthe cytoplasmic domains are homologous to each other and are conserved within the members of the adenylyl cyclase and guanylyl cyclase families.
cyclase (8). Hybridization was performed at 500C in S x SSPE (0.9 M NaCl/50 mM sodium phosphate, pH 7.4/5 mM EDTA)/5x Denhardt's solution/0.1% SDS containing calf thymus DNA (100 jig/ml) (11). Filters were washed with 5 x SSC (0.75 M NaCl/75 mM sodium citrate, pH 7)/0.1% SDS at 500C. Positive inserts were released from the vector by digestion with EcoRI and were subcloned into phage vector M13mpl9. Dideoxynucleotide sequencing was performed with a modified T7 DNA polymerase (Sequenase, United States Biochemical). Northern Blot Analysis. Total RNA was purified and poly(A)+ RNA was selected using an mRNA separator as suggested by the supplier (Clontech). Poly(A)+ RNA was electrophoresed through formaldehyde/agarose gels (11) and transferred to nylon membranes. A full-length type IV adenylyl cyclase cDNA was inserted into the EcoRI site of plasmid pBluescript SK(-) (Stratagene), and the plasmid was linearized at the Hind III site 286 base pairs from the 5' end of the insert. T3 RNA polymerase was then utilized to make a full-length, type IV adenylyl cyclase RNA probe. Hybridization was performed at 650C with 50%o formamide/5x SSC/0.1 M sodium phosphate, pH 7/5x Denhardt's solution/0.1% SDS containing calf thymus DNA (100 ,ug/ml). Filters were washed at 650C with 0.1x SSC/0.1% SDS. Calf liver rRNA (Pharmacia) and a 0.24- to 9.5-kilobase RNA "ladder" (Bethesda Research Laboratories) were used as RNA molecular size standards. Calculated molecular sizes were the average of three experiments. Polymerase Chain Reaction. A GeneAmp RNA polymerase chain reaction kit (Perkin-Elmer/Cetus) was used for all reactions. Two DNA primers specific for type IV adenylyl cyclase were utilized-BG45 (5 '-GCTTGGGTCTGAGGTCA; corresponding to amino acid residues 55-60) and BG35 (5'-AGCCAGCCTACCCAGGTT; nucleotides -86 to -103). The thermal profile used was the same as that for the positive control in the kit, except that the annealing temperature was 70'C instead of 600C. A type IV adenylyl cyclasespecific probe corresponding to amino acid residues 1-15 was used to identify the product (5'-GATCTTCGCTGGGAG-
GAGGCCGGGGACTGAAGAGGCGGGCCATGGATCCA). Hybridization was at 600C in 5 x SSPE/5 x Denhardt's solution/0.2% SDS containing calf thymus DNA
CAGCTCATCAAAGTCAGCAATGATCTCATT-3') (see Fig. 1), which encodes amino acids 901-916 oftype I adenylyl
(100 Ag/ml). Antibodies and Western Blot Analysis. Two peptides, ACIV-1 and ACIV-2, with sequence specific for type IV adenylyl cyclase were synthesized for generation of antibodies. The ACIV-1 sequence is located in the second large cytoplasmic domain (residues 927-940; see Fig. 1) and that for ACIV-2 is at the carboxyl terminus of the protein (residues 1047-1064). Peptides were coupled to the purified protein derivative of tuberculin (PPD), and rabbits were immunized with the coupled peptide as described (12). Prior
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: GTP[yS], guanosine 5'-[y-thio]triphosphate. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M80633).
