Proc. Natl. Acad. Sci. USA Vol. 89, pp. 9809-9813, October 1992
Biochemistry
Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases (cloning/expression/liver/kidney/guanine nucleotide stimulatory protein-stimulated)
RICHARD T. PREMONT, JIANQIANG CHEN, HAI-WEN MA, MADHAVI PONNAPALLI,
AND
RAVI IYENGAR
Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, NY 10029
Communicated by Alfred G. Gilman, July 14, 1992 (received for review March 27, 1992)
from 107 phage (9) of a >4.4-kilobase (kb) size-selected bovine cerebral cortex cDNA library in AZAP 11 (7). The product was subcloned in pBlueScript, sequenced, and used to screen a rat liver cDNA library in AZAP II by using standard methods (10). Positive inserts were recovered from the AZAP II vector by phagemid excision (11), and DNA sequences from double-stranded inserts in pBlueScript were determined by dideoxynucleotide chain-termination technique with specific oligonucleotide primers and T7 DNA polymerase (10). Ambiguous sequences were resequenced with 7-deaza-dGTP or dITP. Extreme 5' end clones were obtained by PCR screening of several rat liver and kidney cDNA libraries in various A phage vectors (9, 12). For each screening step, two nested antisense primers were prepared to known adenylyl cyclase sequences and used in sequential PCR reactions. In the first reactions, an aliquot of a cDNA library (107 phage) was amplified by using an antisense clone primer (21 bp) and one of a pair of primers that flank the virus multiple cloning site (i.e., T3 and T7 17-mer sequencing primers for AZAP II). Amplifications were performed for 35 cycles at 95TC for 1 min, 500C for 1 min, and 720C for 3 min. One microliter of the first reactions was used as template for second reactions using the same vector primer and the nested antisense clone primer (30-mer, containing an EcoRI site). Products of the second reaction (10 1.l) were separated on agarose gels and tested for hybridization to a third adenylyl cyclase sequence probe. Positive bands were subcloned into pBlueScript using EcoRI and sequenced. Sequences obtained from amplified DNAs and irregularities among independent clones (an intron, improper splice sites) were reconfirmed by amplifying and sequencing independent overlapping clones from liver first-strand cDNA with specific primers. Expression. Type 6 adenylyl cyclase clone was reconstructed from two 5' fragments amplified by PCR from liver cDNA (totaling 1 kb) and one clone isolated by hybridization from the liver library (2.5 kb). The type 6 cDNA was inserted into the BamHI and EcoRI sites of pcDNAI vector (Invitrogen, San Diego). Type 2 adenylyl cyclase (4) was inserted into the HindIII and BamHI sites of pcDNA1. Human kidney 293 cells were grown in Eagle's minimal essential medium with 10% horse serum. Cells were transfected with 5 Ag of vector DNA per 3 x 106 cells using DEAE-dextran according to Wong et al. (13). Transfected cells were grown for 24 hr and replated at 15,000-30,000 cells per well in a 24-well plate. The next day the cells were lysed in situ by hypotonic shock with 100 Al of 10 mM sodium Hepes/2 mM EDTA, pH 8.0/100 ,uM phenylmethylsulfonyl fluoride (freshly prepared). Adenylyl cyclase activity of lysates was immediately assayed by conversion of [a-32P]ATP to [32P]cAMP (14). Fifty microliters of a solution
cDNA encoding a hormone- and guanine nuABSTRACT cleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[y-thio~triphosphate, and isoproterenol plus GTP but is not stimulated by 13y subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory proteinregulated adenylyl cyclases. Types 5 and 6 enzymes derme one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission.
