Calmodulin-binding Sites on Adenylyl Cyclase Type VIII*

Chen Gu and Dermot M. F. CooperDagger

From the Neuroscience Program and Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha -helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Mammalian adenylyl cyclases are a diverse group of variously regulated signaling molecules. Details are emerging on some of the molecular features conferring catalytic and regulatory properties on these enzymes. All of the nine cloned adenylyl cyclases are large (1080-1248 amino acids) polypeptides that are proposed to comprise two cassettes of six transmembrane-spanning domains, each cassette being followed by a large cytoplasmic domain (1, 2). The transmembrane domains are not highly conserved among adenylyl cyclases. However, parts of two of the cytoplasmic domains (termed C1a and C2a) are highly conserved, and, when expressed separately, they can combine to display basic catalytic activity (3-8). These molecules have been crystallized, and the combination of C1a and C2a, each consisting of a three layer alpha /beta sandwich, forms an active catalytic core to generate cAMP from ATP (9, 10). The other major cytoplasmic domains of mammalian adenylyl cyclases, the N terminus, the C1b region, and the C2b region, are not conserved at all and are speculated to reflect regulatory features of specific adenylyl cyclases (1, 11-13).

Ca2+ elicits a prominent stimulation of ACI1 and ACVIII, which is mediated by loosely bound calmodulin (3, 11, 14). Although the likely calmodulin-binding domain on ACI has been localized to the C1b region (15, 16), the corresponding regulatory domain has not been identified on ACVIII. Indeed, ACVIII does not possess analogous calmodulin-binding sites in the C1b region. In the case of ACI, peptides corresponding to putative calmodulin-binding domains were used to identify a site in the C1b region as the likely site of calmodulin binding (16). Mutagenesis studies strongly supported this assignment (15, 17). However, no information is yet available on the possible domains that mediate the Ca2+/calmodulin responsiveness of ACVIII. Given that ACI and ACVIII do not share similar C1b domains (they are only ~40% similar at the amino acid level, compared with 80% similarity in the C1a region) and also that their regulation by Ca2+/calmodulin shows distinct properties, it might not be unexpected if different motifs and/or locations were involved.

Identifying calmodulin-binding sites on proteins still mainly depends on experimentation, although some predictive criteria are available to guide experiments. For instance, many known Ca2+-dependent calmodulin-binding proteins possess a region that is often characterized by an amphipathic helix consisting of approximately 20 amino acid residues (18). In these regions, basic amino acids are interspersed among hydrophobic residues, and aromatic amino acids normally appear near either end (16, 18, 19). However, sequence analysis based on these criteria does not always identify calmodulin-binding regions, and indeed, regions of proteins that bind to calmodulin sometimes do not fit these criteria. Another calmodulin-binding motif is the so-called "IQ motif," consensus sequence IQXXXRGXXXR, which often (18) but not always (22, 23) binds calmodulin in a Ca2+-independent manner.

The present studies used a combination of calmodulin overlay assays, mutagenesis, and peptide inhibition studies to locate the calmodulin regulatory domains on ACVIII. Surprisingly, a primary site was located in the C2b region, while an ancillary site that appeared to play a minor autoinhibitory role was located in the N terminus.

    EXPERIMENTAL PROCEDURES

Materials-- Thapsigargin, forskolin, and Ro 20-1724 were from Calbiochem. [2-3H]Adenine, [3H]cAMP, and [alpha -32P]ATP were obtained from Amersham Pharmacia Biotech. The restriction enzymes and other enzymes used in subcloning were from New England Biolabs (Beverly, MA). Other reagents were from Sigma unless stated otherwise.

Production of His-tagged Protein Fragments from ACVIII-- Eight His-tagged fusion proteins were generated for calmodulin overlay experiments and are referred to as follows, with the appropriate ACVIII residues in parentheses: Nt (Met1-Glu179); Nn (Met1-Ser110); Nc (Glu108-Glu179); C1 (Ala346-Asn712); C1a (Ala346-Ser593); C2 (Gly913-Pro1248); C2a (Gly913-Pro1184); C2b (Leu1137-Pro1248). Nt, Nn, C2, C2a, and C2b were constructed by amplifying nucleotides 777-1314, 777-1136, 3513-4520, 3513-4329, and 4185-4520, respectively, by polymerase chain reaction (PCR) and subcloning the PCR products between EcoRI (5'-end) and HindIII (3'-end, blunted) sites of pRSETb (Invitrogen, Carlsbad, CA). C1 and C1a were constructed by amplifying nucleotides 1811-2912 and 1811-2546 by PCR and subcloning the PCR products between NcoI (5'-end) and HindIII (3'-end, blunted) sites of pRSETb. Nc was created from the Nt construct by cutting with EcoRI and BspI, blunting the two ends and ligating them back. Constructs were confirmed by sequencing. These cDNA constructs were transformed into BL21 (DE3) pLys S cells. The cells were grown in Luria's broth containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol at 37 °C until they reached an A600 of 0.8. Expression of these proteins was induced by the addition of 0.5 mM isopropyl beta -D-thiogalactoside and incubation of the cells overnight at 37 °C. The cells were harvested by centrifugation at 6000 × g for 10 min and resuspended in ice-cold extraction buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1% Triton X-100, 1 mM beta -mercaptoethanol, and protease inhibitor mixture). The cell lysate was sonicated (4 × 10 s) and separated by centrifugation at 10,000 × g for 10 min. The pellets were resuspended in equal volumes of 2× sample buffer, boiled, and spun at 13,000 × g for 5 min to eliminate insoluble material. The sample buffer containing bacterial proteins was loaded onto SDS-polyacrylamide gel electrophoresis gel. The expressed fusion proteins could be visualized readily from the gel stained by Coomassie Blue. These protein bands were also confirmed by Western blot probed with RGS-His antibody (Qiagen, Valencia, CA).

Calmodulin Overlay Assay-- The calmodulin overlay assay was performed by fractionating His-tagged fusion proteins by SDS-polyacrylamide gel electrophoresis, transferring to nitrocellulose membranes, and probing with biotinylated calmodulin, as described by the manufacturer (Calbiochem). The blot membranes were stained with India ink.