MATERIALS AND METHODS cDNA Cloning and Sequencing. An oligo(dT)-primed rat testis Agtll cDNA library (Clontech) was screened with oligonucleotide 31C (5'-TTGTAGAAGTCCTTGTCCAT-
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Proc. Natl. Acad. Sci. USA 88 (1991)
cggggcagccagcctacccaggttccctcctggagcctagctttgctac gzigacic tec: .ggagcttaacacaaagcaggcaggggctccgggctggggcgggggatatc
MARLFSPRPPPSEDLFYETYYSLSQOYPLLILLLVIVLCAIVALPAVAWASGRELTSDPS
60
FLITTVLCALGGFSLLLGLASREOQLQRWTRPLSGL;IWAALLALGYGFLFTGGWSAWQV 120 SFFLFI IFTVYAMLPLGMRDAAGOVSSLSHLLVLGLYLGWRPESORDLLPOLAANAVL 180 FLCGNVVGAYHKALMERALRATFREALSSLHSRRRLDTEKKHOEHLLLSI LPAYLAREMK AE IMARLOAGQSSRPENTNNFHSLYVKRHQGVSVLYADIVGFTRLASECSPKELVLMLNE FGKFDOIAKEHECMRIKILGDCYYCVSGLPLSLPDHAINCVRMGLDMCRAIRKLRVATG AYAVERADMEHRDPYLRELGEPTYLVIDPWAEEEDEKGTERGLLSSLEGHTMRPSLLMTR .L:SWAA; F.F AHt SHVDSPASTSTPLPEKAFSPOWSLDRSRTPRGL HDELDTGDAKFFO
V;FQLNSCK;WKQSKDFNLLTLYFREKEMEKQYRLSALPAFKYYAACTFLVFLSNFTIQN
600
LVTTRPPALATTYSITFLLFLLLLFVCFSEHLTKCVQKGPKMLHWL0ALSVLVATRPGLR
660 720 780 840
VALGTATILLVFTMAVVSLLFLPVSSDCPFLAFGVSSVAF®DTSWELPASLPL1SIPYSMH CCVLGFLSCSLFLHMSFELKLLLLLLWLVASCSLFLHSHAWLSDCLIARLYQGSLGSRPG LKL K LMtiA IYF FIlFF FTt LLLARQNE YYCRLDF LWJKKK L RQE REE TETMENVLPAHVA rCL GO PRNREDI.YH jSYECVCVL FAS IPDFKEFY SE SN IN HEOGLECLRL LLE II AD FF| E3)KFSG 0VEKIKTIGSTYMAATGLNATPGODTQQDAERSCSHLGTMVEFAVALGSKLG J'IN KiiSFNN4F'RLRVGLNHu'PVVAGVIGAQKPQYIW0GNTVNVASRMESTGVLGKIQVTEE IA.S T Y SR V I KVKGK LCTY FLNT LTRTGSPSAS tagacacc t gagc t c:c r-t' tr.t i'ntr t caa taaaatgtctccaggcatctg
nucleotide was removed. Activated Ga was incubated with Sf9 cell membranes for 10 min prior to assay. Calmodulin, Ca2+, EGTA, forskolin, or G-protein fry subunit complex was added immediately prior to assay.
240 300 360
420 480 540
VD INMRVGVHSGSVLCGVIGLQKWQYDVWSHDVTLANHMEAGGVPGRVHITGATLALLAG
900 96,0 1020
4n064
FIG. 1. Deduced amino acid sequence of type IV adenylyl cyclase. The sequence corresponding to probe 31C is boxed. Peptides ACIV-1 and ACIV-2, which were used for raising antibodies, are underlined. The two circled asparagine residues are likely glycosylation sites. Shaded sequences indicate potential transmembrane spans. Nucleotide sequences upstream and downstream of the coding region are given in lowercase letters.