Hormone-stimulated adenylyl cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] is one of the best-studied guanine nucleotide-binding protein (G protein)-regulated signaling pathways. Detailed molecular information on the multiplicity of receptors and G proteins has been available for some time, but much less has been known about the effector until quite recently. Major progress was achieved with the development of affinity purification of adenylyl cyclase on forskolin-agarose (1) and the cloning of a calmodulinstimulated bovine brain adenylyl cyclase (2). This work has been followed by the recent cloning and characterization of three additional mammalian adenylyl cyclases (3-5). Hormone-regulated adenylyl cyclases have been functionally divided into two categories, calmodulin-sensitive and calmodulin-insensitive (6). Calmodulin-sensitive adenylyl cyclase activity is found predominantly in the brain, whereas liver and most other peripheral tissues contain only calmodulin-insensitive activity (6). We have had a long-standing interest in the glucagon-stimulated liver adenylyl cyclase system, and we set out to isolate the cDNA encoding the liver adenylyl cyclase. In this article we report on the isolation of cDNA clones encoding two distinct adenylyl cyclase enzymes that are highly related and widely expressed.*
MATERIALS AND METHODS Materials. Size-selected bovine cortex cDNA libraries in AZAP 11 (7), rat liver cDNA libraries in AZAP II (Stratagene) and in Agtll (Clonetech), and rat kidney libraries in AZAP II (Stratagene) and AgtlO (8) were used. Cloning. A 900-base-pair (bp) fragment encoding the final 300 amino acids of type 1 adenylyl cyclase (2) was amplified by PCR using specific primers from the published sequence,
Abbreviations: Gs, stimulatory guanine nucleotide-binding regulatory protein; GTP[yS], guanosine 5'-[y-thio]triphosphate. *The sequences reported in this paper have been deposited in the GenBank data base (accession nos. M%159 and M96160).
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. 9809
9810
Biochemistry: Premont et al.
containing adenylyl cyclase assay reagents (15), 500 ,uM isobutylmethylxanthine, and appropriate stimulators were added directly to the 293 cell lysates in wells to give a final assay volume of 150 pA. The by subunits of G proteins were purified from bovine brain membranes and stored as described for guanine nucleotide-binding regulatory proteins inhibitory and guanine nucleotide-binding regulatory proteins (16). The concentration of fBy subunits was determined by quantitative Coomassie blue staining of SDS/polyacrylamide gels (17). For f3y subunit experiments, cells were lysed in 70 ul, and the fPy subunits or storage buffer was added in 30 pl. Assays were incubated for 20 min at 30TC. Adenylyl cyclase activity was determined as pmol of [32P]cAMP formed per 106 cells. All assays were done with triplicate wells, and values shown are the means of the triplicate determinations. All experiments were repeated at least twice. mRNA Blotting. Total RNA was prepared from rat tissues by the guanidinium thiocyanate/phenol extraction method (10). Poly(A)+ RNAs were purified by using two sequential passages through oligo(dT)-cellulose spin columns (Pharmacia). Ten micrograms of poly(A)+ RNA was electrophoresed through 0.7% agarose-formaldehyde gels and transferred to nitrocellulose by capillary action (10). Probes were prepared by random-primed labeling of coding regions of the type 5 (3.2 kb) and type 6 (3.4 kb) sequences using [a-32P]dCTP (10). Blots were hybridized in 5 x standard saline phosphate/ EDTA (SSPE; lx SSPE is 0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA) in 50%o formamide at 45TC overnight and washed with 0.2x standard saline/citrate buffer at 700C before exposure to x-ray film for the indicated times. PCR-Based Detection of mRNA. One microgram of poly(A)+ RNA was reverse-transcribed in 20 dul by using SuperScript Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL) with 40 units of RNasin (Promega). Two pairs of oligonucleotides were prepared for use as primers for specifically amplifying equivalent 41100-bp fragments of the type 2 and the type 5/6 adenylyl cyclases, encompassing membrane spans 7-12. These primers are as follows: for type 2, 5'-CGACGGAGTCCTCAGCATCTC and 5'-CCTACAGTAATATTCACTCTG; and for types 5 and 6, 5'-CGGAAAGARGAGAAGGCCATG and 5'GCGRGCRGTRGATTCCACCTG (where R = A or G). PCR amplification reactions (9) were done by using 0.1 p.1 of first-strand cDNA from several rat tissues (35 cycles, 95TC for 1 min, 60TC for 1 min, 72TC for 3 min). Aliquots of reactions were incubated with specific restriction enzymes to ensure that products of expected size were digested into expected fragments: type 2, Sca I; type 6, Xho I; and type 5, Sac I. DNA fragments were resolved by electrophoresis. Sequence Analysis. Sequences were analyzed by using the PC/GENE software (IntelliGenetics). Sequence alignments were by the CLUSTAL program, with minor adjustments by hand. Identity and similarity were calculated from adjusted pairwise alignments. Similar residues are defined as A,G,P,S,T; D,E,N,Q; F,Y,W; I,L,M,V; and H,K,R.
