Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8774-8778, September 1992 Biochemistry
Cloning and characterization of a sixth adenylyl cyclase isoform: Types V and VI constitute a subgroup within the mammalian adenylyl cyclase family SHUICHI KATSUSHIKA*, LIANG CHENt, JUN-ICHI KAWABE*, RAMASWAMY NILAKANTANt, NANCY J. HALNON*, CHARLES J. HOMCYtt, AND YOSHIHIRO ISHIKAWA*§ Departments of *Pharmacology and tMedicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032; and tMedical Research Division, American Cyanamid Company, Pearl River, NY 10965
Communicated by Vincent T. Marchesi, June 8, 1992
A sixth member of the mammalian adenylyl ABSTRACT cyclase family has been isolated from a canine cardiac cDNA library. This isoform is more highly homologous to type V than to the other adenylyl cyclase types; sequence similarity is apparent even in the transmembrane regions where the greatest divergence among the types exists. Type VI mRNA expression is most abundant in heart and brain; however, unlike type V, a low level of expression is also observed in a variety of other tissues examined. Type VI adenylyl cyclase can be stimulated by NaF, guanosine 5'-[rthio]triphosphate, and forskolin but not by Ca2+/calmodulin, whereas it is inhibited by adenosine and its analogues. Comparison of both their structural and biochemical properties suggests that ypes V and VI constitute a distinct subgroup of the mammalian adenylyl cyclase family.
MATERIALS AND METHODS Isolation of Clones. Screening and hybridization were performed with an Aat 1-HincIl fragment from type I adenylyl cyclase cDNA as a probe (1, 8). Transient Expression in CMT Cells and Adenylyl Cyclase Assay. A 4.0-kilobase (kb) EcoRI-Ssp I fragment, which was constructed by ligating a 0.9-kb EcoRl-Sph I fiagment from clone 6 and a 3.1-kb Sph I-Ssp I fragment from clone 27 and contained the entire coding sequence, was subcloned into the EcoRIf-EcoRV sites of the polylinker of pcDNA-amp (pcDNAamp 27-6). Transfection into CMT cells (derived from monkey COS cells) (9) and the adenylyl cyclase assay were performed as described (5). Northern Blot Analysis. Poly(A)+ RNA from various tissues and cells was prepared as described (5). The 5.4-kb fragment from clone 27 was used as the probe for the detection of type VI adenylyl cyclase mRNA. Sequence Homology Analysis. All six members of the mammalian adenylyl cyclase family (1-5) were analyzed for their amino acid sequence homology by using the multiplesequence analysis program PILEUP (10). The similarity scores were calculated and used to draw a dendrogram (11). Homology in different regions of the enzyme among the various isoforms was assessed similarly. Pairwise comparisons between various regions of these cyclases were performed with the program GAP (10). The putative cytoplasmic domains were aligned, together with the C-terminal portion of a mammalian membrane-bound type of guanylyl cyclase (6), by using MACVECTOR 3.5 (12).
Since the cloning of the first mammalian adenylyl cyclase cDNA, four additional adenylyl cyclase cDNAs have been isolated (1-5). Although each has a distinct tissue distribution and biochemical characteristics, the proteins they encode share the same topology: tandem repetition of a hydrophobic putative transmembrane domain that spans the membrane six times, followed by a large hydrophilic cytoplasmic domain. The amino acid sequence of the cytoplasmic domain is relatively well conserved among the family members; this region also shows homology to other cyclases, such as guanylyl cyclase (6) or adenylyl cyclases from lower eukaryotes (7). However, the amino acid sequence differs significantly in the transmembrane regions of the different adenylyl cyclase types (4). The degree of this divergence differs among different members; type II adenylyl cyclase is more homologous to type IV than to other types, whereas type I and III share a lesser degree of homology with the others. Two of the adenylyl cyclases (types II and IV) are similar not only in their amino acid sequence but also in their structure and function; both have a truncated C-terminal tail and lack calmodulin sensitivity. Their tissue distribution, however, is different. This greater degree of similarity between types II and IV suggests that certain adenylyl cyclase isoforms might constitute subclasses within the adenylyl cyclase family. We have recently cloned a cardiac adenylyl cyclase (type V), which appears to be limited in its expression to heart and neural tissue (5). We now describe a type VI adenylyl cyclase;¶ it exhibits a much higher degree of homology, both in sequence and structure, with type V adenylyl cyclase than with the other known family members. Furthermore, its tissue distribution and biochemical features are also similar. Types V and VI may therefore form a subclass within the mammalian adenylyl cyclase family.
RESULTS Cloning of Type VI Adenylyl Cyclase cDNA. In the primary screening, using a low-stringency hybridization protocol, we obtained 160 clones that hybridized with the probe. We finally obtained four clones, nos. 8, 25, 27, and 113. Three clones, nos. 8, 25, and 113, encoded type V adenylyl cyclase. However, clone 27, containing a 5.4-kb insert, was clearly different from the other clones albeit highly homologous to type V adenylyl cyclase. Among several hybridizing clones thereafter identified, clone 6 contained an additional 280 base pairs (bp) of 5' sequence. This included the initiator ATG in
the context of a reasonable Kozak consensus sequence, in comparison to other types of mammalian adenylyl cyclase (types I-V), in a long open reading frame (13). The 5' untranslated region is remarkable for its high G+C content
(96%) (Fig. 1).
This isoform was designated as type VI adenylyl cyclase. A hydropathy analysis identified the common motif of tan-
§To whom reprint requests should be addressed. IThe sequence reported in this paper has been deposited in the
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
GenBank data base (accession no. M94968).
