©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Functional Domains of Adenylyl Cyclase Using in Vivo Chimeras (*)

(Received for publication, November 2, 1994; and in revised form, January 20, 1995)

Lonny R. Levin (§) Randall R. Reed (¶)

From the Department of Molecular Biology and Genetics and the Howard Hughes Medical Institute, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Adenylyl cyclase, the effector molecule of the cAMP signaling pathway, is composed of a family of isoforms that differ in their modes of regulation. Many of these modulatory interactions are dependent upon well characterized molecules from various second messenger pathways; however, very little is known about their mechanisms or sites of action on adenylyl cyclase. Chimeras were produced by a novel in vivo mechanism between two differentially modulated adenylyl cyclases to identify their regulatory domains. The basal activity of the type I adenylyl cyclase (AC1) is activated by calcium/calmodulin, inhibited by G protein beta subunits, and insensitive to protein kinase C regulation. In contrast, type II adenylyl cyclase (AC2) is insensitive to calcium/calmodulin regulation and is activated by G protein beta subunits as well as by activated protein kinase C. Expression and biochemical characterization of chimeras between AC1 and AC2 identified a single specific domain of AC1 responsible for calmodulin binding and a small, well defined region near the C terminus of AC2 required for protein kinase C activation.


INTRODUCTION

Adenylyl cyclase synthesizes the second messenger cAMP in response to a variety of extracellular signals. Various hormones and neurotransmitters modulate cAMP production by binding to members of the seven-transmembrane receptor family coupled to stimulatory (G(s)) and inhibitory (G(i)) G proteins. Intracellular cAMP levels are also affected through mechanisms that utilize other signaling pathways to modulate cyclase activity. Ca and protein kinase C, which function in discrete second messenger pathways, can independently modulate the activity of adenylyl cyclase. Ca activation synergizes with G(s) stimulation, allowing adenylyl cyclase to integrate signals from different second messenger pathways. Current biochemical models for associative learning in the sea slug Aplysia and the fruit fly Drosophila suggest that dual activation of adenylyl cyclase is responsible for the integration of independent stimuli. The G protein beta subunits participate in the guanine nucleotide exchange cycle and the transient activation of alpha subunits, but they also regulate adenylyl cyclase directly. This G stimulation may account for the ability of certain G(i)-coupled neurotransmitters and hormones to potentiate the effects of G(s)-coupled agonists in the brain.

To date, seven mammalian isoforms of adenylyl cyclase have been characterized and can be distinguished by their modes of regulation. Three forms, types I, III, and VIII (AC1, (^1)AC3, and AC8), appear to be activated by Ca, through the modulatory protein calmodulin (CaM), but AC1 and AC3 differ in their responses to G(1, 2, 3, 4) . Types V and VI (AC5 and AC6) are the most closely related cyclase isoforms and appear to be similarly regulated; both are inhibited by physiological levels of Ca and insensitive to G protein beta subunit regulation (5, 6, 7, 8, 9) . The activity of AC5 is also stimulated when the protein is phosphorylated by protein kinase C(10) . Adenylyl cyclase types II (AC2) and IV (AC4) are highly related in sequence and display similar biochemical profiles(11, 12) . Both are insensitive to Ca regulation, and their G-stimulated activities are potentiated by G protein beta subunits(2, 11) . AC2 differs from AC4 by being dramatically stimulated by protein kinase C in vivo(13, 14, 15) . The ability of adenylyl cyclase to integrate various signals and to generate different responses to identical inputs provides considerable versatility to this second messenger cascade.

In each case, the mechanism of action of regulation is not known; however, all have been shown to directly modulate adenylyl cyclase activity in vitro(1, 2, 10, 16, 17, 18) . A specific region of AC1 is thought to bind Ca/CaM, leading to activation, but the sites of action of the other known cyclase regulators have not been identified. While protein kinase C has been shown to stimulate AC5 in vitro, its phosphorylation sites and mechanism of action have not yet been determined. Jacobowitz and Iyengar (31) demonstrated that protein kinase C stimulation of AC2 activity following phorbol ester treatment of cells correlated with an increase in AC2 phosphorylation. Whether the protein kinase C activation of AC2 arises from this direct phosphorylation or alternatively through indirect processes remains to be determined. Additionally, nothing is known about the structural requirements for beta subunit regulation of adenylyl cyclases.