RESULTS Cloning of Liver Adenylyl Cydases. A 900-bp fragment encoding the final 300 amino acids of the bovine brain adenylyl cyclase (2) was used to probe a rat liver cDNA library in AZAP II. Positive clones from the liver library were used to identify additional overlapping clones encoding two distinct adenylyl cyclase sequences (types 5 and 6). Screening of >107 phage with various probes led to the identification offour independent type 5 and five type 6 clones but no 5'-end sequences. The sequences of the 5' regions were obtained by screening several rat cDNA libraries with a PCR technique similar to that described by Gibbons et al. (12), using sequential amplifications between vector primers and nested
Proc. Natl. Acad. Sci. USA 89 (1992)
clone primers. The deduced amino acid sequences of the rat type 5 and type 6 adenylyl cyclases are aligned in Fig. 1. The type 6 adenylyl cyclase is predicted to have 1180 amino acids and a molecular mass of 131,978 Da for the protein backbone. The type 5 adenylyl cyclase has >1098 amino acids, but no candidate initiator methionine has yet been found after screening six libraries. Expression of Cloned Adenylyl Cyclases. The cloned type 6 and type 2 (4) adenylyl cyclases were inserted into the mammalian expression vector pcDNA1 under the control of the cytomegalovirus promoter. These constructs were transfected into the human kidney 293 cell line to induce transient expression. Transfection with the type 6 or type 2 constructs leads to increased basal adenylyl cyclase activity (Fig. 2A). This activity is increased several-fold by addition of forskolin or by isoproterenol and GTP, indicating that the type 6 adenylyl cyclase is regulated by receptors via stimulatory guanine nucleotide-binding regulatory protein (Gs). Expression of the type 5 clone (engineered to convert Leu-40 to methionine) also results in enhanced adenylyl cyclase activity in 293 cells (J.C. and R.I., unpublished work). Recently fBy subunits of G proteins have been shown to specifically enhance Gs stimulation of the type 2 and type 4 adenylyl cyclases (5, 19). We tested whether the type 6 enzyme could also be regulated in a similar fashion. Type 6 adenylyl cyclase expressed in 293 cells was stimulated with guanosine 5'-[Iiythio]triphosphate (GTP[yS]) to activate Gs, and the effect of adding a saturating concentration (100 nM) of purified bovine brain Py subunits was assessed. As expected, under the conditions where the type 2 enzyme was highly (=3-fold) stimulated by exogenous fry subunits, the type 6 enzyme was not significantly affected (Fig. 2B). Adenylyl Cyclase Subfile. Mammalian Gs-regulated adenylyl cyclases appear to share a similar overall topology, with two sets of six putative membrane-spanning regions, a large central cytoplasmic loop, and a long cytoplasmic tail (2). Analysis of the type 5 and 6 sequences indicates the presence of at least 12 membrane spans, which are shown as overlines in Fig. 1. Residues 33-53 in the type 5 clone also have a relatively high hydrophobicity (which is absent in the type 6 sequence), which may be an additional membrane span. However, regions in both the first and second conserved domains of the types 1 and 4 enzymes, which are predicted to be cytoplasmic, also have similar hydrophobicity. Whether these regions are truly membrane spanning or are within globular structures will have to be experimentally determined. Both the type 5 and 6 sequences have two predicted sites for N-glycosylation between predicted spans 9 and 10 and another between spans 11 and 12. Comparison of the known mammalian adenylyl cyclase sequences reveals that in the two large cytoplasmic domains there are substantial stretches of near-identity and high overall similarity. Conserved regions among all six sequences (with >60%o similarity) are shown as darkened bars in Fig. 3A. Both of these regions also share similarity with cloned guanylyl cyclases (20) and have been proposed to encode the catalytic site (2). Comparison of the type 5 and type 6 sequences in these two 250-amino acid presumed catalytic regions reveals that they share 96% amino acid similarity in both domains. Additional regions of homology seen between types 2 and 4 and between types 5 and 6 are shown as darkened bars in Fig. 3 B and C, respectively. The sequences were analyzed for predicted protein kinase A phosphorylation sites (Fig. 3D). Both the type 5 and 6 sequences have a conserved putative protein kinase A phosphorylation site at the end of the large central cytoplasmic domain near transmembrane span 7. This site is of potential significance because in analyzing the loci of change during hormone-induced desensitization of the chicken liver (15, 21) and S49 lymphoma cell adenylyl cyclase systems, we had
Biochemistry: Premont et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 1. Comparison of deduced amino acid sequences of the rat type S and 6 adenylyl cyclases. Alignment of rat type 5 and 6 adenylyl cyclase amino acid sequences. Residues identical in both sequences are indicated with 1, except that residues also identical in five other mammalian adenylyl cyclases (2-5, 18) are indicated with *. Positions with conservative substitutions between types 5 and 6 are indicated with A. Predicted transmembrane regions of the type 5 and 6 sequences are indicated by solid lines above the alignment.