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 89 (1992)
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TACCCGACTGTTACTACTGTCTCTGTCCCCCCTCCCACCCCCCGCACATCCACCCCCACTGCTGTCGTACATCGGCGTGCACATGATCGAGGCCATCTC L G D C Y Y C V S G L P e A R A D H A N C C V e N G V D N I e A I S 1501 GCTGGTCGTGAGGTGACAGGTGTGAACGTGAACATCCGTGGGCATCCACAGCGCGTGTGCACTGTGGTGTTTGGCCTGCGGAAATGGCAGTTC L V R C V T G V N V N N R V G I N S G R V H C G V L G L R K Q F 1601 CACCTGTCCTCCAATCACCTGACTCTGCCCAACCATATCGACGCCGCCCCGGCCGGCCGCATCCACATCACCCCGGCCACGCTCCAGTACCTGAACGCGG D V N N D V T L A N N N C A A R A G R I H I T R A T L Q Y L N G 17 01 ACTACGAGGTGGAGCCCGGCCGCGGTGGCGAGCGGAACGCGTACCTCAAGGAGCAGCACATCGAGACCTTCCTCATCCTGGGAGCCAGCCAGAAACGGAA D Y Z V C P G R G C c R N A Y L K C Q H I C T F L I L G A S Q K R K 1501 ACAGGAGAAGCCCATCCTGCCCAAGCTCGCAGCCCACCCGGCCAACTCCATGCAAGCCTGATCCCACCCTCGCTGCCCGACCGCGCCTTCTCCCCGGACC C C K A N L A K L Q R T R A N S N C C L N P R V P D R A F S R T 1901 AAGGACTCCAAGGCTTTCCGCCAGATGGCATTGATGATTCCAGCAAAGACAACCGGGTGCCCAAGATGCCCTGAACCCCGAGGATGAGGTCGATGAGT K D S K A F R Q N G I D D S S K D N R G A Q D A L N P e D E V D E 2001 TCCTGGCCGTGCCATCGATGCCCGCAGCATCGATCAGCTACGGAAGGACCATGTGCCCGCTTCCTGCTCACCTTCCAGAGAGAGGATCTTGAAAAGAA F L C R A I D A R S I D Q L R K D H V R R F L L T F Q R e D L e K K 2101 GTACTCA G AGCCTACGTGTCGCTGTTGGTCTTCTCTTCATCTCTTTACACTCCTCGTCTTCCCACAC Y S R K V D P R F G A Y V A C A L L V F C F I C F I Q L L V F P H 2201 TCAACCGTGATGCTTGGGATCTACGCCAGTATCTGTGTGTTCTGATCACCGTCTGACCTGTGCCGTGTACTCCTGTCTCTCTCTTCCCCAAGG S T V N L G I Y A s I F V L L L I T V L s C A V Y S C G S L F P K 2301 CCCTCCGACGTCTTTCCCGCAGCATCCTCCGCTCTCGGCACACACCACTGTCCTTYGCATITrTCACTCTGCCTACTGTTCACCTCTGCCATCGCCAA A L R R L S R S I V R S R A N S S V V G I F S V L L V F T S A I A N 2401 CATCTTCACCITGAACCACACCCCCATCCGGACCTCGCCACCCCCGATGCTGCATTCAACACCCCCTGACATCACTGCCCTCCACCTCCACCAGCTCAAT N F T C N N T P I R T C A A R N L N V T P A D I T A C H L Q Q L N 2501 TACTCTCTGCCCCTCCATCCTCCGCTCTGTCAGCCCACCGCACCCACTTCCAGCTTCCCTGAGTACTTCCTTGCCAACATCCTCCTCAGTCTCTTGGCCA Y S L G L D A P L C e G T A P T C S r P c Y F V G N N L L S L L A 2601 GCTCTCTTTrCCTCCACATCAGTAGCATCGGCGAMGCTTCCATCATCTTTGTCCTGCGCCTCAITATTTCTGCCTGCTTCTCCTGCGCCCCCCCAGCAC S S V F L N I S S I G K L A N I r V L G L I Y L V L L L L G P P S T 2701 CATCTTTGACAACTATGACCTGCTGCTTGGTGTCATGCTTGCTTCTTCCATGACACCTTTATGGCTGACTGCCCAGCTCGGAGGGTGGCA I F D N Y D L L L C V N G L A S S N D T F D G L D C P A A G R V A 2801 CTGATACATGACCCTGTGATTCTGCTGGTG~rGCCCTCGCTGTATCTGCAGC CAGTGGAATCACTCACGTCTGGACTTCCTCTGGA L K Y N T P V I L L V r A L A L Y L N A Q Q V C S T A R L D F L 2901 AACTCCACGCAACCGGGGACAACGACCAGATCGAGGAGCTCCAGGCCTACAACCCAACCCTCCTGCATAACATTCTGCCTAAGCACGTGGCTGCCCACTT K L Q A S C C K C C N C C L Q A Y N R R L L H N I L P K D V A A H F 3001 CCTGCCCGGGAGCGCCGAATGAGCTCTACTACCAGTCGTGTGAGTG CGTCATGTTaCCTCCATTGCCAACTTTTCTGAGTTCTATGTG L A R C R R N D C L Y Y Q s C c C V A V N F A S I A N F S e F Y V 3101 GACTGGACAAACAATGAGGGTGTCGAGTCCTCGGCTGCTCACGAATATCGCCGATTTGATAATCATCAGGAGCGGTTCCGCAGC e L e A N N e c V e C L R L L N e I I A D F D e I I S e e R F R Q 3201 TGGAGATCAAGACGATCGGTAGCACGTACATGGCTCGTCGGCTGACGCCAGCACCTACGATCAGGCCGGCCGCTCCCACATCACTGCCCTGGC L K I K S I C S S Y N A A s G L N A S T Y D Q A G R S H I T A L A 3301 CGACTATCCATCGGCTCATGGACAGATGAAACACAACGACACTCCTTCAACAACTTCCAATGAATTGGCTGAACATGGGCCCAGTTGTG D Y A N R L N N S N Q N K N I N F N N F K I G L N M G P V V 3401 GCAGCCCTCATTGCCGCTCIGAACCCACACTATGACATCTCCGCGAACACGGTGAAITCTCTCAGCCGTATGGACAGCACGGCGGTTCCTGACCGAATCC A G V I C A R K P r D I N G N S V N V S S R N D S T G V P D R I 3501 ACCTGACCACCGAC,,GTACCAGGTTCTACTCCAAACGTACCAGCTGGAGTGTCGAGGGTGGTCAAGGTGAAGGCAGGAGATGACCACCTA Q V T T D L Y Q V L A A K R Y Q L C R C V V K V K G K G e M T T Y 3601 CTTCCTtAATGGGCCCCCCCAGTTAGCAGA GCTACAAGTTCAcTGTCAGGACCAAWTGGGCATGTGGACTCTGTGCTGGATGG F L N CGC P P S 3701 ACCTCTCCCCCGCCGCACCAAGCCTCCAGACCCTGCCTCCCACACCAACACCCAC CACTTCCTTCCACCATCTCGTCTGCCCTCAGGCTGG 3601 TGAACAAGGGATACCAAGAGGATTATGCAAGTGACTTrTATTTCTAATTGGGGTAGGCsTGGCTGTTCCCTCTTTCCTGCTTTGGGAGCAGGGG 3901 AGGCAGCTGCAGCAGAGGCAGCAGGAGCCCTCCTGCCTGAGGGTTTAAAATGCCAGCTTGCCATGCCTACCCTTTCCCCTGTCTGTCTGGGCAACAGCAT 4001 CCGGGCTCGGCCCTTCCTT*TCCCTCTTTTTCCIGCCAATAITrFCT
223 257 290 323 357 390 423
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FIG. 1. Nucleotide and annotated amino acid sequence of type VI adenylyl cyclase. The entire coding sequence is shown together with part, of the 5' and 3' untranslated regions.
dem repetition of six transmembrane spans followed by a large hydrophilic cytoplasmic domain (14). A dot matrix comparison showed a high sequence homology with type I in the cytoplasmic domains, with a lesser degree in the transmembrane domains. However, when the type VI sequence was compared with that of the type V isoform, the homology was strikingly higher. Sequence Comparison with Other Types. A more extensive analysis of sequence homology among the different types of adenylyl cyclase was performed. The amino acid sequence along different regions of the molecule was compared among the different types by using the GAP program. Such a comparison for the first transmembrane domain and the second cytoplasmic domain is shown in Fig. 2. Both domains are more similar between types II and IV and between types V and VI than for any other set of pairs. There is less homology between the two transmembrane domains within the same molecule (27% in type VI, for example). It is interesting that the homology in the second cytoplasmic domain between
types III and VI is lower than the homology between types
V and VI in the first transmembrane domain despite the fact that the homology is typically highest in the cytoplasmic domains among different types of adenylyl cyclase. The sequence homology is highest in the initial half of each of the cytoplasmic domains. The latter half exhibits a lower degree of homology among the different types or is totally lacking as in types I and III. The amino acid sequence of the second cytoplasmic domain of the different types and that of the C-terminal portion of guanylyl cyclase (6) were aligned for comparison (Fig. 3). Multiple residues in this region are highly conserved among the mammalian cyclases and it is in this region where the highest degree of homology between the cytoplasmic loops is also apparent. A dendrogram based upon the homology score among the various mammalian adenylyl cyclase family members was drawn (Fig. 4). Types I and III clearly diverge from the others and the degree of divergence is somewhat greater in type III. Types II and IV exhibit a high degree of homology with each other, while
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Biochemistry: Katsushika et al.