All known metazoan adenylyl cyclase isozymes share a predicted topological structure(19) . Two cytoplasmic domains (C and C) display homology to each other as well as to the catalytic portion of guanylyl cyclases(20) . These regions exhibit the greatest degree of sequence conservation between isozymes and are thought to be responsible for adenylyl cyclase catalytic activity. Each catalytic domain is preceded by a set of six transmembrane spans. These transmembrane domains share little sequence homology, but are predicted to be structurally similar. Between the first catalytic domain (C) and the second set of transmembrane domains is a nonconserved cytoplasmic region (C) of unknown function.

Functional domains have been identified within families of distinctly regulated, but related proteins by investigating the properties of chimeric molecules. Such studies have been particularly successful in determining the ligand-binding and G protein-coupling domains of the seven transmembrane receptors (21) as well as the beta subunit-binding, receptor-interacting, and effector-activating regions of G subunits(22, 23) . In the case of the G protein-coupled, seven transmembrane receptors, chimeras were produced by swapping independently identified, putative structural domains. Previous studies of adenylyl cyclase (1) indicated that no functional protein could be formed when the cytoplasmic domains of two different isoforms were expressed in the same cell, suggesting a need for an alternative approach. We have utilized a novel method for producing chimeras between two homologous genes that results in a collection of molecules with crossovers scattered throughout their most conserved regions.

We investigated the regulatory domains of two adenylyl cyclase isoforms that differentially respond to each of the known mechanisms of regulation. The basal activity of AC1 is activated by Ca/CaM, unresponsive to protein kinase C, and inhibited by G protein beta subunits. AC2 is unresponsive to Ca/CaM, but is activated by either protein kinase C or G protein beta subunits. Chimeric cyclases were produced between these two isoforms by a variety of methods and have defined domains essential for regulation by Ca/CaM and protein kinase C.


EXPERIMENTAL PROCEDURES

Materials

DNA restriction and DNA-modifying enzymes were purchased from commercial vendors. Oligodeoxynucleotides were synthesized by the Howard Hughes Medical Institute Biopolymer Core Facility at Johns Hopkins School of Medicine on an Applied Biosystems 380B DNA synthesizer. [alpha-P]ATP, [^3H]adenine, and I-calmodulin were purchased from DuPont NEN. N-Ethylmaleimide was purchased from Sigma. alpha(2)AR was kindly provided by Dr. Lee Limbird (Vanderbilt University Medical Center). UK14304 was purchased from RBI.

In Vivo Chimeras

XhoI linkers were ligated to the 4.0-kilobase EcoRI fragment containing bovine AC1(20) , and the resulting fragment was inserted into the XhoI site upstream of AC2 in pRB2B(11) . The resultant plasmid, designated pAC1/AC2, containing the two cyclase genes in a head-to-tail tandem array, was used to isolate AC1-2 chimeras. For the isolation of AC2-1 chimeras, the XhoI-linked 4.0-kilobase EcoRI AC1 fragment was cloned into a vector in which the XhoI site was 3` of the AC2 gene. This plasmid, designated pAC2/AC1, had the AC1 gene downstream of the AC2 coding sequence. The plasmids were digested with HindIII and SalI, each enzyme cutting the plasmid once between the cyclase genes. Approximately 100 ng of each of the linearized DNAs was used to transform bacteria (HB101). Transformants were screened by digestion with EcoRI to distinguish between plasmids containing two cyclase genes (religated) or a single chimeric cyclase gene. Of 72 random colonies screened, 29 contained plasmids with a single cyclase gene. Restriction mapping confirmed that these genes were chimeric and narrowed down the region where the putative changeover occurred. Nucleotide sequencing confirmed all 29 encoded precise chimeras with changeover points corresponding to regions of nucleotide identity between the two cyclases. Chimeras were named to reflect the parental origin of the N- and C-terminal sequences (AC12 or AC21) followed by a unique number that identifies the crossover point. Chimeric genes were excised by digestion with EcoRI and cloned into the mammalian expression vector pGW1 (British Biotechnology).

Targeted Reciprocal Chimeras

Specific restriction sites were introduced at equivalent positions in AC1 and AC2 by site-directed mutagenesis using the oligonucleotides indicated in Table 1. Cyclase activity determined by in vitro adenylyl cyclase assay or in vivo cAMP accumulation assay was not altered by amino acid substitutions arising from restriction site introduction (data not shown). The modified vectors, containing the specific restriction sites, were digested, and corresponding fragments from the other cyclase were exchanged. Chimeras pGW/212x(76/56), pGW/212x(78/56), and pGW/212x(80/56) were constructed by introducing their particular HindIII sites into pGW/12x56 using AC1-specific oligonucleotides. To construct pGW/212x(15/81), the XbaI site at position 1057 was introduced into pGW/21x15, and the C-terminal fragment was replaced with the corresponding region from AC2.