to electrophoresis, membranes were treated as described by Tang et al. (13); the Western blotting procedure was as described (14). Production of Recombinant Baculovirus. The full length type IV adenylyl cyclase cDNA was released from the pBluescript SK(-) plasmid and was inserted into the Bgl II and Sma I sites of baculovirus vector pVL1393 (15). Spodoptera frugiperda (Sf9) cells (2 x 106) were cotransfected (using calcium phosphate) with 1 ,ug of wild-type viral DNA and 18 ,ug of baculovirus vector containing type IV adenylyl cyclase cDNA (15). Screening and purification of recombinant virus were as described (13). Sf9 Cell Membrane Preparation and Adenylyl Cyclase Assays. Sf9 cells were infected with recombinant baculovirus at a multiplicity of infection of 1 plaque-forming unit per cell. Cells were usually harvested 48 hr after infection and were lysed by nitrogen cavitation at 550 psi (1 psi = 6.89 kPa) for 30 min in 20 mM Na Hepes, pH 8/1 mM EDTA/1 mM EGTA/150 mM NaCl/2 mM dithiothreitol. Membranes were prepared as described by Tang et al. (13). Adenylyl cyclase assays were performed in the presence of 10 mM MgCl2 for 20 min (4). Recombinant (Escherichia coli-derived) G. was purified as described (16); the protein was activated with guanosine 5'-[y-thio]triphosphate (GTP[yS]) and unbound
RESULTS cDNA Cloning and Protein Structure. Screening of -300,000 clones from a rat testis cDNA library with oligonucleotide 31C yielded 24 positives; 5 of these clones were sequenced and 1 was homologous to type I adenylyl cyclase. The deduced amino acid sequence of this clone, designated type IV adenylyl cyclase, is shown in Fig. 1. The first potential translation initiation codon is preceded by 109 nucleotides, including an in-frame stop codon, and is followed by an open reading frame encoding 1064 amino acid residues. The nucleotides surrounding the first ATG codon are characteristic of those found in eukaryotic translation initiation sequences (17). The hydrophobicity profile of type IV adenylyl cyclase resembles those of types 1 (8), 11 (9), and III (10), suggesting that all of these proteins contain two large hydrophobic regions, each consisting of six putative transmembrane helices. The rest of the sequence is relatively hydrophilic and is presumed to be cytoplasmic. The entire sequence of the type IV protein was divided into seven regions for more detailed comparisons. As shown in Table 1, regions C1a and C2a, comprising the bulk of each of the two large cytoplasmic domains, are the most conserved portions of the four adenylyl cyclases; the similarities between type II and type IV adenylyl cyclase are particularly high in these regions (76% and 79% amino acid identity). These two domains are also similar to each other and to portions of the catalytic domains of several guanylyl cyclases (18), and they have limited homology with the adenylyl cyclase from Saccharomyces cerevisiae (19). There is little or no sequence similarity among adenylyl cyclases in the regions designated N1, Clb, and C2b. The length of the amino-terminal portions of the proteins (N1 and N2) is quite variable (e.g., 28 residues for type IV, 77 residues for type III). The carboxyl-terminal region designated C2b is present only in the type I and type III proteins. In general, sequence homologies are also modest among the hydrophobic domains, although the type II and type IV proteins again show the greatest similarities. There are four consensus sequences for N-linked glycosylation within the type IV sequence. Two ofthese sites (N595 and N927) are presumed to be intracellular; the other two (N694 and N701; circled in Fig. 1) are located between transmembrane spans 9 and 10. There is one such site in types I and III adenylyl cyclase and two in the type II protein in the same
Table 1. Comparisons of different adenylyl cyclases
Types compared
M1 M2
% identity
N2
Ml
Cia
Clb
10179
M2
C2a
C2b
28 56 17 58 35 28 43 79 31 76 52 56 IV/Il N 2 NH 51 32 54 28 33 IV/III 52 NH NH 58 33 18 II/I 25 56 27 54 35 19 II/Ill NH NH 54 NH 54 NH 35 III/I Ct1a C1b C2a The structural model of mammalian adenylyl cyclases is shown at right. N denotes the amino-terminal region of the proteins; the N1 domain is largely absent from type IV adenylyl cyclase. M1 and M2 are the first and second membrane-spanning domains. C1 and C2 are the predicted cytoplasmic domains. C2b is present only in types I and III adenylyl cyclase. The tabulated numbers are the percentage of identical amino acid residues in each region for the indicated pairs of adenylyl cyclase. Gaps were introduced to obtain the best alignment. NH (nonhomologous) indicates that the percentage identity score is less than or equal to 15. The residues in each region are as follows: type I, N2 = 46-63, M1 = 64-237, Cla = 238-432, Clb = 433-588, M2 = 589-806, C2a = 807-1070, C2b = 1071-1134; type II, N2 = 28-44, Ml = 45-211, Cl. = 212-441, Clb = 442-595, M2 = 596-821, C2a = 822-1088; type III, N2 = 61-77, M1 = 78-247, Cia = 248-471, Clb = 472-624, M2 = 625-858, C2a = 859-1122, C2b = 1123-1144; type IV, N2 = 11-28; M1 = 29-195; Cia = 1%-425; Clb = 426-577; M2 = 578-805; C2a = 806-1064.