Mutagenesis-- Deletion mutants of ACVIII were generated by using different strategies. Mutants C1Delta 588-619, C1Delta 620-644, C1Delta 588-644, and the Phe158-Arg169 deletion of NDelta 1-106, Delta 158-169 were constructed by a PCR-based strategy (24). The deleted ACVIII residues of the mutants above are shown in Table I. Mutants NDelta 1-106 and NDelta 1-49 were constructed by cutting pcDNA3/ACVIII plasmid with BspI and EcoR47, respectively, blunting the ends with T4 DNA polymerase, cutting again with NotI, and subcloning the fragments into the EcoRI (blunted with T4 DNA polymerase) and NotI sites of pcDNA3/Hisb (Invitrogen, Carlsbad, CA) in frame. C2Delta 1126-1248 was generated by cutting pcDNA3/ACVIII plasmid with BamHI and NotI, polishing the ends with T4 DNA polymerase, and religating them back together. Mutant C2Delta 1184-1248 was constructed by cutting the C2a fragment in pRSET with BamHI and BglII (blunt) and subcloning into the BamHI and NotI (blunt) sites of pcDNA3/ACVIII. Deletions NC1Delta 1-106, Delta 635-700, NC1Delta 1-106, Delta 588-619, and NC1Delta 1-106, Delta 620-644 were constructed by cutting C1Delta 635-700, C1Delta 588-619, and C1Delta 620-644 with EcoRV and SfiI and subcloning the fragments into the EcoRV and SfiI sites of NDelta 1-106 plasmid. Mutants NC2Delta 1-106, Delta 1184-1248, NC2Delta 1-106, Delta 158-169, Delta 1184-1248, and NC1C2Delta 1-106, Delta 635-700, Delta 1184-1248 were constructed by cutting NDelta 1-106, NDelta 1-106, Delta 158-169, and NC1Delta 1-106, Delta 635-700 with EcoRV and ApaI and subcloning the fragments into EcoRV and ApaI sites of mutant C2Delta 1184-1248. All of the mutated cDNA constructs were confirmed by sequencing.

Measurement of cAMP Accumulation-- HEK 293 cells were maintained and transfected as described previously (25). cAMP accumulation in intact cells expressing different ACVIII mutant constructs was measured according to the method of Evans et al. (1984) as described previously (26, 27) with some modifications. HEK 293 cells on 12-well plates were incubated in minimal essential medium (60 min, 37 °C) with [2-3H]adenine (1.5 µCi/well) to label the ATP pool. The cells were then washed twice and incubated with a nominally Ca2+-free Krebs buffer (900 µl/well) containing 120 mM NaCl, 4.75 mM KCl, 1.44 mM MgSO4, 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin (fraction V) adjusted to pH 7.4 with 2 M Tris base. The use of Ca2+-free Krebs buffer in experiments denotes the addition of 0.1 mM EGTA to the nominally Ca2+-free Krebs buffer. All experiments were carried out at 37 °C in the presence of the phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (500 µM) and Ro 20-1724 (100 µM), which were preincubated with the cells for 10 min prior to a 1-min assay. Unless stated otherwise, cells were preincubated for 10 min with the Ca2+-ATPase inhibitor thapsigargin at a final concentration of 100 nM. This has the effect of passively emptying the Ca2+ stores, establishing a low basal [Ca2+]i, and priming the cells for capacitative Ca2+ entry (28). Assays were terminated by the addition of 5% (w/v final concentration) trichloroacetic acid. Unlabeled cAMP (100 µl, 10 mM), ATP (10 µl, 65 mM), and [alpha -32P]ATP(~7000 cpm) were added to monitor recovery of cAMP and ATP. After pelleting, the [3H]ATP and [3H]cAMP content of the supernatant were quantified according to the standard Dowex/alumina methodology (29). The accumulation of cAMP is expressed as the percentage of conversion of [3H]ATP into [3H]cAMP; means ± S.D. of triplicate determinations are indicated. The basal activity of some constructs was high, with the result that significant amounts of cAMP had accumulated before the beginning of the 1-min incubation period. In such cases, the Krebs buffer bathing the cells was exchanged with buffer containing the same concentrations of 3-isobutyl-1-methylxanthine/Ro20-1724, EGTA, and thapsigargin, at the beginning of the 1-min assay, as indicated in the figure legends.

Preparation of Plasma Membranes from Transfected HEK 293 Cells-- HEK 293 cells, transfected with pcDNA3 vector alone, ACVIII, or ACVIII mutant plasmids, were lysed using a modification of a glycerol-stabilized lysis method (30, 31). The lysate from six 75-cm2 flasks was adjusted to 3% sucrose, layered on top of linear sucrose gradients (5-45%), and centrifuged at 34,000 rpm (1 h, 4 °C, SW40 rotor, Beckman Instruments, Palo Alto, CA). The plasma membranes appeared as a clear band around 32-38% sucrose as described previously (32). The plasma membrane protein band was pelleted in 20 mM HEPES (pH 7.4) buffer and resuspended in the assay buffer (200 mM Tris pH 7.4, 800 µM EGTA, and 0.25% bovine serum albumin (fraction V)).

Western Blotting Experiments-- Two antibodies were used in Western blot experiments. Ab VIII-A 1229-1248 was raised against amino acids 1229-1248 of ACVIII at the very end of the C terminus; the other, Ab VIII-A 666-682, was raised against amino acids 666-682 in the C1b region of ACVIII (33). The experimental procedure was described previously (33). 10-µg membrane proteins were loaded on each lane of a 10% polyacrylamide gel. After electrophoresis, the contents of the polyacrylamide gel were transferred to polyvinylidene difluoride membranes (Micron Separations Inc.), which were incubated with TBST (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk, for 1 h at room temperature. The primary antibody (1:20,000 dilution) was then added, and the membrane was incubated for another 1 h. After washing in TBST, the membrane was incubated in TBST containing 5% nonfat dry milk with goat anti-rabbit IgG horseradish peroxidase conjugate (1:1000 dilution; Bio-Rad) at room temperature for 1 h. The immune complex was detected by enhanced chemiluminescence (manufacturer's protocol; Amersham Pharmacia Biotech).

Adenylyl Cyclase Activity Measurements-- Determination of adenylyl cyclase activity in vitro was performed as described previously (34) on isolated transfected HEK 293 cell membranes. The adenylyl cyclase activity of the HEK 293 cells was measured in the presence of the following components: 12 mM phosphocreatine, 2.5 units of creatine phosphokinase, 0.1 mM cAMP, 1 mM MgCl2, 0.1 mM ATP, 70 mM Tris buffer, pH 7.4, 0.04 mM GTP, 1 µCi of [alpha -32P]ATP, calmodulin (1 µM), and forskolin (20 µM), as indicated. Free Ca2+ concentrations were established from a series of CaCl2 solutions buffered with 200 µM EGTA in the assay (34). The reaction mixture (final volume, 100 µl) was incubated at 30 °C for 20 min. Reactions were terminated with sodium lauryl sulfate (0.5%); [3H]cAMP was added as a recovery marker, and the [32P]cAMP formed was quantitated as described previously (29). Data points are presented as mean activities ± S.D. of triplicate determinations.