C-terminal AC2-1-2 Chimeras

Chimeras incorporating specific cyclase sequences were constructed using polymerase chain reaction (PCR). Oligonucleotides encoding different blocks of divergent amino acids were used for PCR with the indicated cyclase gene serving as template (Table 2). PCR products spanned approximately 120 nucleotides and included a 5`-BamHI site and a 3`-XbaI site. The 111-base pair BamHI/XbaI-digested PCR fragments were cloned into the BamHI/XbaI backbone from pGW/212x(15/81). The sequences of the introduced fragments were confirmed by nucleotide sequencing.



Cyclase Assay

Assays of adenylyl cyclase activity were performed on membranes from transfected HEK293 cells as described(24) . Briefly, adenylyl cyclase expression plasmids were transfected into HEK293 cells by CaPO(4) precipitation. After 3 days, cells were suspended in 200 µl of lysis buffer (50 mM Tris (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 0.1 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) and disrupted by sonication (cup sonicator for 1 min on ice). Extracts were centrifuged at 100,000 times g for 10 min, and pelleted membranes were resuspended in 400 µl of lysis buffer by repeated passage through a 22-gauge needle. Adenylyl cyclase assays were performed, in duplicate, on approximately 20 µl (1/20 of transfected plate) of resuspended membranes. Specific activity was determined and expressed as picomoles of cAMP formed per minute/milligram of protein.

cAMP Accumulation Assay

In vivo cAMP accumulation was determined by the procedure of Federman et al.(25) . Two days after transfection, cells were labeled overnight with 2 µCi of [^3H]adenine. Labeled cells were preincubated with 1 mM isobutylmethylxanthine in Hepes-buffered Dulbecco's modified Eagle's medium for 30 min and then treated for 30 min with either 100 nM PMA or 1 µM UK14304 in the presence of isobutylmethylxanthine. Cells were lysed with cold 5% trichloroacetic acid, and adenine nucleotides were fractionated by the method of Salomon(26) . Accumulation of cAMP is presented as a percentage of total adenine nucleotides to normalize for labeling variability.

CaM Overlay

Three days after transfection, HEK293 cells were collected, and 100 µg of whole cell sonicate was separated by 8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P transfer membranes (Millipore) and incubated with 5 µCi of I-calmodulin as described(27) . Membrane was exposed to film for approximately 3 days with an intensifying screen.


RESULTS

Identification of the Calmodulin-binding Domain of AC1

Chimeras were created between AC1 and AC2 using a novel in vivo method in which Escherichia coli correction/repair systems generated a large number of independent products with changeover points scattered throughout the conserved catalytic domains (see ``Experimental Procedures''). Briefly, the two cyclase genes were cloned in tandem, in a head-to-tail orientation, in the same plasmid. The plasmid was linearized between the two genes and introduced into bacteria by CaCl(2) transformation. Transformants, derived from plasmids recircularized in vivo or not cleaved in the initial restriction enzyme digestion, were recovered at a frequency of approximately 10^2/µg of DNA. Recircularization likely occurs by repair pathways at regions of nucleotide identity shared between the two genes, resulting in chimeric cyclase coding regions.

Chimeras were expressed from the cytomegalovirus promoter in HEK293 cells and assayed for adenylyl cyclase activity. The basal cyclase activity of AC1-transfected cell membranes was further stimulated by the addition of exogenous Ca/CaM and inhibited by the Ca chelator EGTA, reflecting activation by endogenous Ca/CaM (Fig. 1). In contrast, cells transfected with calcium-insensitive AC2 (11) are unaffected by Ca/CaM or EGTA addition. One particular AC1-2 chimera (AC12x56) displayed an activity profile similar to that of AC1. Activity was increased by exogenous Ca/CaM and inhibited by EGTA, indicating that AC12x56 retains responsiveness to Ca/CaM. This chimera defined the CaM-responsive domain to within the N-terminal two-thirds of AC1 (Fig. 2). An AC2-1 chimera (AC21x15) displayed an activity profile similar to that of AC2 (Fig. 1), indicating that it is unresponsive to Ca/CaM regulation.