found that one component appeared to be the cAMPdependent decrease in the activity of adenylyl cyclase itself (22). Mouse S49 cells contain type 6 adenylyl cyclase with this conserved protein kinase A site, whereas chicken liver contains both types 5 and 6 enzymes (22). This putative protein kinase A site was not conserved in any other subfamily. The type 4 enzyme has no putative protein kinase A sites, whereas the others (types 1-3) have one or two predicted sites. Four of the six mammalian adenylyl cyclases appear to group into two distinct subfamilies, composed of types 2 and 4 and types 5 and 6 (Fig. 3E). There is substantial (70-75%) overall homology between members of each of these two subfamilies, but the divergence between these two subfamilies is as extensive as their divergence from the type 1 and 3 enzymes (=50%). Thus, it appears that there are at least four distinct subfamilies of mammalian adenylyl cyclases. In addition, a partial adenylyl cyclase-related sequence from human brain (18), which contains the conserved region in the large cytoplasmic tail indicative that it is probably an adenylyl cyclase, could represent an additional subfamily. Tissue Distribution of Type 5 and 6 Adenylyl Cyclases. The size of the mRNAs encoding the type 5 and 6 adenylyl cyclases were determined by blotting 10 pg of poly(A)+ RNAs [after two sequential oligo(dT) chromatographies] with type 5 and type 6 cDNA probes. With the type 6 probe, a major 6.9-kb message was seen in all three tissues, with lowest abundance in the liver (Fig. 4A). Fainter bands at 5.5 and 4.2 kb were also observed. With the type 5 probe, several
distinct messages were apparent in brain and kidney of 8.3, 7.4, 6.4, and 5.6 kb (Fig. 4B). The brain showed the highest abundance of all type 6 messages, whereas liver showed only very faint signals after 5-day exposure. The significance of these multiple messages is currently not known. An extensive survey of the type 5 and type 6 mRNA distributions was undertaken by use of a sensitive PCR-based mRNA detection method. Specific primers were used to amplify a fragment of an expected size from tissue first-strand cDNAs, and the products were identified by electrophoresis. The primers were chosen so as to amplify a divergent region in the adenylyl cyclase sequences, which contained a unique restriction site allowing the amplified products to be verified by digestion with the appropriate restriction enzyme. The conditions for amplification were such that the type 2 sequence could only be detected in brain and lung, as had been demonstrated by mRNA blotting (4). Under the same conditions, both the type 5 and type 6 sequences were found in all tissues tested (Fig. 4C). These observations indicate that the type 5 and 6 enzymes may be widely expressed.
DISCUSSION Prior functional studies had predicted some adenylyl cyclase diversity (6). Molecular cloning studies have demonstrated a surprisingly greater extent of structural diversity. Of the cloned adenylyl cyclases, the calmodulin-sensitive form (type 1) was expected (2). Existence of the olfactory-specific (type 3) enzyme (3) is also not entirely surprising because
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Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Premont et al.