type I 100
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Proc. Nad. Acad. Sci. USA 89 (1992)
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FiG. 2. Sequence homology among mammalian adenylyl cyclase
(TM1) and the initial half of the second cytoplasmic domain (CPD2) were compared for percent identity as described in Materials and Methods (10). The residues used for comparison from each type are as indicated: type I, 64-237 for TM1 and 807-1070 for CPD2; type 45-211 forTMl and 822-1088 forCPD2; type III, 79-247 forTMl and 859-1132 for CPD2; type IV, 29-195 for TM1 and 930-1184 for CPD2; type V, 165-322 for TM1 and 930-1184 for CPD2; and type VI, 151-308 for TM1 and 912-1165 for CPD2. 11,
types V and VI share an even higher degree of homology. Thus types II/IV and V/VI distinguish themselves as homologous pairs. V
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FIG. 4. Similarity relationship among the different types of mammalian adenylyl cyclase. The amino acid similarity scores were calculated as described in Materials and Methods (11). The calculated values indicate the position that divides the branches into subfamilies. Callular ExpressIon and Measurement of Catalytic Activity. Type VI adenylyl cyclase was transiently expressed in CMT cells. There was a concentration-dependent increase in adenylyl cyclase catalytic activity in transfected cell membranes when the amount of plasmid DNA was varied from 0 to 20 A.g (data not shown). Control cells were mocktransfected and similarly induced. Significantly higher catalytic activity in the transfected as compared to control membranes was obtained under either basal or stimulated (NaF, guanosine 5'-[(thio]triphosphate, or forskolin) con-
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FiG. 3. Comparison of the amino acid sequences of mammalian adenylyl cyclases (types I-VI) and guanylyl cyclase (GC) (6). Sequences were aligned for comparison by using MACVECTOR 3.5 (12). Amino acid residues are numbered at the right of each line. The amino acid sequences of a portion of the putative second cytoplasmic loop, type I (805-1063), type 11 (820-1081), type II (857-1125), type IV (804-1057), type V (928-1182), and type VI (910-1165), as well as the sequence of the C-terminal portion of guanylyl cyclase (810-1053), were compared.
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Katsushika et al. Table 1. Expression of type VI adenylyl cyclase in CMT cells Activity, pmol/(min-mg of protein) Forskolin Cells Basal NaF GTP[yS] Control 29 ± 5 59 ± 10 4 ± 0.6 12 ± 3 45 ± 5 110 ± 11 211 ± 25 Transfected 9 ± 0.8 Adenylyl cyclase activity in membranes (control cells versus cells transfected with pcDNAamp 27-6) was measured in the absence (basal) or presence of various activators: NaF (10 mM), guanosine 5'-[L-thio]triphosphate (GTP[yS], 100 jtM), forskolin (100 ,uM). Values are means ± SEM. For each condition (basal or activated), activity in control membranes was significantly lower than that in membranes from transfected cells (P < 0.001). Similar results were obtained in three independent experiments. For each experiment, the results represent the average obtained from six independently transfected plates of CMT cells.
ditions, with the forskolin-stimulated activity showing the greatest increase (Table 1). Adenylyl cyclase has been shown to be inhibited by adenosine and its analogues through an allosteric or P site (5, 15). We found that adenylyl cyclase activity in the transfected cells was inhibited in a concentration-dependent manner by adenosine and its analogues (Fig. 5). The order of potency was identical for types V and VI: 2'-deoxy-3'-AMP > 3'AMP > 2'-deoxyadenosine > adenosine. The Ca2+/ calmodulin sensitivity of type VI adenylyl cyclase was also examined in the transfected CMT cell system. Like type V adenylyl cyclase, type VI was also inhibited in a concentration-dependent manner by the addition of 0-1 mM Ca2+. Calmodulin (200 nM) did not alter this inhibition (Fig. 6). Northern Blotting. The tissue distribution of type VI mRNA was examined by analysis of poly(A)+ RNA prepared from various tissues (Fig. 7). A single message of -6 kb was widely distributed among all the tissues examined. Brain and heart expressed 4- to 7-fold more message than the other tissues as assessed by densitometry. Cell lines such as S49 (mouse lymphoma), A10 (rat smooth muscle), and GH4 (rat pituitary tumor) also expressed type VI adenylyl cyclase mRNA. Thus, in all tissues and cell types that we examined, a low level of type VI adenylyl cyclase mRNA was apparent except for heart and brain, where the level of expression was significantly greater. 100 90
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Ca2+(mM) FIG. 6. Effect of Ca2+/calmodulin on type VI adenylyl cyclase activity. Adenylyl cyclase activity in membranes (from CMT cells transfected with pcDNAamp 27-6 or mock-transfected control) was measured in the presence of various concentrations of Ca2+ (0-1 mM). Membrane preparations were washed with EGTA prior to assay (16). Similar results were obtained in three independent experiments. e, Transfectant membrane; o, transfectant membrane with calmodulin (200 nM); o, control membrane; *, control membrane with calmodulin (200 nM).
DISCUSSION Six adenylyl cyclase family members have thus far been cloned and characterized (1-5). They share a motif of two tandem repeats of six transmembrane spans separated by a large cytoplasmic loop and terminating in a similarly large cytoplasmic tail. This motif also characterizes a larger superfamily of transporters including the cystic fibrosis gene product and the multiple-drug-resistance gene product (17, 18). Based on homology comparisons, certain of the six adenylyl cyclase family members can be further segregated into discrete groups. Two sets of pairs, types II/IV and V/VI, become apparent when a relatively rigorous comparison of sequence identity is utilized as the index of homology. Types V and VI share the highest degree of homology and are kb
40
iI
9.49 7.46 -
5% 30 20
10t
4.4 20
40
60
P-site inhibitors
80
100
(A.M)
FIG. 5. Effect of adenosine and its analogues on type VI adenylyl cyclase activity. Membranes prepared from cells (transfected with pcDNAamp 27-6 or mock-transfected control) were preincubated with 5 mM Mn2+ and 100 gM forskolin for 10 min prior to the assay. *, Adenosine; o, 2'-deoxyadenosine; *, 3'-AMP; O, 2'-deoxy-3'AMP. Similar results were obtained in three independent experiments. Each point is the average of duplicate determinations. A similar degree of inhibition was obtained with type V adenylyl cyclase cDNA.
1.35-
H
B
T
S
K
L
FIG. 7. Northern blot analysis of mRNA from canine heart (H), brain (B), testis (T), skeletal muscle (S), kidney (K), and lung (L). Ten micrograms of poly(A)+ RNA was used per lane. An EcoRI 5.4-kb insert from clone 27 was used as a probe.