IV/I
t~~~Cb
10180
Proc. Natl. Acad Sci. USA 88 (1991)
Biochemistry: Gao and Gilman
A
B
k7.
.
28 S ....--
S.0
la
@
-.
s
* -- 283bp
4
a
i8S--
FIG. 2. (A) Distribution of mRNA for type IV adenylyl cyclase. Nine micrograms of poly(A)+ RNA from brain, kidney, and testis and 10 j&g of 28S and 18S ribosomal RNA were electrophoresed in a formaldehyde/0.9% agarose gel. The blot was hybridized with an RNA probe corresponding to type IV adenylyl cyclase. Each preparation of poly(A)+ RNA contained approximately the same amount of actin mRNA (data not shown). (B) One microgram of poly(A)+ RNA from each tissue was used for polymerase chain reaction as described in Materials and Methods. The integrity of each preparation of poly(A)+ RNA was tested with actin-specific primers (data not shown). There is an intervening sequence of -200 base pairs (bp) in the portion of the type IV adenylyl cyclase gene that is flanked by the two primers used for the polymerase chain reaction. Thus, the RNA preparations were not contaminated with genomic DNA.
presumed extracellular loop. There is evidence for glycosylation of types I and III adenylyl cyclase (10, 13); glycosylation of the type I protein appears to occur in the carboxylterminal half of the molecule (13). The hydrophobic domains of type IV adenylyl cyclase contain some curious amino acid sequences, all involving leucine residues. There is a leucine-rich region in the adenylyl cyclase of S. cerevisiae (19). This region is composed of tandem leucine-rich repeats of 23 amino acids that span about 600 amino acid residues. There are 7 hydrophobic amino acids in the repeat, including 4 leucines. This structure, which is highly amphipathic, is also found in the leucine-rich
A__ MO _'__
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Adenylyl Cyclase 140 100 pmo' min
275 1200
7
2
647
34 400
67
37
.mg
FIG. 3. Western blot analysis ofaccumulation of type IV adenylyl cyclase after infection of Sf9 cells with recombinant baculovirus. Membrane proteins (2.7 Ag) were electrophoresed through a 9O acrylamide gel. The antibody was against peptide ACIV-1. Adenylyl cyclase specific activities (assayed with 5 mM MnCl2 and 100 ,uM forskolin) are shown at the bottom. For lane 48*, cells were infected with wild-type (nonrecombinant) baculovirus. Molecular masses (kDa) are shown at left.
Table 2. Adenylyl cyclase activities of Sf9 cell membranes containing the type IV or type I enzyme % maximal activity Type I Type IV Assay condition 5.2 1.7 Mg2+ 14 4.4 Mn2+ 68 20 Mn2 , forskolin 18 5.1 Mn2+, GTP[yS] 46 39 G,,-GTP[yS] 74 39 Mn2+, G. GTP[yS] 100 100 Mn2+, G.,GTP[yS], forskolin 85 24 GsGTP[yS], forskolin 30 1.7 Ca , CaM 9 1.7 Ca CaM, EGTA In each case, the maximal activity was observed in the presence of Mn2 , G~.-GTP[yS], and forskolin: type IV, 6 nmol min- mg 1; type I, 16 nmol min-l mg-1. The concentrations of effectors were as follows: Mn2+, 5 mM; forskolin, 100 ALM; GTP[yS], 10 tiM; G.-GTP[yS], 50 nM; EGTA, 100 ,M; Ca2+, 50 nM; and calmodulin (CaM), 100 nM.