Protein Secondary Structure Prediction and Synthetic Peptide Experiments-- The protein sequence was analyzed with DNASIS 2.5 (Hitachi Software Engineering Co. Ltd., San Bruno, CA). The secondary structures of the N-terminal fragment and the C2b region were predicted using the Chou and Fasman program. Two peptides were synthesized (Genemed Synthesis Inc., South San Francisco, CA). One was 21 residues, called 8Ncam (RPQRLLWQTAVRHITEQRFIH), corresponding to amino acids 32-52 of ACVIII; the other one was 25 residues, called 8Ccam (YSLAAVVLGLVQSLNRQRQKQLLNE), corresponding to amino acids 1186-1211 of ACVIII. The N termini of the two peptides were protected by acetylation, and the C termini were protected by amidation, to prevent self-circulation and to mimic the in vivo conformation. Both peptides were purified above 95%. The molecular weight for 8Ncam and 8Ccam is 2686 and 2885, respectively. The purity and molecular weight of the peptides were confirmed by high pressure liquid chromatography and mass spectroscopy, respectively (Genemed Synthesis). The peptide (CamkII, LKKFQARRKLKGAILTTMLA) of the CAM kinase II calmodulin-binding domain was purchased from Calbiochem. The peptide 8CT (TPSGPEPGAQAEGTDKSDLP) has 20 residues, corresponding to amino acids 1229-1248 of ACVIII, which was originally synthesized for raising antibody Ab VIII-A 1229-1248 (33). Stock solutions (2 mM) were made in water for all four peptides, which were used in in vitro adenylyl cyclase assays as described above, to generate competition and inhibition curves. The inhibition curves were fitted by the program Inplot 4.04 (GraphPad Software Inc.) with the following equation: Y = A + (B - A)/(1 + (X/C)), where Y represents the adenylyl cyclase activity relative to the activity in the absence of peptides and X is the peptide concentration. The maximal value (A), the minimal value (B), and IC50 (C) were obtained by curve fitting. The IC50 for each peptide was calculated from three independent experiments.

    RESULTS

Calmodulin Overlay Assays-- Overlay assays were an initial strategy adopted to identify putative regions for calmodulin regulation of ACVIII. It seemed reasonable to assume that a sizable molecule (~19 kDa) such as calmodulin, which is dissociable from ACVIII by washing in EGTA-containing buffers (14), should bind somewhere in the three large intracellular loops. Consequently, eight fusion proteins including the regions comprising these three loops were generated, designated Nt, Nn, Nc, C1, C1a, C2, C2a, and C2b (Fig. 1A) as described under "Experimental Procedures." Western blotting with an anti-RGS-His antibody indicated that most of the expressed fusion proteins were in the pellets from the Escherichia coli cell lysates (data not shown). Therefore, cell lysate pellets solubilized with sample buffer were used in calmodulin overlay assays. Following transfer from SDS-polyacrylamide gel electrophoresis, nitrocellulose membranes with fractionated E. coli proteins were incubated with biotinylated calmodulin, and the bands that bound calmodulin were detected by horseradish peroxidase-streptavidin incubation and enhanced chemiluminescence (see "Experimental Procedures"). The N-terminal fragments, Nt and Nn, yielded strong signals for calmodulin binding (Fig. 1, C and E). This signal was Ca2+-dependent and was removable by washing the membrane with EGTA-containing buffer (data not shown). No significant signal was detected for any other domains (Fig. 1, C and E). Two other fusion proteins including the C1b region were also generated, neither of which showed positive results in calmodulin overlay assays (data not shown). Thus, these data indicate that the N-terminal exclusively binds calmodulin. However, since proteins on nitrocellulose membranes are denatured, it is quite possible that a site binding to calmodulin in the native state would not bind calmodulin in an overlay assay. Therefore, mutagenesis studies and functional experiments were considered critical to evaluate the significance of any apparent interactions derived from overlay assays.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 1.   Calmodulin overlay assays. A, diagram of protein fragments from ACVIII cytoplasmic domains. Five amino acids and their positions are marked in the one-dimensional structure diagram of ACVIII from left (N terminus) to right (C terminus). Vertical black boxes represent the predicted transmembrane domains; two thick lines represent the highly conserved C1a and the C2a region. The positions of eight cytoplasmic fragments (horizontal black boxes) are shown below, which were generated into His-tagged fusion proteins as described under "Experimental Procedures." B-E, calmodulin overlay assays were performed as described under "Experimental Procedures" in the presence of 0.5 mM CaCl2. The fusion proteins loaded on the gel are indicated at the top of the panels. The molecular weight is marked at the left. B and D, transferred membrane blots stained with India ink. C and E, calmodulin binding shown in the x-ray film from the membranes of B and D, respectively. The positions of the fusion proteins are indicated (arrowheads), which were confirmed by Western blot probed with anti-RGS His antibody. Nonspecific binding to horseradish peroxidase-streptavidin is also indicated (asterisk). When 0.5 mM EGTA was substituted for CaCl2 in the overlay assay, the calmodulin-binding bands in C and E were no longer evident (not shown).

Ca2+ Sensitivity of ACVIII Mutants in Vivo-- The C1a and C2a regions, which are the catalytic domains (4, 6, 35) are highly conserved across the mammalian adenylyl cyclase family. These domains have already been crystallized and well characterized (5, 9, 10, 36, 37). A putative calmodulin-binding site was not revealed in these highly conserved regions by either sequence analysis or earlier experimental attempts (15, 16). On the other hand, the N terminus, C1b, and C2b regions are poorly conserved and are the regions where type-specific regulatory domains would be expected to reside. Therefore, a group of deletions was made, concentrating on these three areas (Table I). These mutant constructs were expressed in HEK 293 cells by transient transfection, and 48 h later, cAMP accumulation was measured in the intact HEK 293 cells in response to capacitative Ca2+ entry. The cells were pretreated with 100 nM thapsigargin to deplete the intracellular Ca2+ stores and prime the cells for capacitative Ca2+ entry, which selectively regulates the activities of Ca2+-sensitive adenylyl cyclases (25, 38, 39). Thus, the Ca2+ sensitivity of adenylyl cyclases can be determined by comparing the cAMP accumulation in cells pretreated with thapsigargin in the presence or absence of Ca2+ during the assay. Surprisingly, the N terminus deletion of ACVIII, NDelta 1-106 (mutant 1 in Fig. 2A) remained sensitive to Ca2+, although this deleted region bound calmodulin in the overlay assay (Fig. 1). The -fold stimulation by Ca2+ for NDelta 1-106 and two other N terminus deletions, NDelta 1-49 (Fig. 2A, mutant 2) and NDelta 1-106, Delta 158-169 (mutant 3) was only about half of that for wild type ACVIII; nevertheless, clear Ca2+ regulation was retained. These data strongly suggested that the N-terminal region was not the only functionally significant calmodulin-binding region within ACVIII.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Mutant constructs of ACVIII