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>50% similar even in the putative transmembrane regions. It has been speculated that the poor conservation of amino acid sequence in the transmembrane regions among the other adenylyl cyclase isoforms is related to the likelihood that molecules of this class no longer function as transporters (4). Thus, the maintenance of homology in types V and VI, even in the transmembrane domains, is consistent with their being more closely related family members. Whether some functional activity, possibly shared by these adenylyl cyclase types, has also been maintained remains to be addressed. The greater degree of similarity between types II/IV and between types V/VI is also evident when certain shared structural features are compared. The length of the N-terminal tail is similarly shorter in types II/IV (44 and 28 residues, respectively) or longer in types V/VI (164 and 149 residues, respectively). Type I (63 residues) and type III (77 residues) possess an N-terminal tail that is intermediate in length. Although there is some homology in the N-terminal tail in the segment adjacent to the transmembrane domain (types V and VI, for example), the homology is generally low. Types V/VI contain consensus sequences for N-linked glycosylation at identical positions in the extracellular loop linking membranes spans 9 and 10. In types II/IV, these sites are similar in context but have clearly diverged in types I and III. Based on identity of sequence and these structural similarities, a pattern emerges wherein type III appears to have diverged at an early stage, thereafter types I/V/VT and types II/IV. Types II/IV and V/VI diverged from each other more recently (Fig. 4). In addition to differences in primary structure, the pattern of tissue distribution varies markedly among the adenylyl cyclase types. First, certain tissues appear to express several different types of adenylyl cyclase. For example, heart contains mRNA for types IV, V, and VI. The brain contains every isoform (types I-VI). By in situ hybridization (unpublished data), we have detected mRNA for types V and VI in cardiocytes but little in cardiac fibroblasts. Although type IV mRNA is also present in heart, its exact cellular distribution has not been reported. Finally, it is interesting that S49 lymphoma cells (16), which have been extensively used to study the G protein-adenylyl cyclase signal transduction pathway, express type VI adenylyl cyclase. The functional consequences of one cell type expressing more than one isoform are not known. Types V and VI, both of which are expressed in cardiocytes, exhibit profiles of biochemical properties that are quite similar. It may be that these isoforms will show distinct patterns of responsiveness to G-protein activation. Alternatively, these isoforms (types V and VI) may be differentially expressed in development or in response to physiologic and pathophysiologic stimuli. Several studies have indicated that adenylyl cyclase responsiveness, independent of abnormalities at the level of the (3-adrenergic receptor or G. protein, is impaired in heart failure (19, 20). Clarifying how these two closely related
Proc. Nad. Acad. Sci. USA 89 (1992)
isoforms, types V and VI, contribute to the abnormal pattern of adenylyl cyclase responsiveness in pathophysiologic states may identify factors that, either pre- or posttranslationally, impact differentially in regulating their expression and activity in the heart. We thank Drs. A. G. Gilman (University of Texas Southwestern Medical Center) and R. Reed (Johns Hopkins University) for providing us with adenylyl cyclase cDNAs, Dr. B. F. Hoffman (Columbia University) for his advice and encouragement, and Dr. S. Wrenn (Lederle Laboratories) for helpful discussions. We are grateful to Drs. D. Cooper (University of Colorado) and R. Iyengar (Mount Sinai Medical School) for sharing sequences of their cloned type V and VI adenylyl cyclases prior to publication. This work was supported in part by National Institutes of Health Grant HL38070; Joint Research Grants 63044118 and 01044120 from the International Scientific Research Program for the Ministry of Education, Science, and Culture of Japan; and a grant from Lederle Laboratories. 1. 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. 2. Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Natl. Acad. Sci. USA 88, 10173-10177. 3. Bakalyar, H. A. & Reed, R. R. (1990) Science 250,1403-1406. 4. Gao, B. & Gilman, A. G. (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182. 5. Ishikawa, Y., Katsushika, S., Chen, L., Halnon, N. J. & Homcy, C. J. (1992) J. Biol. Chem. 267, 13553-13557. 6. Chinkers, M., Garbers, D. L., Chang, M. S., Lowe, D. G., Chin, H., Goeddel, D. V. & Schulz, S. (1989) Nature (London) 338, 78-83. 7. Kataoka, T., Broek, D. & Wigler, M. (1985) Cell 43, 493-505. 8. Ishikawa, Y., Bianchi, C., Nadal-Ginard, B. & Homcy, C. J.
(1990) J. Biol. Chem. 265, 8458-8462. 9. Gerard, R. D. & Gluzman, Y. (1985) Mol. Cell. Biol. 5, 32313240. 10. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395. 11. Feng, D. R. & Doolittle, R. F. (1987) J. Mol. Evol. 25, 351-360. 12. Pustell, J. M. (1988) Nucleic Acids Res. 16, 1813-1820. 13. Kozak, M. (1989) J. Cell Biol. 108, 229-241. 14. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. 15. Tang, W.-J., Krupinski, J. & Gilman, A. G. (1991) J. Biol.
Chem. 266, 8595-8603.
16. Bourne, H. R., Beiderman, B., Steinberg, F. & Brothers, V. M. (1982) Mol. Pharmacol. 22, 204-210. 17. Rommens, J. M., Iannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J. R., Tsui, L. C. & Collins, F. S. (1989) Science 245, 1059-1065. 18. Gros, P., Croop, J. & Housman, D. (1986) Cell 47, 371-380. 19. Homcy, C. J., Vatner, S. F. & Vatner, D. E. (1991) Annu. Rev. Physiol. 53, 137-159. 20. Feldman, M. D., Copelas, L., Gwathmey, J. K., Philips, P., Warren, S. E., Schoen, F. J., Grossman, W. & Morgan, J. P. (1987) Circulation 75, 331-339.
Cloning and characterization of a sixth adenylyl cyclase isoform: Types V and VI constitute a subgroup within the mammalian adenylyl cyclase family SHUICHI KATSUSHIKA*, LIANG CHENt, JUN-ICHI KAWABE*, RAMASWAMY NILAKANTANt, NANCY J. HALNON*, CHARLES J. HOMCYtt, AND YOSHIHIRO ISHIKAWA*§ Departments of *Pharmacology and tMedicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032; and tMedical Research Division, American Cyanamid Company, Pearl River, NY 10965
Communicated by Vincent T. Marchesi, June 8, 1992
A sixth member of the mammalian adenylyl ABSTRACT cyclase family has been isolated from a canine cardiac cDNA library. This isoform is more highly homologous to type V than to the other adenylyl cyclase types; sequence similarity is apparent even in the transmembrane regions where the greatest divergence among the types exists. Type VI mRNA expression is most abundant in heart and brain; however, unlike type V, a low level of expression is also observed in a variety of other tissues examined. Type VI adenylyl cyclase can be stimulated by NaF, guanosine 5'-[rthio]triphosphate, and forskolin but not by Ca2+/calmodulin, whereas it is inhibited by adenosine and its analogues. Comparison of both their structural and biochemical properties suggests that ypes V and VI constitute a distinct subgroup of the mammalian adenylyl cyclase family.
MATERIALS AND METHODS Isolation of Clones. Screening and hybridization were performed with an Aat 1-HincIl fragment from type I adenylyl cyclase cDNA as a probe (1, 8). Transient Expression in CMT Cells and Adenylyl Cyclase Assay. A 4.0-kilobase (kb) EcoRI-Ssp I fragment, which was constructed by ligating a 0.9-kb EcoRl-Sph I fiagment from clone 6 and a 3.1-kb Sph I-Ssp I fragment from clone 27 and contained the entire coding sequence, was subcloned into the EcoRIf-EcoRV sites of the polylinker of pcDNA-amp (pcDNAamp 27-6). Transfection into CMT cells (derived from monkey COS cells) (9) and the adenylyl cyclase assay were performed as described (5). Northern Blot Analysis. Poly(A)+ RNA from various tissues and cells was prepared as described (5). The 5.4-kb fragment from clone 27 was used as the probe for the detection of type VI adenylyl cyclase mRNA. Sequence Homology Analysis. All six members of the mammalian adenylyl cyclase family (1-5) were analyzed for their amino acid sequence homology by using the multiplesequence analysis program PILEUP (10). The similarity scores were calculated and used to draw a dendrogram (11). Homology in different regions of the enzyme among the various isoforms was assessed similarly. Pairwise comparisons between various regions of these cyclases were performed with the program GAP (10). The putative cytoplasmic domains were aligned, together with the C-terminal portion of a mammalian membrane-bound type of guanylyl cyclase (6), by using MACVECTOR 3.5 (12).