Figure 1: Ca/CaM-responsive activity of adenylyl cyclase chimeras. Adenylyl cyclase activity is shown normalized to vector-transfected HEK293 cells in the absence of any additions (blackbars), in the presence of 30 µM CaCl(2) and 50 µg/ml CaM (stripedbars), or in the presence of 1 mM EGTA (graybars). Bars represent the average of multiple membrane preparations derived from independent transfections, each assayed in duplicate (AC1, 10 transfections; AC2, nine transfections; AC12x56, eight transfections; AC12x75, three transfections; and AC21x15, six transfections).




Figure 2: Summary of Ca/CaM responsiveness and I-CaM binding of chimeric adenylyl cyclases. Selected adenylyl cyclase chimeras are depicted diagrammatically. Light-gray portions are derived from AC1, and dark-gray portions are derived form AC2. Cyclases are shown divided into putative structural domains as described in the text. Ca/CaM-responsive chimeras (as determined from Fig. 1) are indicated by plus signs. Inactive chimeras are indicated by emptyparentheses. Chimeras that bind CaM (as determined from Fig. 3A) are indicated by plus signs; those that do not are indicated by minussigns. TM, transmembrane.




Figure 3: Calmodulin binding of adenylyl cyclase chimeras. A, I-CaM overlay of whole cell extracts from HEK293 cells transfected with the indicated cyclases or chimeras or with vector alone (pGW1). Molecular weight markers are indicated on the left. The dots indicate the position of AC1. B, I-CaM overlay of transfected HEK293 extracts treated with N-ethylmaleimide (NEM; leftpanel) or untreated (rightpanel) prior to denaturation in sample buffer.



Several in vivo chimeras with changeover points within the first catalytic domain contained no detectable activity. Cyclase activity in extracts from cells transfected with these chimeras was indistinguishable from vector alone-transfected cells under basal and forskolin-stimulated conditions (data not shown). Additional chimeras were produced by site-directed mutagenesis and domain swapping. One of these targeted AC1-2 chimeras (AC12x75) was responsive to Ca/CaM (Fig. 1); however, tripartite chimeras made within the context of the CaM-responsive AC12x56 chimera were inactive. Direct assessment of the calmodulin binding using an overlay technique (28) delineated the CaM-binding domain of the active as well as the enzymatically inactive proteins. Whole cell extracts of transfected cells were incubated with I-CaM in the presence of calcium. Cells transfected with AC1 contained a CaM-binding protein at approximately 110 kDa that was absent in vector-transfected and AC2-transfected cells (Fig. 3A). The CaM-responsive chimeras (AC12x56 and AC12x75) also displayed this specific I-CaM binding. Among the inactive chimeras, only those containing the C domain of AC1 (Fig. 2) were able to bind I-CaM (Fig. 3A). Expression of adenylyl cyclase protein by all the chimeric constructs was confirmed by Western blotting with antibodies raised against the C terminus of either AC1 or AC2 (data not shown). These results define C as the only domain of AC1 responsible for CaM binding.

N-Ethylmaleimide Sensitivity of CaM Binding

A peptide derived from sequences within the C region has been shown to bind CaM with high affinity(29) . This sequence encompasses two cysteine residues, and treatment of extracts from cells expressing AC1 with sulfhydryl-modifying reagents specifically attenuated the Ca/CaM activation of AC1(30) . We investigated whether cysteine modification directly inhibits CaM binding by AC1. In an overlay experiment, N-ethylmaleimide treatment prevented the I-CaM binding of AC1 and selected chimeras while not altering CaM binding by endogenous proteins (Fig. 3B). These results directly demonstrate that at least one cysteine residue is in or near the only CaM-binding site of AC1 and suggest that the region defined by the CaM-binding peptide within the C domain is the only binding site for CaM in AC1.

Identification of a Region of AC2 Required for Protein Kinase C Activation

Activation of protein kinase C by phorbol ester treatment of cells markedly stimulated the basal activity of AC2(13, 14, 15, 31) , but not of AC1. We investigated the phorbol ester responsiveness of the chimeras using an in vivo cAMP accumulation assay to identify sequences within AC2 responsible for activation by protein kinase C. AC2-transfected 293 cells accumulated approximately 4-fold more cAMP in the presence of the phorbol ester PMA than in its absence, while the level of cAMP in AC1-transfected cells was unaffected by PMA treatment (Fig. 4A). Two in vivo generated chimeras, the Ca/CaM-responsive AC1-2 chimera (AC12x56) and an AC2-1 chimera (AC21x15), were also unresponsive to PMA stimulation (Fig. 4A). AC21x15 is identical to AC2 except for its final 50 amino acids, which are replaced by the C terminus of AC1. This chimera was enzymatically active in an in vitro cyclase assay (Fig. 1) and resembled AC2 in its responses to alpha(2)AR (Fig. 4B). Agonist-dependent activation of alpha(2)AR results in the stimulation of a variety of G proteins, including G(s), G(i), and G(q)(32) , each of which could stimulate AC2 via different mechanisms. Stimulation of AC21x15 by alpha(2)AR is sensitive to pertussis toxin (data not shown), implying that this chimera, like AC2, is responsive to G(i) protein beta subunits.