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2.   The sensitivity of ACVIII mutants to capacitative Ca2+ entry. Assays (1 min) were performed in intact, transfected cells pretreated with 3-isobutyl-1-methylxanthine/Ro20-1724, EGTA and thapsigargin, as described under "Experimental Procedures." A, the effect of capacitative Ca2+ entry on cAMP accumulation in cells transfected with vector alone (pcDNA3.0), wild type (AC8), and mutants. Mutant numbers as identified in Table I are used. cAMP accumulation was measured under three assay conditions: basal (including 0.2% Me2SO; black bars); forskolin (20 µM; white bars); and forskolin (20 µM) plus external CaCl2 (4 mM; gray bars). The data shown are representative of at least three similar experiments. B, a modified in vivo assay protocol was used as described under "Experimental Procedures" to diminish the influence of extracellular cAMP accumulation in the preincubation. Cells transfected with vector alone (pcDNA3.0), ACVIII wild type, C2Delta 1184-1248, and NC2Delta 1-106, Delta 1184-1248, were assayed. 10% trichloroacetic acid was added at the beginning of a 1-min assay to one group of transfected cells to obtain a T0 value. This value (0.1% for vector; 0.5% for ACVIII; 1.34% for C2Delta 1184-1248; 7.8% for NC2Delta 1-106, Delta 1184-1248) was subtracted from the subsequent values, in which cAMP accumulation was measured in the indicated groups for 1 min at basal condition (dark gray bars), 4 mM external CaCl2 (white bars), 20 µM forskolin (black bars), or 20 µM forskolin and 4 mM external CaCl2 (light gray bars), as indicated.

Deletions in the C1b region were next evaluated. Deleting the entire C1b region (Pro587-Ser701) fully inactivated ACVIII (data not shown). The mutant C1Delta 635-700 (Fig. 2A, mutant 4), which is a naturally occurring splice variant of ACVIII (the "C form" (33), which lacks 66 amino acids in the C1b region), was stimulated by Ca2+. A deletion C1Delta 588-644 (Fig. 2A, mutant 7), missing the remaining part of the C1b region, appeared to lose its sensitivity to Ca2+, but its activity was very low. Two related deletions, C1Delta 588-619 (Fig. 2A, mutant 5) and C1Delta 620-644 (mutant 6), were similar to the wild type ACVIII in terms of their -fold stimulation by Ca2+, although the forskolin- and Ca2+-stimulated activity of C1Delta 620-644 was much higher than that of C1Delta 588-619. These results suggest that the C1b region is important for ACVIII activity, and, consequently, deletions in the C1b region could modify the Ca2+ regulation and the catalytic activity of ACVIII. However, it seems unlikely that this region is involved in the direct regulation by calmodulin. A double deletion, NDelta 1-106, Delta 588-619 (Fig. 2A, mutant 10), lacking approximately two-thirds of the N terminus and a major part of the C1b region, was also Ca2+-stimulable. This mutant further confirmed that the N terminus and the C1b region are unlikely to be the calmodulin-binding region responsible for stimulation by Ca2+.

Finally, the C2b region of ACVIII was explored. An extensive C2b deletion, C2Delta 1126-1248 (Fig. 2A, mutant 8) was inactive. Another C2b mutant, C2Delta 1184-1248 (mutant 9) showed extremely high basal activity, which was comparable with wild type ACVIII activity when the latter was stimulated by forskolin and Ca2+. Forskolin could further stimulate C2Delta 1184-1248 activity by about 50%, but Ca2+ could evoke no stimulation (Fig. 2A). An additional deletion of C2Delta 1184-1248 in the N terminus, NC2Delta 1-106, Delta 1184-1248 (Fig. 2A, mutant 13), resulted in an even higher basal cAMP accumulation. The high basal cAMP accumulation of NC2Delta 1-106, Delta 1184-1248 coupled with its insensitivity to stimulation suggests the removal of inhibitory influences. Two other multiple deletions, including the C2b region, NC2Delta 1-106, Delta 158-169, Delta 1184-1248 (Fig. 2A, mutant 14) and NC1C2Delta 1-106, Delta 158-169, Delta 636-702, Delta 1184-1248 (mutant 15), were also insensitive to forskolin and Ca2+, although the basal activity of both of these multiple deletions was much lower than that of NC2Delta 1-106, Delta 1184-1248.

In the assays described above, the basal activity of adenylyl cyclase is the cAMP that accumulates both inside and outside the cells before (10 min) and during the 1-min assay, in the presence of PDE inhibitors to prevent the breakdown of cAMP. Basal accumulation of cAMP is normally trivial during the preincubation period; however, where basal activities of some of these constructs are high (particularly C2b deletions), a considerable accumulation of cAMP can occur prior to the onset of the 1-min assay. Therefore, to clearly examine the activity of NC2Delta 1-106, Delta 184-1248 within the 1-min assay period, we modified our in vivo assay protocol. The medium was exchanged with new buffer just before the 1-min assay to minimize the influence of accumulated extracellular cAMP. In addition, the intracellular cAMP that had accumulated was measured by exchanging the medium and lysing one group of cells with trichloroacetic acid at the beginning of the 1-min assay. When HEK 293 cells transfected with pcDNA3, ACVIII, C2Delta 1184-1248, or NC2Delta 1-106, Delta 1184-1248 were assayed using this modified protocol, the cAMP accumulation of C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248 in all of the conditions was much reduced. However, stimulation by Ca2+ of wild type ACVIII was evident even in the basal state and was strikingly evident when activity was also stimulated by forskolin (Fig. 2B). A clear stimulation of NC2Delta 1-106, Delta 1184-1248 by forskolin was also revealed (Fig. 2B), while in the original in vivo assay no significant forskolin stimulation was detectable, probably due to masking by the high extracellular cAMP accumulation (Fig. 2A). Most significantly, again, in keeping with the original in vivo assay results (Fig. 2A), both C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248 were quite insensitive to Ca2+ (Fig. 2B). Overall, these data strongly indicate that the C2b region of ACVIII is critical to the Ca2+ stimulation. The activity of these deletions was also assessed in in vitro assays to confirm or disprove the conclusions from the intact cell assays.