Since the cloning of the first mammalian adenylyl cyclase cDNA, four additional adenylyl cyclase cDNAs have been isolated (1-5). Although each has a distinct tissue distribution and biochemical characteristics, the proteins they encode share the same topology: tandem repetition of a hydrophobic putative transmembrane domain that spans the membrane six times, followed by a large hydrophilic cytoplasmic domain. The amino acid sequence of the cytoplasmic domain is relatively well conserved among the family members; this region also shows homology to other cyclases, such as guanylyl cyclase (6) or adenylyl cyclases from lower eukaryotes (7). However, the amino acid sequence differs significantly in the transmembrane regions of the different adenylyl cyclase types (4). The degree of this divergence differs among different members; type II adenylyl cyclase is more homologous to type IV than to other types, whereas type I and III share a lesser degree of homology with the others. Two of the adenylyl cyclases (types II and IV) are similar not only in their amino acid sequence but also in their structure and function; both have a truncated C-terminal tail and lack calmodulin sensitivity. Their tissue distribution, however, is different. This greater degree of similarity between types II and IV suggests that certain adenylyl cyclase isoforms might constitute subclasses within the adenylyl cyclase family. We have recently cloned a cardiac adenylyl cyclase (type V), which appears to be limited in its expression to heart and neural tissue (5). We now describe a type VI adenylyl cyclase;¶ it exhibits a much higher degree of homology, both in sequence and structure, with type V adenylyl cyclase than with the other known family members. Furthermore, its tissue distribution and biochemical features are also similar. Types V and VI may therefore form a subclass within the mammalian adenylyl cyclase family.
RESULTS Cloning of Type VI Adenylyl Cyclase cDNA. In the primary screening, using a low-stringency hybridization protocol, we obtained 160 clones that hybridized with the probe. We finally obtained four clones, nos. 8, 25, 27, and 113. Three clones, nos. 8, 25, and 113, encoded type V adenylyl cyclase. However, clone 27, containing a 5.4-kb insert, was clearly different from the other clones albeit highly homologous to type V adenylyl cyclase. Among several hybridizing clones thereafter identified, clone 6 contained an additional 280 base pairs (bp) of 5' sequence. This included the initiator ATG in
the context of a reasonable Kozak consensus sequence, in comparison to other types of mammalian adenylyl cyclase (types I-V), in a long open reading frame (13). The 5' untranslated region is remarkable for its high G+C content
(96%) (Fig. 1).
This isoform was designated as type VI adenylyl cyclase. A hydropathy analysis identified the common motif of tan-
§To whom reprint requests should be addressed. IThe sequence reported in this paper has been deposited in the
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
GenBank data base (accession no. M94968).
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 89 (1992)
8775
CGGCCGGGCGGGCTGCGGGCGGCGAGGCTCCGCCGGGGCGCGGGCGGCGGGGGGCGCGGGGCGGCCGGCCGGGCCGGAGCCCGGGGGGCGGCGGGGCGGGG 101 TCCGGGGCGGCGCGGAGCGGGGCCGGCAGC)ATGTCGTGGTTAGTGGCCTCCTGGTCCCCAAAGTGGATGAACGGAAGACAGCCTGGGGTGAACGCAATG N S N F S G L L V P K V D E R K T A N G E R N
201 201 GCCACAACCGSCCACCCCCGCCGACSCGCACCACTGCCCTCTCCACCCCCCGCTATATCACCSCCCTCCGCCATGCGCAGCCCCCCAGSCCCACCCCTGC G Q K R P R R G T R T S G F C T P R Y N S C L R D A Q P P S P T P A 301
R
P
P
C
P
Q
e
D
A
R
I
F
R
G
G
P
X
C
G
C
T
G
L
R
L
A
A
V
G
L
F
90
GAGGACACTGAGGCCATCTCAGCCCTTCGCGCAGCTCCAGCTCGCCCTGACGTCACCCCCGCCAGTAGCCCATCCTGCTGGCGCCGTCTGCCCCAGGTGT e
C
T
D
A
S
N
G
V
A
A
G
A
G
G
P
D
T
V
G
P
R
S
R
R
C
S
R
L
A
123
V
Q
501
TCCAGCCCAACCAGTTCCCCTCGCCAACCTGGAGCGCCTGTACCAGCGTACTCTI-TCACATCAACCACAGCACCCTGACGCTGCTCATCGCCGTGCT
601
GGTGCTGCCSACAGCCCTGCTGCTAGCCTTCCATCCTGCACCTGCCCCCCCTCAGCCTCCCTACGSCCCCCTCCTCCCCTGTCCCGCCACCCTCTTCGTG
Q
F
S
V
701
57
GGCTCCCCCTCGGTGCCCCTGCCAGGATGAGGCCTTCATCCGGAGACCGGCCCGGGCAAGGGCACGGAGCTGGGGCTGCGGGCCCTGGCCCTGGGCTTC A
401
23
Q
K
L
L
T
R
F
A
A
S
V
L
e
L
K
L
A
F
R A
N
Q
Y
L A
P
R
R
A
Y
F
Q
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N
N
A
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Y
V
S
0
A
S
T
L
L
L
A
L
L A
C
A
N
T
A
L
L
V V
F
157 190
GCGCTCATGGTGGTCTGTAACCGCCACAGCTTTCCCCAGGACTCCATGTGGGTTGAGCTACGTCCTGCTGCTGGCCATCCTGGCAGCCGTTCAGGTTCfGG A
L
N
V
V
C
N
R
N
S
F
R
Q D
S
N
V
V
S
Y
V
V
L
G
I
L
A
A
V
Q
V
G