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Biochemistry
Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases (cloning/expression/liver/kidney/guanine nucleotide stimulatory protein-stimulated)
RICHARD T. PREMONT, JIANQIANG CHEN, HAI-WEN MA, MADHAVI PONNAPALLI,
AND
RAVI IYENGAR
Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, NY 10029
Communicated by Alfred G. Gilman, July 14, 1992 (received for review March 27, 1992)
from 107 phage (9) of a >4.4-kilobase (kb) size-selected bovine cerebral cortex cDNA library in AZAP 11 (7). The product was subcloned in pBlueScript, sequenced, and used to screen a rat liver cDNA library in AZAP II by using standard methods (10). Positive inserts were recovered from the AZAP II vector by phagemid excision (11), and DNA sequences from double-stranded inserts in pBlueScript were determined by dideoxynucleotide chain-termination technique with specific oligonucleotide primers and T7 DNA polymerase (10). Ambiguous sequences were resequenced with 7-deaza-dGTP or dITP. Extreme 5' end clones were obtained by PCR screening of several rat liver and kidney cDNA libraries in various A phage vectors (9, 12). For each screening step, two nested antisense primers were prepared to known adenylyl cyclase sequences and used in sequential PCR reactions. In the first reactions, an aliquot of a cDNA library (107 phage) was amplified by using an antisense clone primer (21 bp) and one of a pair of primers that flank the virus multiple cloning site (i.e., T3 and T7 17-mer sequencing primers for AZAP II). Amplifications were performed for 35 cycles at 95TC for 1 min, 500C for 1 min, and 720C for 3 min. One microliter of the first reactions was used as template for second reactions using the same vector primer and the nested antisense clone primer (30-mer, containing an EcoRI site). Products of the second reaction (10 1.l) were separated on agarose gels and tested for hybridization to a third adenylyl cyclase sequence probe. Positive bands were subcloned into pBlueScript using EcoRI and sequenced. Sequences obtained from amplified DNAs and irregularities among independent clones (an intron, improper splice sites) were reconfirmed by amplifying and sequencing independent overlapping clones from liver first-strand cDNA with specific primers. Expression. Type 6 adenylyl cyclase clone was reconstructed from two 5' fragments amplified by PCR from liver cDNA (totaling 1 kb) and one clone isolated by hybridization from the liver library (2.5 kb). The type 6 cDNA was inserted into the BamHI and EcoRI sites of pcDNAI vector (Invitrogen, San Diego). Type 2 adenylyl cyclase (4) was inserted into the HindIII and BamHI sites of pcDNA1. Human kidney 293 cells were grown in Eagle's minimal essential medium with 10% horse serum. Cells were transfected with 5 Ag of vector DNA per 3 x 106 cells using DEAE-dextran according to Wong et al. (13). Transfected cells were grown for 24 hr and replated at 15,000-30,000 cells per well in a 24-well plate. The next day the cells were lysed in situ by hypotonic shock with 100 Al of 10 mM sodium Hepes/2 mM EDTA, pH 8.0/100 ,uM phenylmethylsulfonyl fluoride (freshly prepared). Adenylyl cyclase activity of lysates was immediately assayed by conversion of [a-32P]ATP to [32P]cAMP (14). Fifty microliters of a solution
cDNA encoding a hormone- and guanine nuABSTRACT cleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[y-thio~triphosphate, and isoproterenol plus GTP but is not stimulated by 13y subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory proteinregulated adenylyl cyclases. Types 5 and 6 enzymes derme one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission.