Figure 4: Phorbol ester response of chimeras. A, cAMP accumulation in HEK293 cells transfected with the indicated cyclases or vector alone (0) either in the presence (stripedbars) or absence (solidbars) of 100 nM PMA. Shown is a representative assay performed in duplicate. Errorbars indicate standard deviation of the mean. B, HEK293 cells cotransfected with the indicated cyclases and alpha(2)AR. cAMP accumulation is measured in the presence (stripedbars) or absence (solidbars) of the alpha(2)AR-specific agonist UK14304 (10 µM).



Targeted chimeras constructed by site-directed mutagenesis and domain swapping (AC21x82 and AC212x(15/81)) confined the region required for protein kinase C responsiveness to 39 amino acids, of which 19 are identical between AC1 and AC2 (Fig. 5A). There are no putative targets for protein kinase C phosphorylation within this region of AC2. Additional chimeras were generated using a PCR-based strategy to identify the specific residues within this region required for PMA stimulation. Each resulting protein still responded to alpha(2)AR stimulation (Fig. 4B), and as summarized in Fig. 5B, a four-amino acid stretch of AC2 is required for responsiveness to phorbol ester treatment. The corresponding region of AC1 exchanged for these residues does conform to a consensus protein kinase C phosphorylation site (RRGSYR), but the data presented here suggest that introduction of the putative protein kinase C target sequence results in loss of phorbol ester responsiveness. Models for this site's importance posit a negative regulatory modification in AC1 that can be introduced into AC2. This does not appear to be the case as mutation of serine 1035 to alanine in AC21x15 did not render the enzyme phorbol ester-responsive (data not shown).


Figure 5: Summary of phorbol ester responsiveness of chimeric adenylyl cyclases. A, adenylyl cyclase chimeras are depicted diagrammatically as described in the legend to Fig. 2, except this time, the C domain is drawn out of proportion to depict the C-terminal chimeras. Phorbol ester response is indicated to the right of each chimera. B, shown are the amino acid sequences of the C terminus of AC2 (11) and the corresponding regions of the PCR-generated chimeras (as outlined under ``Experimental Procedures'' and in Table 2): AC1(20) , AC3(34) , AC4 (12) , AC5(6, 8, 9) , AC6(5, 6, 7) , and AC8(4) . Amino acids differing from AC2 are shaded. The average -fold stimulation in the presence of PMA is indicated to the right (averages derived from at least 10 distinct cAMP accumulation assays).




DISCUSSION

We have described a novel method for rapidly producing chimeric molecules with changeover points randomly scattered throughout the most conserved regions of homologous genes. The method is recA-independent and likely involves circularization of linear DNA molecules by natural bacterial repair systems. Presumably, one strand of the transformed linear DNA is preferentially digested by single-stranded exonucleases, revealing regions of nucleotide identity on opposite strands of the homologous genes. Pairing of the two single-stranded regions at small patches of sequence homology shared between related genes results in DNA polymerase-mediated repair using the two genes as templates for extension in opposing directions. This bacterial repair mechanism results in the conversion to one gene sequence on one side of the initial annealed region and to the other homologous gene on the other side, resulting in a single chimeric gene. The crossover point of the chimera is defined by the stretch of identical nucleotide sequence that initially annealed in vivo.

This method has many advantages over more traditional procedures for producing chimeras. Crossover points are generated at the most similar regions between genes, and the resulting linear sequence contains only naturally occurring amino acid neighbors. Additionally, there is no experimenter bias in selecting crossover sites based on predetermined, but potentially inappropriate structural domains. The changeover points are therefore less apt to alter important structural motifs or functional domains. Generating in vivo chimeras from two starting plasmids, with the homologous genes in the alternative head-to-tail positions, will produce reciprocal pairs of chimeras; however, it may require extensive screening to isolate every partner. This method efficiently produces a large number of chimeras randomly distributed throughout the conserved regions of two genes. A single transformation generates a large number of chimeras that can be rapidly screened and mapped by analysis of simple restriction digests. Finally, this method does not require the time and cost of oligoncleotide synthesis and site-directed mutagenesis.