601 GTGCCCTGGCACCAACCCCCGCAGCCCCTCTGTGGGCCTCTGGTGCCCTGTGTTTTTTGTCTACATCACCTACACGCTCCTACCCATCCGCATGCGGC G A L A A N P R S P V G L C P V F F V Y I T Y T L L P I R N R A 901 AGCTGTCTTCAGTGGCCTGGCCTGTCCACCCTGCATTTGATCTTGGCCTGGCAACTCAACCGCGGTGACGCCTTCCTCTGGAAGCAGCTCGGTGCCAAC A V F S G L G L S T L N L I L A K Q L G A N Q L N R G D A F L 1001 ATCTGCTGTTCCTCTCACCAACGTCATTGCATCTGCACACACTACCCACTGAGGTCTCTCAGCGCCAGCCTTCAGGAGACCCGCGGTTACATC L L F L C T N V I G I C S N Y P A e V S 0 R Q A F Q e T R G Y s N 1101 AGGCCCGGCTGCACCTGCCAGATGACCCAGCAGGAACGCTCTGCTGTGTGTTGCCCAGCATGTTGCCATGGAGATGAAAGAAGATATCAA Q A R L N L P D e N R Q Q R L L L S V L P Q N V A N e N K E D I N 1201 C AAGCATGTGCCACAAGATCTACATCCAGAAGCATGACAATGTCACATCCTGTTTGCAGACATTGAAGGCTTCACCACCTGGCG T K K e D N N F N K I Y I Q K H D N V S I L F A D I E G F T S L A 1301 TCCCAGTCACCGCGCAG T CTGAACGAGCTC~GA GCCCGTGAAGCTGGCTCGGAAAATCACTGCCTGAGGATCAAGATCT S Q C T A Q e L V N T L N e L F A R F D K L A A E N H C L R I K I 1401 TACCCGACTGTTACTACTGTCTCTGTCCCCCCTCCCACCCCCCGCACATCCACCCCCACTGCTGTCGTACATCGGCGTGCACATGATCGAGGCCATCTC L G D C Y Y C V S G L P e A R A D H A N C C V e N G V D N I e A I S 1501 GCTGGTCGTGAGGTGACAGGTGTGAACGTGAACATCCGTGGGCATCCACAGCGCGTGTGCACTGTGGTGTTTGGCCTGCGGAAATGGCAGTTC L V R C V T G V N V N N R V G I N S G R V H C G V L G L R K Q F 1601 CACCTGTCCTCCAATCACCTGACTCTGCCCAACCATATCGACGCCGCCCCGGCCGGCCGCATCCACATCACCCCGGCCACGCTCCAGTACCTGAACGCGG D V N N D V T L A N N N C A A R A G R I H I T R A T L Q Y L N G 17 01 ACTACGAGGTGGAGCCCGGCCGCGGTGGCGAGCGGAACGCGTACCTCAAGGAGCAGCACATCGAGACCTTCCTCATCCTGGGAGCCAGCCAGAAACGGAA D Y Z V C P G R G C c R N A Y L K C Q H I C T F L I L G A S Q K R K 1501 ACAGGAGAAGCCCATCCTGCCCAAGCTCGCAGCCCACCCGGCCAACTCCATGCAAGCCTGATCCCACCCTCGCTGCCCGACCGCGCCTTCTCCCCGGACC C C K A N L A K L Q R T R A N S N C C L N P R V P D R A F S R T 1901 AAGGACTCCAAGGCTTTCCGCCAGATGGCATTGATGATTCCAGCAAAGACAACCGGGTGCCCAAGATGCCCTGAACCCCGAGGATGAGGTCGATGAGT K D S K A F R Q N G I D D S S K D N R G A Q D A L N P e D E V D E 2001 TCCTGGCCGTGCCATCGATGCCCGCAGCATCGATCAGCTACGGAAGGACCATGTGCCCGCTTCCTGCTCACCTTCCAGAGAGAGGATCTTGAAAAGAA F L C R A I D A R S I D Q L R K D H V R R F L L T F Q R e D L e K K 2101 GTACTCA G AGCCTACGTGTCGCTGTTGGTCTTCTCTTCATCTCTTTACACTCCTCGTCTTCCCACAC Y S R K V D P R F G A Y V A C A L L V F C F I C F I Q L L V F P H 2201 TCAACCGTGATGCTTGGGATCTACGCCAGTATCTGTGTGTTCTGATCACCGTCTGACCTGTGCCGTGTACTCCTGTCTCTCTCTTCCCCAAGG S T V N L G I Y A s I F V L L L I T V L s C A V Y S C G S L F P K 2301 CCCTCCGACGTCTTTCCCGCAGCATCCTCCGCTCTCGGCACACACCACTGTCCTTYGCATITrTCACTCTGCCTACTGTTCACCTCTGCCATCGCCAA A L R R L S R S I V R S R A N S S V V G I F S V L L V F T S A I A N 2401 CATCTTCACCITGAACCACACCCCCATCCGGACCTCGCCACCCCCGATGCTGCATTCAACACCCCCTGACATCACTGCCCTCCACCTCCACCAGCTCAAT N F T C N N T P I R T C A A R N L N V T P A D I T A C H L Q Q L N 2501 TACTCTCTGCCCCTCCATCCTCCGCTCTGTCAGCCCACCGCACCCACTTCCAGCTTCCCTGAGTACTTCCTTGCCAACATCCTCCTCAGTCTCTTGGCCA Y S L G L D A P L C e G T A P T C S r P c Y F V G N N L L S L L A 2601 GCTCTCTTTrCCTCCACATCAGTAGCATCGGCGAMGCTTCCATCATCTTTGTCCTGCGCCTCAITATTTCTGCCTGCTTCTCCTGCGCCCCCCCAGCAC S S V F L N I S S I G K L A N I r V L G L I Y L V L L L L G P P S T 2701 CATCTTTGACAACTATGACCTGCTGCTTGGTGTCATGCTTGCTTCTTCCATGACACCTTTATGGCTGACTGCCCAGCTCGGAGGGTGGCA I F D N Y D L L L C V N G L A S S N D T F D G L D C P A A G R V A 2801 CTGATACATGACCCTGTGATTCTGCTGGTG~rGCCCTCGCTGTATCTGCAGC CAGTGGAATCACTCACGTCTGGACTTCCTCTGGA L K Y N T P V I L L V r A L A L Y L N A Q Q V C S T A R L D F L 2901 AACTCCACGCAACCGGGGACAACGACCAGATCGAGGAGCTCCAGGCCTACAACCCAACCCTCCTGCATAACATTCTGCCTAAGCACGTGGCTGCCCACTT K L Q A S C C K C C N C C L Q A Y N R R L L H N I L P K D V A A H F 3001 CCTGCCCGGGAGCGCCGAATGAGCTCTACTACCAGTCGTGTGAGTG CGTCATGTTaCCTCCATTGCCAACTTTTCTGAGTTCTATGTG L A R C R R N D C L Y Y Q s C c C V A V N F A S I A N F S e F Y V 3101 GACTGGACAAACAATGAGGGTGTCGAGTCCTCGGCTGCTCACGAATATCGCCGATTTGATAATCATCAGGAGCGGTTCCGCAGC e L e A N N e c V e C L R L L N e I I A D F D e I I S e e R F R Q 3201 TGGAGATCAAGACGATCGGTAGCACGTACATGGCTCGTCGGCTGACGCCAGCACCTACGATCAGGCCGGCCGCTCCCACATCACTGCCCTGGC L K I K S I C S S Y N A A s G L N A S T Y D Q A G R S H I T A L A 3301 CGACTATCCATCGGCTCATGGACAGATGAAACACAACGACACTCCTTCAACAACTTCCAATGAATTGGCTGAACATGGGCCCAGTTGTG D Y A N R L N N S N Q N K N I N F N N F K I G L N M G P V V 3401 GCAGCCCTCATTGCCGCTCIGAACCCACACTATGACATCTCCGCGAACACGGTGAAITCTCTCAGCCGTATGGACAGCACGGCGGTTCCTGACCGAATCC A G V I C A R K P r D I N G N S V N V S S R N D S T G V P D R I 3501 ACCTGACCACCGAC,,GTACCAGGTTCTACTCCAAACGTACCAGCTGGAGTGTCGAGGGTGGTCAAGGTGAAGGCAGGAGATGACCACCTA Q V T T D L Y Q V L A A K R Y Q L C R C V V K V K G K G e M T T Y 3601 CTTCCTtAATGGGCCCCCCCAGTTAGCAGA GCTACAAGTTCAcTGTCAGGACCAAWTGGGCATGTGGACTCTGTGCTGGATGG F L N CGC P P S 3701 ACCTCTCCCCCGCCGCACCAAGCCTCCAGACCCTGCCTCCCACACCAACACCCAC CACTTCCTTCCACCATCTCGTCTGCCCTCAGGCTGG 3601 TGAACAAGGGATACCAAGAGGATTATGCAAGTGACTTrTATTTCTAATTGGGGTAGGCsTGGCTGTTCCCTCTTTCCTGCTTTGGGAGCAGGGG 3901 AGGCAGCTGCAGCAGAGGCAGCAGGAGCCCTCCTGCCTGAGGGTTTAAAATGCCAGCTTGCCATGCCTACCCTTTCCCCTGTCTGTCTGGGCAACAGCAT 4001 CCGGGCTCGGCCCTTCCTT*TCCCTCTTTTTCCIGCCAATAITrFCT
223 257 290 323 357 390 423
457 490 523 557 590 623 657 690
723 757
790
823
857 890 923 957 990
1023
1057 1090
1123 1157 1165
FIG. 1. Nucleotide and annotated amino acid sequence of type VI adenylyl cyclase. The entire coding sequence is shown together with part, of the 5' and 3' untranslated regions.