a2-glycoprotein of human serum (20). A similar sequence is found in a presumed intracellular loop between membrane spans 8 and 9 in type IV adenylyl cyclase (residues 640-662). Membrane spans 10 and 11 each contain the 7-amino acid sequence SCSLFLH. Sixteen amino acid residues, including a stretch of 6 leucines, separate this repeat. There is a periodic repeat of leucine residues at every seventh position (residues 55, 62, 69, 76) in the region encompassed by membrane span 2. Tissue Distribution of Type IV Adenylyl Cyclase. An RNA probe for type IV adenylyl cyclase was used to detect the mRNA that encodes the protein. A single band of -3.5 kilobases was detected in poly(A)+ RNA from brain and kidney (Fig. 2A). Since this band was difficult to detect, the polymerase chain reaction was utilized to determine the distribution of mRNA in other tissues. Positive signals were detected in brain, heart, intestine (minor), kidney, liver, and lu.ng, but not testis (Fig. 2B). The failure to detect mRNA in testis is curious in view of the fact that the clone was isolated from a commercial testis cDNA library. Type IV adenylyl cyclase appears to be widely distributed. Expression and Characterization of Type IV Adenylyl Cyclase in Sf9 Cells. The type IV adenylyl cyclase cDNA was inserted into the baculovirus genome under the control of the strong polyhedron promoter, and Sf9 cells were infected with the recombinant virus. There was a notable increase in adenylyl cyclase activity 2 days after viral infection. The maximal specific activity achieved was 1.2 nmol min-'-mg(assayed in the presence of Mn2' and forskolin) (Fig. 3). Enzymatic activities in uninfected cells or in cells infected with wild-type virus were lower by a factor of -10. Antiserum from a rabbit immunized with peptide ACIV-1 was used to detect the expressed protein. A diffuse band with an apparent molecular mass of 110 kDa was present in cells infected with the recombinant baculovirus (Fig. 3). We presume that the diffuseness of the band was due to varying degrees of glycosylation or other modifications, including proteolysis. An antiserum raised against the ACIV-2 peptide recognized the same band (data not shown). The intensity of the signal on immunoblots suggested that the level of expression of type IV adenylyl cyclase was considerably greater than that seen previously for the type I enzyme (13). This was verified by examination of Coomassie blue-stained gels, where a distinct protein band at 110 kDa was evident 2 days after infection. Since the specific activity of the type-IV adenylyl cyclase in Sf9 cell membranes was less than that seen with type I, we assume that a considerable amount of the protein was denatured and/or aggregated. Adenylyl cyclase
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Gao and Gilman
2 CD
0
E
0~~~~~~~0
too===
20 o Is
Ec C)
CU
a) C. co
CZ
a)
60
10181
of Ca2+ and calmodulin. This is true even when membranes have been washed extensively with EGTA and then assayed in the presence of calmodulin and Ca2+ at concentrations ranging from 50 nM to 500 AM (data not shown). The effects of G. and forskolin on type IV adenylyl cyclase activity are interactive and are dependent on divalent cations. In the presence of Mn2+, the effects of G, and forskolin are greater than additive (Table 2; Fig. 4). This is not true of type I adenylyl cyclase (Table 2; ref. 13). In the absence of Mn2 , forskolin actually inhibits activity in the presence of maximal concentrations of G.", while the diterpene enhances activity synergistically with low concentrations of G, and increases the apparent affinity of adenylyl cyclase for the G-protein a subunit (Fig. 4). G-protein jBy subunit complex inhibits the activity of stimulated (G,, or calmodulin) type I adenylyl cyclase (13, 21, 22). The pfy subunit complex has the opposite effect on type II adenylyl cyclase, stimulating the enzyme markedly if G~a is also present (22). Type IV adenylyl cyclase behaves much like the type II protein (Fig. 5). The f3y subunit complex has no effect on type IV adenylyl cyclase when tested alone. However, in the presence of activating concentrations of G.", enzymatic activity is increased roughly 5-fold by nanomolar concentrations of fry.
DISCUSSION
Gs., nM FIG. 4. Activation of type IV adenylyl cyclase by forskolin and G,,GTP[,yS]. Adenylyl cyclase activity of membranes from Sf9 cells infected with recombinant baculovirus was assayed in the presence (A) or absence (B) of 5 mM Mn2+. The forskolin concentration was 0 (0), 1 ,uM (A), 10 AuM (o), or 100 AM (K).