Hormone-stimulated adenylyl cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] is one of the best-studied guanine nucleotide-binding protein (G protein)-regulated signaling pathways. Detailed molecular information on the multiplicity of receptors and G proteins has been available for some time, but much less has been known about the effector until quite recently. Major progress was achieved with the development of affinity purification of adenylyl cyclase on forskolin-agarose (1) and the cloning of a calmodulinstimulated bovine brain adenylyl cyclase (2). This work has been followed by the recent cloning and characterization of three additional mammalian adenylyl cyclases (3-5). Hormone-regulated adenylyl cyclases have been functionally divided into two categories, calmodulin-sensitive and calmodulin-insensitive (6). Calmodulin-sensitive adenylyl cyclase activity is found predominantly in the brain, whereas liver and most other peripheral tissues contain only calmodulin-insensitive activity (6). We have had a long-standing interest in the glucagon-stimulated liver adenylyl cyclase system, and we set out to isolate the cDNA encoding the liver adenylyl cyclase. In this article we report on the isolation of cDNA clones encoding two distinct adenylyl cyclase enzymes that are highly related and widely expressed.*
MATERIALS AND METHODS Materials. Size-selected bovine cortex cDNA libraries in AZAP 11 (7), rat liver cDNA libraries in AZAP II (Stratagene) and in Agtll (Clonetech), and rat kidney libraries in AZAP II (Stratagene) and AgtlO (8) were used. Cloning. A 900-base-pair (bp) fragment encoding the final 300 amino acids of type 1 adenylyl cyclase (2) was amplified by PCR using specific primers from the published sequence,
Abbreviations: Gs, stimulatory guanine nucleotide-binding regulatory protein; GTP[yS], guanosine 5'-[y-thio]triphosphate. *The sequences reported in this paper have been deposited in the GenBank data base (accession nos. M%159 and M96160).
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containing adenylyl cyclase assay reagents (15), 500 ,uM isobutylmethylxanthine, and appropriate stimulators were added directly to the 293 cell lysates in wells to give a final assay volume of 150 pA. The by subunits of G proteins were purified from bovine brain membranes and stored as described for guanine nucleotide-binding regulatory proteins inhibitory and guanine nucleotide-binding regulatory proteins (16). The concentration of fBy subunits was determined by quantitative Coomassie blue staining of SDS/polyacrylamide gels (17). For f3y subunit experiments, cells were lysed in 70 ul, and the fPy subunits or storage buffer was added in 30 pl. Assays were incubated for 20 min at 30TC. Adenylyl cyclase activity was determined as pmol of [32P]cAMP formed per 106 cells. All assays were done with triplicate wells, and values shown are the means of the triplicate determinations. All experiments were repeated at least twice. mRNA Blotting. Total RNA was prepared from rat tissues by the guanidinium thiocyanate/phenol extraction method (10). Poly(A)+ RNAs were purified by using two sequential passages through oligo(dT)-cellulose spin columns (Pharmacia). Ten micrograms of poly(A)+ RNA was electrophoresed through 0.7% agarose-formaldehyde gels and transferred to nitrocellulose by capillary action (10). Probes were prepared by random-primed labeling of coding regions of the type 5 (3.2 kb) and type 6 (3.4 kb) sequences using [a-32P]dCTP (10). Blots were hybridized in 5 x standard saline phosphate/ EDTA (SSPE; lx SSPE is 0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA) in 50%o formamide at 45TC overnight and washed with 0.2x standard saline/citrate buffer at 700C before exposure to x-ray film for the indicated times. PCR-Based Detection of mRNA. One microgram of poly(A)+ RNA was reverse-transcribed in 20 dul by using SuperScript Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL) with 40 units of RNasin (Promega). Two pairs of oligonucleotides were prepared for use as primers for specifically amplifying equivalent 41100-bp fragments of the type 2 and the type 5/6 adenylyl cyclases, encompassing membrane spans 7-12. These primers are as follows: for type 2, 5'-CGACGGAGTCCTCAGCATCTC and 5'-CCTACAGTAATATTCACTCTG; and for types 5 and 6, 5'-CGGAAAGARGAGAAGGCCATG and 5'GCGRGCRGTRGATTCCACCTG (where R = A or G). PCR amplification reactions (9) were done by using 0.1 p.1 of first-strand cDNA from several rat tissues (35 cycles, 95TC for 1 min, 60TC for 1 min, 72TC for 3 min). Aliquots of reactions were incubated with specific restriction enzymes to ensure that products of expected size were digested into expected fragments: type 2, Sca I; type 6, Xho I; and type 5, Sac I. DNA fragments were resolved by electrophoresis. Sequence Analysis. Sequences were analyzed by using the PC/GENE software (IntelliGenetics). Sequence alignments were by the CLUSTAL program, with minor adjustments by hand. Identity and similarity were calculated from adjusted pairwise alignments. Similar residues are defined as A,G,P,S,T; D,E,N,Q; F,Y,W; I,L,M,V; and H,K,R.