dem repetition of six transmembrane spans followed by a large hydrophilic cytoplasmic domain (14). A dot matrix comparison showed a high sequence homology with type I in the cytoplasmic domains, with a lesser degree in the transmembrane domains. However, when the type VI sequence was compared with that of the type V isoform, the homology was strikingly higher. Sequence Comparison with Other Types. A more extensive analysis of sequence homology among the different types of adenylyl cyclase was performed. The amino acid sequence along different regions of the molecule was compared among the different types by using the GAP program. Such a comparison for the first transmembrane domain and the second cytoplasmic domain is shown in Fig. 2. Both domains are more similar between types II and IV and between types V and VI than for any other set of pairs. There is less homology between the two transmembrane domains within the same molecule (27% in type VI, for example). It is interesting that the homology in the second cytoplasmic domain between
types III and VI is lower than the homology between types
V and VI in the first transmembrane domain despite the fact that the homology is typically highest in the cytoplasmic domains among different types of adenylyl cyclase. The sequence homology is highest in the initial half of each of the cytoplasmic domains. The latter half exhibits a lower degree of homology among the different types or is totally lacking as in types I and III. The amino acid sequence of the second cytoplasmic domain of the different types and that of the C-terminal portion of guanylyl cyclase (6) were aligned for comparison (Fig. 3). Multiple residues in this region are highly conserved among the mammalian cyclases and it is in this region where the highest degree of homology between the cytoplasmic loops is also apparent. A dendrogram based upon the homology score among the various mammalian adenylyl cyclase family members was drawn (Fig. 4). Types I and III clearly diverge from the others and the degree of divergence is somewhat greater in type III. Types II and IV exhibit a high degree of homology with each other, while
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type I 100
I II III IV V VI TM1
100 22 26 29 28 30 I
Proc. Nad. Acad. Sci. USA 89 (1992)
II 55
III 54
IV 56
V 59
100
52 100
76 52 100
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CPD2
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58 56
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52
51
57 100
59 92 100
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100 31
100
25
100 28 23 26
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III
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48 19
-Type V 'Type VI
-Type III
VI
type
FiG. 2. Sequence homology among mammalian adenylyl cyclase
(TM1) and the initial half of the second cytoplasmic domain (CPD2) were compared for percent identity as described in Materials and Methods (10). The residues used for comparison from each type are as indicated: type I, 64-237 for TM1 and 807-1070 for CPD2; type 45-211 forTMl and 822-1088 forCPD2; type III, 79-247 forTMl and 859-1132 for CPD2; type IV, 29-195 for TM1 and 930-1184 for CPD2; type V, 165-322 for TM1 and 930-1184 for CPD2; and type VI, 151-308 for TM1 and 912-1165 for CPD2. 11,
types V and VI share an even higher degree of homology. Thus types II/IV and V/VI distinguish themselves as homologous pairs. V
IV III II I
II
I
0.6
0.8
I 1.0
Similarity Score
types I-VI. Amino acid sequences from the first transmembrane
VI
-Type II Type IV
FIG. 4. Similarity relationship among the different types of mammalian adenylyl cyclase. The amino acid similarity scores were calculated as described in Materials and Methods (11). The calculated values indicate the position that divides the branches into subfamilies. Callular ExpressIon and Measurement of Catalytic Activity. Type VI adenylyl cyclase was transiently expressed in CMT cells. There was a concentration-dependent increase in adenylyl cyclase catalytic activity in transfected cell membranes when the amount of plasmid DNA was varied from 0 to 20 A.g (data not shown). Control cells were mocktransfected and similarly induced. Significantly higher catalytic activity in the transfected as compared to control membranes was obtained under either basal or stimulated (NaF, guanosine 5'-[(thio]triphosphate, or forskolin) con-
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FiG. 3. Comparison of the amino acid sequences of mammalian adenylyl cyclases (types I-VI) and guanylyl cyclase (GC) (6). Sequences were aligned for comparison by using MACVECTOR 3.5 (12). Amino acid residues are numbered at the right of each line. The amino acid sequences of a portion of the putative second cytoplasmic loop, type I (805-1063), type 11 (820-1081), type II (857-1125), type IV (804-1057), type V (928-1182), and type VI (910-1165), as well as the sequence of the C-terminal portion of guanylyl cyclase (810-1053), were compared.
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Katsushika et al. Table 1. Expression of type VI adenylyl cyclase in CMT cells Activity, pmol/(min-mg of protein) Forskolin Cells Basal NaF GTP[yS] Control 29 ± 5 59 ± 10 4 ± 0.6 12 ± 3 45 ± 5 110 ± 11 211 ± 25 Transfected 9 ± 0.8 Adenylyl cyclase activity in membranes (control cells versus cells transfected with pcDNAamp 27-6) was measured in the absence (basal) or presence of various activators: NaF (10 mM), guanosine 5'-[L-thio]triphosphate (GTP[yS], 100 jtM), forskolin (100 ,uM). Values are means ± SEM. For each condition (basal or activated), activity in control membranes was significantly lower than that in membranes from transfected cells (P < 0.001). Similar results were obtained in three independent experiments. For each experiment, the results represent the average obtained from six independently transfected plates of CMT cells.
ditions, with the forskolin-stimulated activity showing the greatest increase (Table 1). Adenylyl cyclase has been shown to be inhibited by adenosine and its analogues through an allosteric or P site (5, 15). We found that adenylyl cyclase activity in the transfected cells was inhibited in a concentration-dependent manner by adenosine and its analogues (Fig. 5). The order of potency was identical for types V and VI: 2'-deoxy-3'-AMP > 3'AMP > 2'-deoxyadenosine > adenosine. The Ca2+/ calmodulin sensitivity of type VI adenylyl cyclase was also examined in the transfected CMT cell system. Like type V adenylyl cyclase, type VI was also inhibited in a concentration-dependent manner by the addition of 0-1 mM Ca2+. Calmodulin (200 nM) did not alter this inhibition (Fig. 6). Northern Blotting. The tissue distribution of type VI mRNA was examined by analysis of poly(A)+ RNA prepared from various tissues (Fig. 7). A single message of -6 kb was widely distributed among all the tissues examined. Brain and heart expressed 4- to 7-fold more message than the other tissues as assessed by densitometry. Cell lines such as S49 (mouse lymphoma), A10 (rat smooth muscle), and GH4 (rat pituitary tumor) also expressed type VI adenylyl cyclase mRNA. Thus, in all tissues and cell types that we examined, a low level of type VI adenylyl cyclase mRNA was apparent except for heart and brain, where the level of expression was significantly greater. 100 90
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Ca2+(mM) FIG. 6. Effect of Ca2+/calmodulin on type VI adenylyl cyclase activity. Adenylyl cyclase activity in membranes (from CMT cells transfected with pcDNAamp 27-6 or mock-transfected control) was measured in the presence of various concentrations of Ca2+ (0-1 mM). Membrane preparations were washed with EGTA prior to assay (16). Similar results were obtained in three independent experiments. e, Transfectant membrane; o, transfectant membrane with calmodulin (200 nM); o, control membrane; *, control membrane with calmodulin (200 nM).