Ca2+ Sensitivity of ACVIII Mutants in Vitro-- Plasma membranes were prepared from transfected HEK 293 cells, and adenylyl cyclase activity was assayed with either 20 µM forskolin or 20 µM forskolin plus 2 µM free Ca2+. All of the mutants in the N terminus and the C1b regions were sensitive to Ca2+ except C1Delta 588-644, whose activity was close to the background (Fig. 3). Mutants NDelta 1-106 and C1Delta 620-644 showed higher -fold stimulation by Ca2+ than did the wild type (Fig. 3). Three double deletions, NC1Delta 1-106, Delta 635-700, NC1Delta 1-106, Delta 588-619 (Fig. 3, mutant 11), and NC1Delta 1-106, Delta 620-644 (mutant 12), could also still be stimulated by Ca2+. As anticipated from the in vivo studies, deletion of the C2b region (C2Delta 1184-1248), resulted in a loss of the Ca2+ sensitivity (Fig. 3). The activity of the double deletion, NC2Delta 1-106, Delta 1184-1248, was inhibited (rather than stimulated) approximately 20% by 2 µM free Ca2+ (Fig. 3). Although the activity of this latter construct was lower than those of the other mutants, it was still significantly higher than the control (130 versus 20 pmol/mg/min; Fig. 3). Thus, the Ca2+ sensitivity, from both the in vivo and in vitro assays, of all of the mutants agree well and confirm that the C2b region is critical for the Ca2+ sensitivity of ACVIII. However, it is notable that the activities of C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248 are lower than that of the wild type in vitro (Fig. 3), while in the intact cells in the presence of forskolin, their activity is much higher than the wild type activity (Fig. 2B). This discrepancy could result from (among other things) misdirection of the adenylyl cyclase constructs to inappropriate membrane locations. Therefore, to confirm the expression of all of the mutants and to estimate the amount of protein being expressed in the plasma membrane, Western blot experiments were performed on the membranes that were used in in vitro assays.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   In vitro measurement of the Ca2+ sensitivity of various mutants. Adenylyl cyclase activity was determined in plasma membranes (5 µg/reaction) prepared from cells transfected with either vector alone (pcDNA3.0, as control), wild type ACVIII (AC8), or the mutants indicated as described under "Experimental Procedures." Activity was measured in the presence of calmodulin (1 µM) and forskolin (20 µM), with (white bars) or without (black bars) 2 µM free Ca2+. Mutant numbers from Table I are used. Values shown are from an experiment that was repeated at least three times with similar results.

Expression of Mutants Assessed by Western Blot-- Ab VIII-A 1229-1248 was used to recognize the mutants with intact C termini (Fig. 4A), while Ab VIII-A 666-682 was used to recognize the mutants lacking C termini but with intact C1b regions (Fig. 4B). Mutants 1-13 were all expressed, although in varying amounts (Fig. 4, A and B). Mutants NDelta 1-106, NDelta 1-49, NDelta 1-106, Delta 158-169, C1Delta 620-644, and NC1Delta 1-106, Delta 620-644 were expressed well (Fig. 4A), which coincided with their approximately equivalent in vitro activity. On the other hand, C2Delta 1126-1248 was well expressed as determined by Western blot but only had background level activity, indicating that this molecule is totally inactive (Fig. 4B). C1Delta 635-700 and NC1Delta 1-106, Delta 635-700 were also well expressed (Fig. 4A), but their in vitro activity was low, underlining their low intrinsic activity. Deletion mutants C1Delta 588-619, NC1Delta 1-106, Delta 588-619, C2Delta 1184-1248, and NC2Delta 1-106, Delta 1184-1248 were shown to have low expression in the plasma membrane (Fig. 4, A and B), which corresponded to their low activity in vitro. Based on these Western blot results, it is conceivable that the discrepancy between the activities of C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248, which were high in vivo but low in vitro, could reflect mistargeting of these two mutated proteins to subcellular fractions not banding as the plasma membrane in our preparation.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   Western blots of various constructs. The same membrane proteins assayed in Fig. 3 were loaded on SDS-polyacrylamide gel (10 µg/lane) and probed as described under "Experimental Procedures." Mutant numbers (Table I) are indicated at the top of the corresponding lanes. The molecular masses are marked in kDa on the left. A, Ab VIII 1229-1248 antibody was used to probe the mutants with intact C termini. B, Ab VIII-A 666-682 was used to probe the mutants with intact C1b regions but with a deleted C terminus. This result is representative of two experiments yielding similar results.

Characterization of the Three Deletions, NDelta 1-106, C2Delta 1184-1248, and NC2Delta 1-106, Delta 1184-1248-- From the foregoing data, three of the apparently more informative mutants, NDelta 1-106, C2Delta 1184-1248, and NC2Delta 1-106, Delta 1184-1248, were chosen for more detailed analysis. Ca2+ and calmodulin concentration-response curves were generated for these three mutants and compared with the wild type ACVIII. For wild type ACVIII, activity began to increase at 0.4 µM Ca2+, reached a plateau between 1 and 10 µM, and declined when the free Ca2+ concentration exceeded 10 µM (Fig. 5A). The decrease of ACVIII activity at high [Ca2+]free is considered to reflect Ca2+ competing with Mg2+ at an allosteric regulatory site (1, 40, 41). This inhibition by high [Ca2+] is a property of all mammalian adenylyl cyclases, apart from AC3, regardless of their response to Ca2+ in the submicromolar range (25, 41, 42). The Ca2+ concentration-response curve of NDelta 1-106 is similar to that of the wild type, except that the Ca2+-stimulated activity is somewhat higher (Fig. 5A). On the other hand, the C2Delta 1184-1248 construct was unaffected by low [Ca2+]free, although its activity started to decline when [Ca2+]free exceeded 10 µM (Fig. 5A). Ca2+ also did not stimulate, but rather inhibited, the activity of NC2Delta 1-106, Delta 1184-1248 when concentrations surpassed 1 µM (Fig. 5A).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5.   Ca2+ and calmodulin concentration-response curves of three deletion mutants, NDelta 1-106, C2Delta 1184-1248, and NC2Delta 1-106, Delta 1184-1248, and wild type ACVIII. Adenylyl cyclase activities were determined in plasma membranes prepared from cells transfected with wild type ACVIII (), NDelta 1-106 (open circle ), C2Delta 1184-1248 (black-down-triangle ), or NC2Delta 1-106, Delta 1184-1248 (down-triangle). A, Ca2+ concentration-response curves in the presence of 2 µM forskolin, 1 µM calmodulin, and the indicated free Ca2+ concentrations. -Fold stimulation of adenylyl cyclase is indicated for each Ca2+ concentration relative to activity measured in the absence of Ca2+. B, as A except that exogenous calmodulin was not included in the reactions. C, calmodulin concentration-response curves in the presence of 2 µM forskolin, 22.4 µM free Ca2+ and the indicated exogenous calmodulin concentrations. -Fold stimulation of adenylyl cyclase is indicated for each calmodulin concentration relative to activity measured in the absence of calmodulin.