We used this method to generate chimeras between AC1 and AC2, two genes that are <60% identical within their most conserved catalytic domains. Of 72 miniplasmid DNAs screened by restriction analysis, 29 contained an insert of the appropriate size for a single chimeric cyclase gene. Further restriction analysis determined that these were composed of elements from each of the starting cyclase genes, and nucleotide sequencing confirmed that all were precise chimeras. Chimeric genes were expressed in HEK293 cells, and each produced protein of the predicted molecular weight (data not shown).

Many of the in vivo chimeras had no detectable catalytic activity, and almost all of the directed chimeric cyclases, generated by swapping supposedly separate domains, were inactive. Additionally, every construct representing a reciprocal of an active chimera was enzymatically inactive (data not shown). Our difficulty in producing chimeras and the behavior of reciprocal molecules are consistent with previous attempts at producing chimeras between adenylyl cyclase isoforms. Coexpression of mixtures of structural domains from different cyclase isoforms in insect cells revealed a bias between the putative catalytic portions of AC1 and AC2(1) . The first catalytic domain of AC1 in combination with the second catalytic domain of AC2, similar to the active AC12x75 chimera described here, resulted in at least partial activity. In contrast, the reciprocal arrangement, the first catalytic domain of AC2 coexpressed with the second catalytic domain of AC1, resulted in no detectable activity. This arrangement is analogous to the inactive AC21x76 and AC21x78 chimeras we produced.

Both active and inactive chimeras were useful in defining the CaM-binding domain of AC1 to the nonconserved cytoplasmic portion between the first catalytic domain and the second set of transmembrane domains (C). CaM binding by chimeras 212x(78/56) and 12x79 demonstrates that the C domain of AC1 is sufficient to confer CaM binding, while the inability of chimeras 12x77 and 21x80 to bind indicates that it encompasses the only detectable CaM-binding site in AC1. Computer programs predicted that a specific sequence within this domain comprises a CaM-binding site, and a synthetic peptide corresponding to that sequence was able to bind CaM with high affinity(29) . Additionally, amino acid substitutions within this sequence specifically affected CaM activation of AC1(33) . These results and the identification of the C domain as being both necessary and sufficient for CaM binding confirm that this region (and presumably the sequence defined by the peptide) mediates the Ca/CaM activation of AC1.

A small region near the C terminus of AC2 was found to be required for PMA responsiveness; however, it does not contain any potential protein kinase C phosphorylation sites. Phorbol ester treatment has been shown to increase AC2 phosphorylation, which was correlated with stimulation of cyclase activity(31) . This suggests that the C terminus of AC2 plays a more general catalytic role and interacts with specific phosphorylation site(s) to increase cyclase activity. AC1-2 reciprocal constructs of the PMA-insensitive chimeras (AC21x15 and AC21x81) were inactive (data not shown), but the presence of this region in the CaM-responsive AC1-2 chimeras, which were insensitive to phorbol ester treatment (Fig. 4), demonstrated that this region was not sufficient to confer PMA responsiveness. The amino acid sequence of the required region is not conserved between AC2 and the only other adenylyl cyclase isoform (AC5) whose activity is known to be directly modulated by protein kinase C phosphorylation(10) . In fact, the only cyclase displaying any conservation in this region is AC4, but its activity is not affected by phorbol ester activation of protein kinase C(14) .


FOOTNOTES

*
This work was supported by grants from the National Institute of Mental Health and the Human Frontiers Science Program (to R. R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pharmacology, Cornell University Medical College, New York, NY 10021.

To whom correspondence should be addressed: Dept. of Molecular Biology and Genetics, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-4631; Fax: 410-614-0827.

(^1)
The abbreviations used are: AC, adenylyl cyclase; CaM, calmodulin; alpha(2)AR, alpha(2)-adrenergic receptor; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We thank Drs. Wei-Jen Tang and Al Gilman (University of Texas, Southwestern Medical Center) for providing affinity-purified antibodies against AC1 and AC2 and Dr. Lee Limbird for providing alpha(2)AR. We also thank Dr. Bruce Conklin (University of California, San Francisco) and Dr. Ravi Iyengar (Mt. Sinai School of Medicine) for communicating results prior to publication.


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