DISCUSSION Six adenylyl cyclase family members have thus far been cloned and characterized (1-5). They share a motif of two tandem repeats of six transmembrane spans separated by a large cytoplasmic loop and terminating in a similarly large cytoplasmic tail. This motif also characterizes a larger superfamily of transporters including the cystic fibrosis gene product and the multiple-drug-resistance gene product (17, 18). Based on homology comparisons, certain of the six adenylyl cyclase family members can be further segregated into discrete groups. Two sets of pairs, types II/IV and V/VI, become apparent when a relatively rigorous comparison of sequence identity is utilized as the index of homology. Types V and VI share the highest degree of homology and are kb
40
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40
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100
(A.M)
FIG. 5. Effect of adenosine and its analogues on type VI adenylyl cyclase activity. Membranes prepared from cells (transfected with pcDNAamp 27-6 or mock-transfected control) were preincubated with 5 mM Mn2+ and 100 gM forskolin for 10 min prior to the assay. *, Adenosine; o, 2'-deoxyadenosine; *, 3'-AMP; O, 2'-deoxy-3'AMP. Similar results were obtained in three independent experiments. Each point is the average of duplicate determinations. A similar degree of inhibition was obtained with type V adenylyl cyclase cDNA.
1.35-
H
B
T
S
K
L
FIG. 7. Northern blot analysis of mRNA from canine heart (H), brain (B), testis (T), skeletal muscle (S), kidney (K), and lung (L). Ten micrograms of poly(A)+ RNA was used per lane. An EcoRI 5.4-kb insert from clone 27 was used as a probe.
8778
Biochemistry: Katsushika et al.
>50% similar even in the putative transmembrane regions. It has been speculated that the poor conservation of amino acid sequence in the transmembrane regions among the other adenylyl cyclase isoforms is related to the likelihood that molecules of this class no longer function as transporters (4). Thus, the maintenance of homology in types V and VI, even in the transmembrane domains, is consistent with their being more closely related family members. Whether some functional activity, possibly shared by these adenylyl cyclase types, has also been maintained remains to be addressed. The greater degree of similarity between types II/IV and between types V/VI is also evident when certain shared structural features are compared. The length of the N-terminal tail is similarly shorter in types II/IV (44 and 28 residues, respectively) or longer in types V/VI (164 and 149 residues, respectively). Type I (63 residues) and type III (77 residues) possess an N-terminal tail that is intermediate in length. Although there is some homology in the N-terminal tail in the segment adjacent to the transmembrane domain (types V and VI, for example), the homology is generally low. Types V/VI contain consensus sequences for N-linked glycosylation at identical positions in the extracellular loop linking membranes spans 9 and 10. In types II/IV, these sites are similar in context but have clearly diverged in types I and III. Based on identity of sequence and these structural similarities, a pattern emerges wherein type III appears to have diverged at an early stage, thereafter types I/V/VT and types II/IV. Types II/IV and V/VI diverged from each other more recently (Fig. 4). In addition to differences in primary structure, the pattern of tissue distribution varies markedly among the adenylyl cyclase types. First, certain tissues appear to express several different types of adenylyl cyclase. For example, heart contains mRNA for types IV, V, and VI. The brain contains every isoform (types I-VI). By in situ hybridization (unpublished data), we have detected mRNA for types V and VI in cardiocytes but little in cardiac fibroblasts. Although type IV mRNA is also present in heart, its exact cellular distribution has not been reported. Finally, it is interesting that S49 lymphoma cells (16), which have been extensively used to study the G protein-adenylyl cyclase signal transduction pathway, express type VI adenylyl cyclase. The functional consequences of one cell type expressing more than one isoform are not known. Types V and VI, both of which are expressed in cardiocytes, exhibit profiles of biochemical properties that are quite similar. It may be that these isoforms will show distinct patterns of responsiveness to G-protein activation. Alternatively, these isoforms (types V and VI) may be differentially expressed in development or in response to physiologic and pathophysiologic stimuli. Several studies have indicated that adenylyl cyclase responsiveness, independent of abnormalities at the level of the (3-adrenergic receptor or G. protein, is impaired in heart failure (19, 20). Clarifying how these two closely related
Proc. Nad. Acad. Sci. USA 89 (1992)
isoforms, types V and VI, contribute to the abnormal pattern of adenylyl cyclase responsiveness in pathophysiologic states may identify factors that, either pre- or posttranslationally, impact differentially in regulating their expression and activity in the heart. We thank Drs. A. G. Gilman (University of Texas Southwestern Medical Center) and R. Reed (Johns Hopkins University) for providing us with adenylyl cyclase cDNAs, Dr. B. F. Hoffman (Columbia University) for his advice and encouragement, and Dr. S. Wrenn (Lederle Laboratories) for helpful discussions. We are grateful to Drs. D. Cooper (University of Colorado) and R. Iyengar (Mount Sinai Medical School) for sharing sequences of their cloned type V and VI adenylyl cyclases prior to publication. This work was supported in part by National Institutes of Health Grant HL38070; Joint Research Grants 63044118 and 01044120 from the International Scientific Research Program for the Ministry of Education, Science, and Culture of Japan; and a grant from Lederle Laboratories. 1. 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. 2. Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Natl. Acad. Sci. USA 88, 10173-10177. 3. Bakalyar, H. A. & Reed, R. R. (1990) Science 250,1403-1406. 4. Gao, B. & Gilman, A. G. (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182. 5. Ishikawa, Y., Katsushika, S., Chen, L., Halnon, N. J. & Homcy, C. J. (1992) J. Biol. Chem. 267, 13553-13557. 6. Chinkers, M., Garbers, D. L., Chang, M. S., Lowe, D. G., Chin, H., Goeddel, D. V. & Schulz, S. (1989) Nature (London) 338, 78-83. 7. Kataoka, T., Broek, D. & Wigler, M. (1985) Cell 43, 493-505. 8. Ishikawa, Y., Bianchi, C., Nadal-Ginard, B. & Homcy, C. J.
(1990) J. Biol. Chem. 265, 8458-8462. 9. Gerard, R. D. & Gluzman, Y. (1985) Mol. Cell. Biol. 5, 32313240. 10. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395. 11. Feng, D. R. & Doolittle, R. F. (1987) J. Mol. Evol. 25, 351-360. 12. Pustell, J. M. (1988) Nucleic Acids Res. 16, 1813-1820. 13. Kozak, M. (1989) J. Cell Biol. 108, 229-241. 14. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. 15. Tang, W.-J., Krupinski, J. & Gilman, A. G. (1991) J. Biol.
Chem. 266, 8595-8603.
16. Bourne, H. R., Beiderman, B., Steinberg, F. & Brothers, V. M. (1982) Mol. Pharmacol. 22, 204-210. 17. Rommens, J. M., Iannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J. R., Tsui, L. C. & Collins, F. S. (1989) Science 245, 1059-1065. 18. Gros, P., Croop, J. & Housman, D. (1986) Cell 47, 371-380. 19. Homcy, C. J., Vatner, S. F. & Vatner, D. E. (1991) Annu. Rev. Physiol. 53, 137-159. 20. Feldman, M. D., Copelas, L., Gwathmey, J. K., Philips, P., Warren, S. E., Schoen, F. J., Grossman, W. & Morgan, J. P. (1987) Circulation 75, 331-339.