Ca2+ concentration-response experiments were also performed in the absence of calmodulin. The wild type ACVIII was stimulated by approximately 50% with supramicromolar Ca2+ (Fig. 5B). This stimulation apparently resulted from residual calmodulin in the plasma membrane, although the preparation was washed twice with assay buffer containing 800 µM EGTA. Such persistence of calmodulin with adenylyl cyclase has been long encountered (41). It had been considered to reflect the persistent association of calmodulin with plasma membranes or the presence of calmodulin in other assay components, e.g. albumin or creatine phosphokinase (43). However, it is conceivable that there is a tight binding site for calmodulin on adenylyl cyclases. The latter possibility is supported by two observations: (i) the Ca2+ stimulation of wild type ACVIII observed without the addition of exogenous calmodulin can be abolished by the peptide 8Ncam, which binds to calmodulin (data not shown), and (ii) without exogenous calmodulin, Ca2+ has little effect on NDelta 1-106 (Fig. 5B). Thus, the deletion of amino acids 1-106 appears to result in no residual calmodulin. As expected, no stimulation was seen with either C2Delta 1184-1248 or NC2Delta 1-106, Delta 1184-1248, and activities started to decline at 1 µM free Ca2+ concentration (Fig. 5B).

Calmodulin concentration-response experiments were performed on the mutants in the presence of 22.4 µM free Ca2+. (A high [Ca2+] was chosen to saturate high concentrations of calmodulin.) NDelta 1-106 elicited a similar stimulatory profile as wild type ACVIII, although the -fold stimulation (approximately 6-fold) was higher than that of the wild type (approximately 4-fold; Fig. 5C). Deletions C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248 were relatively insensitive to calmodulin when the concentration was lower than 1 µM (Fig. 5C). However, when the calmodulin concentration was increased to 10 µM, the activities of these two mutants were about 2-fold higher than the activity in the absence of calmodulin (Fig. 5C). This increase in the activities of C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248, in the presence of a high concentration of calmodulin, most likely reflected a reversal of the inhibitory effects of high (22.4 µM) Ca2+ on these mutants (see Fig. 5B) due to calmodulin chelation of Ca2+.

Peptide Competition Studies-- The calmodulin overlay assay data and the mutagenesis screen identified two putative calmodulin-binding sites on ACVIII; one is in the N terminus, amino acids Met1 to Ser110, and one is in the C terminus, amino acids Pro1184 to Pro1248. Using the Chou and Fasman secondary structure prediction program, we located only one putative site in the N terminus that could form a helical structure - from Gln34 to His52. This site resembles a classic Ca2+-dependent calmodulin-binding site (Fig. 6A; see Ref. 18), in that it has basic amino acids (net charge, +4) and two aromatic amino acids, Trp38 and Phe50. The hydrophobic amino acid distribution pattern is also similar to some known Ca2+-dependent calmodulin-binding sites, such as the death-associated protein kinase (a serine/threonine kinase associated with mediation of interferon-induced cell death (19)), the human erythrocyte Ca2+ pump (20), a Ca2+-regulated nitric-oxide synthase (16), rabbit skeletal muscle myosin light chain kinase (21), the inositol 1,4,5-trisphosphate type I receptor, and Bacillus anthracis adenylyl cyclase (Fig. 6A; Ref. 18). At the C terminus of ACVIII, between amino acids Pro1184 and Pro1248, the best candidate for a calmodulin-binding site would be from amino acid Tyr1187 to Glu1211. This site is similar to the IQ motif (18), although it lacks one glycine residue (Fig. 6B). It also tends to form a helical structure as predicted by the Chou and Fasman analysis and shares homology with other calmodulin-binding sites, such as the ones in inositol 1,4,5-trisphosphate receptor (18), the epsilon -subunit of protein kinase C (18), and the beta -subunit of cyclic nucleotide-gated channel (Ref. 23; Fig. 6B). This site would be an atypical calmodulin-binding site if it indeed bound calmodulin, since the IQ motif normally binds calmodulin independently of Ca2+, although some studies have shown that the IQ motif can also bind to calmodulin in a Ca2+-dependent manner (22, 23). Two peptides from ACVIII were synthesized for competition studies, one corresponding to amino acids Gln34 to His52, termed 8Ncam, the other corresponding to amino acids Tyr1187-Glu1211, termed 8Ccam. The N termini of the two peptides were protected by acetylation, and the C termini were protected by amidation to prevent self-circulation of the peptide and to mimic the in vivo conformation.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Two calmodulin-binding sites located on ACVIII by structure prediction and synthetic peptide studies. A, prediction of a putative calmodulin-binding site in the N-terminal fragment of ACVIII, which had bound calmodulin in overlay assays (Fig. 1). Amino acid numbers are placed above the sequence. The predicted secondary structure was obtained using the Chou and Fasman program. The putative calmodulin-binding site, called 8Ncam, is indicated in boldface type. The sequence of 8Ncam is aligned with those of calmodulin-binding sites from death-associated protein (DAP) kinase (a serine/threonine kinase associated with mediation of interferon-induced cell death), the human erythrocyte Ca2+ pump, Ca2+-regulated nitric-oxide synthase (NO synthase), rabbit skeletal muscle myosin light chain kinase (MLCK), and B. anthracis adenylyl cyclase. Their hydrophobic amino acid patterns and net charges are compared. B, prediction of a putative calmodulin-binding site in the C terminus of ACVIII whose binding to calmodulin was suggested by mutagenesis studies. The secondary structure of amino acids 1182-1248 of ACVIII was predicted using the Chou and Fasman program. The putative calmodulin-binding site is indicated with boldface type. The sequences of IQ motif, 8Ccam, IP3 receptor, protein kinase C (epsilon -subunit), and CNG channel (beta -subunit) are aligned, and important amino acids are compared. C, the effects of peptides on the calmodulin responsiveness of ACVIII. Plasma membranes from HEK 293 cells transfected with wild type ACVIII were assayed in the presence of 2 µM forskolin, 7.74 µM free Ca2+, and the indicated calmodulin concentrations, along with vehicle (water, ), 8Ncam (4 µM, open circle ), CamkII (4 µM, black-down-triangle ), 8Ccam (4 µM, down-triangle), or 8CT (4 µM, black-square). (ACVIII activities in the presence of different peptides, in the absence of Ca2+ and exogenous calmodulin, are also shown.) D, inhibition profiles of various peptides. Plasma membranes from HEK 293 cells transfected with ACVIII. Activities were assayed in the presence of 2 µM forskolin, 7.74 µM free Ca2+, 0.3 µM calmodulin, and the peptides at the indicated concentrations (8Ncam, open circle ; CamkII, black-down-triangle ; 8Ccam, down-triangle; and 8CT, black-square). IC50 values, extracted from the fitted inhibition curves as described under "Experimental Procedures," were 1.15 ± 0.25 µM for 8Ncam, 0.65 ± 0.17 µM for 8Ccam, and 0.15 ± 0.1 µM for CamkII.