activity in Sf9 cell membranes decreased after 2 days of infection, although the amount of immunoreactive material was relatively constant for an additional 1-2 days. This, too, was presumably a result of denaturation or aggregation. At these times immunoreactive material was seen at the top of the gel. Type IV adenylyl cyclase activity in Sf9 cell membranes can be activated by Mn2 , forskolin, or Ga (Table 2). However, in contrast to the type I enzyme, there is no effect
The family of membrane-bound adenylyl cyclases appears to be a relatively large one. cDNAs for four of these proteins have now been cloned, and additional members of the group have been detected with strategies based on the polymerase chain reaction (J. Krupinski, personal communication). A certain number of common features are obvious, and it is beginning to be possible to correlate different structural features with different functional properties. All of the adenylyl cyclases cloned to date share the same general topology with regard to the membrane and contain conserved, duplicated cytoplasmic domains. Preliminary evidence suggests that interactions between these domains may be necessary for catalysis (13). It is thus particularly interesting that the soluble guanylyl cyclase is a heterodimer, with each subunit containing nonidentical domains homologous to
3i
.0
2
a) E C
>aC a)
1
Gsa(xnM)
py(nM)
0 20 20 20 20 20 20 0 20 20*1 00 1 00*
0 100100 100 100100 1 00 0 1 00 1 00* 300300*
FIG. 5. Effect of G-protein fry subunit complex on activation of type IV adenylyl cyclase by Gsa GTP[-yS]. Sf9 cell membranes containing type IV adenylyl cyclase were incubated with 20 nM (A) or 100 nM (B) GoGTP[yS]. The indicated concentration of 8ry or boiled fry (*) was added at the start of the assay.
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Biochemistry: Gao and Gilman
the C1a and Cu domains of adenylyl cyclase. Coexpression of both subunits is necessary for catalytic activity (23). Membrane-bound guanylyl cyclases have a single subunit that contains one such domain. Perhaps this protein functions as a dimer. The possibility that adenylyl cyclase can function with two identical Cia or Cu domains should be tested. If interactions between the cytosolic domains is crucial, it seems reasonable to suggest that the primary role of the two sets of transmembrane spans and of activators of the enzyme is to promote interaction between the two cytosolic portions of the protein. The most obvious functional differences noted to date among the cloned adenylyl cyclases are their sensitivity to stimulatory effects of calmodulin (types I and III) and their capacity to be inhibited (type I), unaffected (type III), or stimulated (in the presence of G..) (types II and IV) by the G-protein f3ry subunit complex. Type II and type IV adenylyl cyclase also show potentiative interactions between G. and forskolin, whereas type I does not. Types II and IV adenylyl cyclase are the most similar in terms of sequence, and, based on this general criterion, it is perhaps not surprising that their regulatory properties are also similar. They are also similar structurally in lacking the C2b domain at the carboxyl terminus. The general structure of mammalian adenylyl cyclases resembles those of membrane channels or transporters, although there is no amino acid sequence homology between the cyclases and their topographical homologs. This structural similarity originally led to the speculation that adenylyl cyclases might have dual roles, as both enzymes and transporters. This hypothesis now seems less likely in view of the poor conservation of amino acid sequence in the transmembrane spans of the various adenylyl cyclases. Although it is possible that each cyclase might have a distinct role as a transporter, there are no obvious hypotheses as to what group of molecules might serve as substrates for these proteins. It is difficult to fathom the evolutionary pattern of regulated adenylyl cyclase systems. Adenylyl cyclases in both S. cerevisiae and higher organisms are activated by G proteins. In Saccharomyces, however, the G protein is RAS and the cyclase bears little structural resemblance to the mammalian enzymes (19). This is the case despite the fact that yeast contain heterotrimeric G proteins that mediate the response to mating hormones. Thus, the basic regulatory relationship between GTP and adenylyl cyclase has been retained from yeast, but the molecules that transduce the information are totally different. The functions of the G proteins have changed, and structurally distinct adenylyl cyclase molecules have evolved.
Proc. NatL Acad. Sci. USA 88 (1991) We thank Marian Stanzel and Lisa Ortlepp for superb technical assistance, Dr. Wei-Jen Tang for membranes containing type I adenylyl cyclase and for help with the baculovirus expression system, Dr. Barbara Barylko for bovine brain calmodulin, Dr. Maurine Linder for recombinant G,~,, and Ethan Lee for the bovine brain (3'y subunit complex. This work was supported by United States Public Health Service Grant GM34497, American Cancer Society Grant BE-30M, The Raymond and Ellen Willie Chair of Molecular Neuropharmacology, The Lucille P. Markey Charitable Trust, and the Perot Family Foundation.
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