RESULTS Cloning of Liver Adenylyl Cydases. A 900-bp fragment encoding the final 300 amino acids of the bovine brain adenylyl cyclase (2) was used to probe a rat liver cDNA library in AZAP II. Positive clones from the liver library were used to identify additional overlapping clones encoding two distinct adenylyl cyclase sequences (types 5 and 6). Screening of >107 phage with various probes led to the identification offour independent type 5 and five type 6 clones but no 5'-end sequences. The sequences of the 5' regions were obtained by screening several rat cDNA libraries with a PCR technique similar to that described by Gibbons et al. (12), using sequential amplifications between vector primers and nested
Proc. Natl. Acad. Sci. USA 89 (1992)
clone primers. The deduced amino acid sequences of the rat type 5 and type 6 adenylyl cyclases are aligned in Fig. 1. The type 6 adenylyl cyclase is predicted to have 1180 amino acids and a molecular mass of 131,978 Da for the protein backbone. The type 5 adenylyl cyclase has >1098 amino acids, but no candidate initiator methionine has yet been found after screening six libraries. Expression of Cloned Adenylyl Cyclases. The cloned type 6 and type 2 (4) adenylyl cyclases were inserted into the mammalian expression vector pcDNA1 under the control of the cytomegalovirus promoter. These constructs were transfected into the human kidney 293 cell line to induce transient expression. Transfection with the type 6 or type 2 constructs leads to increased basal adenylyl cyclase activity (Fig. 2A). This activity is increased several-fold by addition of forskolin or by isoproterenol and GTP, indicating that the type 6 adenylyl cyclase is regulated by receptors via stimulatory guanine nucleotide-binding regulatory protein (Gs). Expression of the type 5 clone (engineered to convert Leu-40 to methionine) also results in enhanced adenylyl cyclase activity in 293 cells (J.C. and R.I., unpublished work). Recently fBy subunits of G proteins have been shown to specifically enhance Gs stimulation of the type 2 and type 4 adenylyl cyclases (5, 19). We tested whether the type 6 enzyme could also be regulated in a similar fashion. Type 6 adenylyl cyclase expressed in 293 cells was stimulated with guanosine 5'-[Iiythio]triphosphate (GTP[yS]) to activate Gs, and the effect of adding a saturating concentration (100 nM) of purified bovine brain Py subunits was assessed. As expected, under the conditions where the type 2 enzyme was highly (=3-fold) stimulated by exogenous fry subunits, the type 6 enzyme was not significantly affected (Fig. 2B). Adenylyl Cyclase Subfile. Mammalian Gs-regulated adenylyl cyclases appear to share a similar overall topology, with two sets of six putative membrane-spanning regions, a large central cytoplasmic loop, and a long cytoplasmic tail (2). Analysis of the type 5 and 6 sequences indicates the presence of at least 12 membrane spans, which are shown as overlines in Fig. 1. Residues 33-53 in the type 5 clone also have a relatively high hydrophobicity (which is absent in the type 6 sequence), which may be an additional membrane span. However, regions in both the first and second conserved domains of the types 1 and 4 enzymes, which are predicted to be cytoplasmic, also have similar hydrophobicity. Whether these regions are truly membrane spanning or are within globular structures will have to be experimentally determined. Both the type 5 and 6 sequences have two predicted sites for N-glycosylation between predicted spans 9 and 10 and another between spans 11 and 12. Comparison of the known mammalian adenylyl cyclase sequences reveals that in the two large cytoplasmic domains there are substantial stretches of near-identity and high overall similarity. Conserved regions among all six sequences (with >60%o similarity) are shown as darkened bars in Fig. 3A. Both of these regions also share similarity with cloned guanylyl cyclases (20) and have been proposed to encode the catalytic site (2). Comparison of the type 5 and type 6 sequences in these two 250-amino acid presumed catalytic regions reveals that they share 96% amino acid similarity in both domains. Additional regions of homology seen between types 2 and 4 and between types 5 and 6 are shown as darkened bars in Fig. 3 B and C, respectively. The sequences were analyzed for predicted protein kinase A phosphorylation sites (Fig. 3D). Both the type 5 and 6 sequences have a conserved putative protein kinase A phosphorylation site at the end of the large central cytoplasmic domain near transmembrane span 7. This site is of potential significance because in analyzing the loci of change during hormone-induced desensitization of the chicken liver (15, 21) and S49 lymphoma cell adenylyl cyclase systems, we had