These peptides were used in in vitro assays in competition studies. A calmodulin concentration-response curve was generated for ACVIII, in the presence or absence of added peptides (at 4 µM). CamkII, the peptide corresponding to the calmodulin-binding site of CAM kinase II, was used as a positive control. 8CT, the peptide corresponding to the C terminus of ACVIII, from Thr1229 to Pro1248, was used as a negative control. In the presence of 1 µM calmodulin and 7.74 µM free Ca2+, 8CT had no effect on the calmodulin concentration-response curve, while CamkII suppressed the ACVIII activity and shifted the curve to the right (Fig. 6C). Both the 8Ncam and 8Ccam peptides also suppressed the calmodulin stimulated activity of ACVIII, although not quite as efficaciously as the CamkII peptide (Fig. 6C).

Peptide inhibition experiments were also performed for these four peptides in the presence of a fixed calmodulin concentration (0.3 µM; and 7.74 µM free Ca2+). Again, 8CT had no effect on ACVIII activity even at its highest concentration (Fig. 6D). CamkII began to inhibit ACVIII activity when its concentration exceeded 0.1 µM (Fig. 6D; IC50, 0.15 ± 0.1 µM; n = 3). 8Ncam began to inhibit ACVIII activity at 0.3 µM, (IC50, 1.15 ± 0.25 µM; n = 3; Fig. 6D). 8Ccam was a little more potent than 8Ncam (IC50, 0.65 ± 0.17 µM; n = 3; Fig. 6D). These experiments clearly demonstrate that both peptide sequences derived from the N and C termini of ACVIII are bona fide calmodulin-binding sequences and that the most potent peptide is also the one that is of most functional significance.

    DISCUSSION

This study has explored the calmodulin-binding sites on ACVIII. The related Ca2+-stimulable ACI binds calmodulin in the C1b region of the molecule. However, ACVIII and ACI are very dissimilar (only 40% homologous) in this region. Therefore, it might not have been unexpected that different sites would mediate the Ca2+ stimulation of ACVIII. Calmodulin overlay assays revealed one putative Ca2+-dependent calmodulin-binding site in the N terminus of ACVIII (Fig. 1). However, without this region the enzyme was still sensitive to Ca2+ (Figs. 2 and 3). On the other hand, using mutagenesis and functional assays, only those mutants lacking the C2b region, such as C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248, could not be stimulated by Ca2+ either in vivo or in vitro (Figs. 2 and 3). This suggests that the C-terminal region is responsible for the Ca2+/calmodulin stimulation of ACVIII. Moreover, the high basal activities of C2Delta 1184-1248 and NC2Delta 1-106, Delta 1184-1248 suggests the removal of autoinhibitory domains (the C2b region playing the major role), which suppress the activity of wild type ACVIII. The binding of Ca2+/calmodulin to the autoinhibitory domain apparently relieves the inhibitory binding and activates the enzyme. Such a disinhibitory mechanism is employed in a number of Ca2+/calmodulin-activated enzymes, such as Ca2+-regulated nitric-oxide synthase (44), Ca2+/calmodulin-dependent protein kinases (45-47), a Na+/H+ exchanger (48), and calcineurin (49). It appears as though most of the C2b region might participate as the inhibitory binding domain, since the two synthetic peptides 8Ccam (25 residues) and 8CT (20 residues) from the C2b region did not inhibit ACVIII activity in the absence of Ca2+ and calmodulin (Fig. 6C). This disinhibition mechanism of Ca2+/calmodulin stimulation is different from that of forskolin, which is thought to stabilize the C1/C2 heterodimer to activate the adenylyl cyclase activity (9, 10). The latter suggestion is supported by the fact that forskolin and Ca2+/calmodulin synergistically stimulate ACVIII.

The putative calmodulin-binding site in the C terminus has the signature sequence of an IQ motif, which generally reflects Ca2+-independent calmodulin binding. It is possible that the binding of calmodulin is Ca2+-independent and that Ca2+ binding changes the conformation of bound calmodulin to activate the enzyme. Alternatively, the binding of calmodulin may be Ca2-dependent as is the case with the beta -subunit of rod photoreceptor cyclic nucleotide-gated channel (23). It is of some interest that the peptide synthesized from this region (8Ccam) is only 5 times less effective than 8CamkII, which is a conventional calmodulin-binding peptide. This observation underscores how much we still need to learn about the molecular characteristics of calmodulin-binding sequences.

The putative calmodulin-binding site for the N terminus of ACVIII is a conventional Ca2+-dependent calmodulin-binding site, which is reinforced by the results of overlay assays. The fact that the double deletion NC2Delta 1-106, Delta 1184-1248 has higher activity in vivo (Fig. 2) and can be inhibited by a lower concentration of Ca2+ (Fig. 5A) than C2Delta 1184-1248 suggests that this site contributes to the Ca2+ stimulation of ACVIII, although this contribution must be minor.

The fact that there is apparently more residual calmodulin in ACVIII wild type membrane preparations than in those of NDelta 1-106 (Fig. 5B) might suggest a role of the N terminus of ACVIII as a Ca2+-independent calmodulin trap, notwithstanding the apparently conflicting evidence of the Ca2+-dependent manner of the N-terminal calmodulin-binding site from overlay assays.