Biochemistry: Premont et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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found that one component appeared to be the cAMPdependent decrease in the activity of adenylyl cyclase itself (22). Mouse S49 cells contain type 6 adenylyl cyclase with this conserved protein kinase A site, whereas chicken liver contains both types 5 and 6 enzymes (22). This putative protein kinase A site was not conserved in any other subfamily. The type 4 enzyme has no putative protein kinase A sites, whereas the others (types 1-3) have one or two predicted sites. Four of the six mammalian adenylyl cyclases appear to group into two distinct subfamilies, composed of types 2 and 4 and types 5 and 6 (Fig. 3E). There is substantial (70-75%) overall homology between members of each of these two subfamilies, but the divergence between these two subfamilies is as extensive as their divergence from the type 1 and 3 enzymes (=50%). Thus, it appears that there are at least four distinct subfamilies of mammalian adenylyl cyclases. In addition, a partial adenylyl cyclase-related sequence from human brain (18), which contains the conserved region in the large cytoplasmic tail indicative that it is probably an adenylyl cyclase, could represent an additional subfamily. Tissue Distribution of Type 5 and 6 Adenylyl Cyclases. The size of the mRNAs encoding the type 5 and 6 adenylyl cyclases were determined by blotting 10 pg of poly(A)+ RNAs [after two sequential oligo(dT) chromatographies] with type 5 and type 6 cDNA probes. With the type 6 probe, a major 6.9-kb message was seen in all three tissues, with lowest abundance in the liver (Fig. 4A). Fainter bands at 5.5 and 4.2 kb were also observed. With the type 5 probe, several
distinct messages were apparent in brain and kidney of 8.3, 7.4, 6.4, and 5.6 kb (Fig. 4B). The brain showed the highest abundance of all type 6 messages, whereas liver showed only very faint signals after 5-day exposure. The significance of these multiple messages is currently not known. An extensive survey of the type 5 and type 6 mRNA distributions was undertaken by use of a sensitive PCR-based mRNA detection method. Specific primers were used to amplify a fragment of an expected size from tissue first-strand cDNAs, and the products were identified by electrophoresis. The primers were chosen so as to amplify a divergent region in the adenylyl cyclase sequences, which contained a unique restriction site allowing the amplified products to be verified by digestion with the appropriate restriction enzyme. The conditions for amplification were such that the type 2 sequence could only be detected in brain and lung, as had been demonstrated by mRNA blotting (4). Under the same conditions, both the type 5 and type 6 sequences were found in all tissues tested (Fig. 4C). These observations indicate that the type 5 and 6 enzymes may be widely expressed.
DISCUSSION Prior functional studies had predicted some adenylyl cyclase diversity (6). Molecular cloning studies have demonstrated a surprisingly greater extent of structural diversity. Of the cloned adenylyl cyclases, the calmodulin-sensitive form (type 1) was expected (2). Existence of the olfactory-specific (type 3) enzyme (3) is also not entirely surprising because
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Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Premont et al.
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FIG. 2. Expression of cloned type 6 adenylyl cyclase in 293 cells. (A) Forskolin and hormone stimulation of type 6 adenylyl cyclase. Transfected cells [pcDNA1 only (m), type 2 adenylyl cyclase (u), or type 6 adenylyl cyclase (u)] were lysed and assayed with 10 mM MgCl2 in the presence of no stimulator (basal), 10 ,uM (each) isoproterenol and GTP, or 10 AM forskolin, as indicated. (B) Effects of exogenous fry subunits on GTP[yS]-stimulated adenylyl cyclase activity. Transfected 293 cells were lysed and assayed in the absence or presence of 10 MM GTP[yS] and 100 nM purified fPy subunits as indicated. Values are means of triplicate determinations. Coefficient of variance was