Unlike the two regulatory domains (the N terminus and the C2b region) discussed above, the C1b region does not have a free end, which suggests that the disruptions on this region could more easily change the activity of adenylyl cyclases. However, continuous deletions in the C1b region of ACVIII could not eliminate the Ca2+ stimulation of ACVIII (Fig. 2A), while, by contrast, a point mutation in this region of ACI abolished its Ca2+ sensitivity (17). The different calmodulin-binding sites on ACVIII and ACI are underlined by some differences in their regulation by Ca2+/calmodulin; for instance, ACI is more sensitive to lower concentrations of Ca2+ than is ACVIII, and ACVIII is more stimulable by Ca2+/calmodulin than ACI (25). The calmodulin-binding site in ACI is rich in basic amino acids (net charge is +7), and the binding is Ca2+-dependent (15, 16). Since no hyperactivity was observed by mutating the C1b region on ACI (15, 17) and the movement of the C1b region is likely to be more restrained than those of the N terminus and C terminus, the mechanism of Ca2+/calmodulin regulation of ACI might be to stabilize the C1/C2 heterodimer, as has been proposed for forskolin and Gsalpha a (9, 10), unlike the disinhibitory mechanism we have proposed for ACVIII.

In conclusion, two calmodulin-binding sites exist on ACVIII, one (at the C terminus) is of profound regulatory significance, whereas the other (at the N terminus) plays a more minor role. Whether these two domains of ACVIII physically interact to share the same molecule of calmodulin remains to be determined in future studies. Given that the C1a and C2a regions clearly interact for catalytic activity (4, 5, 7, 9, 10, 35), the tantalizing possibility that adenylyl cyclase could adopt a transporter-like structure (11, 50) would be greatly strengthened by interactions between the N and C termini.

    ACKNOWLEDGEMENTS

We thank Drs. J. J. Cali and J. Krupinski for the cDNAs of three isoforms of ACVIII and two antibodies, Ab VIII-A 1229-1248 and Ab VIII-A 666-682, and Drs. J. W. Karpen, K. A. Fagan, and M. Yoshimura for comments on the manuscript.

    FOOTNOTES

* These studies were supported by National Institutes of Health Grant GM 32483.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Box C-236, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-8964; Fax: 303-315-7097; E-mail: cooperd{at}essex.uchsc.edu.

    ABBREVIATIONS

The abbreviations used are: ACI, adenylyl cyclase, type I; ACVIII, adenylyl cyclase, type VIII; PCR, polymerase chain reaction; Ab, antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Sunahara, R. K., Dessauer, C. W., and Gilman, A. G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 461-480[CrossRef][Medline] [Order article via Infotrieve]
  2. Cooper, D. M. F., Mons, N., and Karpen, J. W. (1995) Nature 374, 421-424[CrossRef][Medline] [Order article via Infotrieve]
  3. Tang, W.-J., Krupinski, J., and Gilman, A. G. (1991) J. Biol. Chem. 266, 8595-8603[Abstract/Free Full Text]
  4. Tang, W.-J., and Gilman, A. G. (1995) Science 268, 1769-1772[Medline] [Order article via Infotrieve]
  5. Whisnant, R. E., Gilman, A. G., and Dessauer, C. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6621-6625[Abstract/Free Full Text]
  6. Yan, S.-Z., Hahn, D., Huang, Z.-H., and Tang, W.-J. (1996) J. Biol. Chem. 271, 10941-10945[Abstract/Free Full Text]
  7. Sunahara, R. K., Dessauer, C. W., Whisnant, R. E., Kleuss, C., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22265-22271[Abstract/Free Full Text]
  8. Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277[Abstract/Free Full Text]
  9. Zhang, G., Liu, Y., Ruoho, A. E., and Hurley, J. H. (1997) Nature 386, 247-253[CrossRef][Medline] [Order article via Infotrieve]
  10. Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916[Abstract/Free Full Text]
  11. Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R., and Gilman, A. G. (1989) Science 244, 1558-1564[Medline] [Order article via Infotrieve]
  12. Cooper, D. M. F., Mons, N., and Fagan, K. A. (1994) Cell. Signalling 6, 823-840[Medline] [Order article via Infotrieve]
  13. Krupinski, J., and Cali, J. J. (1998) Adv. Second Messengers Phosphoprotein Res. 32, 53-79[Medline] [Order article via Infotrieve]
  14. Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269, 12190-12195[Abstract/Free Full Text]
  15. Levin, L. R., and Reed, R. R. (1995) J. Biol. Chem. 270, 7573-7579[Abstract/Free Full Text]
  16. Vorherr, T., Knopfel, L., Hofmann, F., Mollner, S., Pfeuffer, T., and Carafoli, E. (1993) Biochemistry 32, 6081-6088[Medline] [Order article via Infotrieve]
  17. Wu, Z., Wong, S. T., and Storm, D. R. (1993) J. Biol. Chem. 268, 23766-23768[Abstract/Free Full Text]
  18. Rhoads, A. R., and Friedberg, F. (1997) FASEB J. 11, 331-340[Abstract/Free Full Text]
  19. Deiss, L. P., Feinstein, E., Berissi, H., Cohen, O., and Kimchi, A. (1995) Genes Dev. 9, 15-30[Abstract]
  20. Enyedi, A., Vorherr, T., James, P., McCormick, D. J., Filoteo, A. G., Carafoli, E., and Penniston, J. T. (1989) J. Biol. Chem. 264, 12313-12321[Abstract/Free Full Text]
  21. Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191[Abstract]
  22. Munshi, H. G., Burks, D. J., Joyal, J. L., White, M. F., and Sacks, D. B. (1996) Biochemistry 35, 15883-15889[CrossRef][Medline] [Order article via Infotrieve]
  23. Weitz, D., Zoche, M., Muller, F., Beyermann, M., Korschen, H. G., Kaupp, U. B., and Koch, K.-W. (1998) EMBO J. 17, 2273-2284[Abstract/Free Full Text]
  24. Hughes, M. J., and Andrews, D. W. (1996) BioTechniques 20, 192-196
  25. Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444[Abstract/Free Full Text]
  26. Evans, T., Smith, M. M., Tanner, L. I., and Harden, T. K. (1984) Mol. Pharmacol. 26, 395-404[Abstract]
  27. Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155[Abstract/Free Full Text]
  28. Putney, J. W., Jr. (1992) Adv. Second Messenger Phosphoprotein Res. 26, 143-160[Medline] [Order article via Infotrieve]
  29. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548[Medline] [Order article via Infotrieve]
  30. Phillips, D. R. (1972) Biochemistry 11, 4582-4588[Medline] [Order article via Infotrieve]
  31. Caldwell, K. K., Newell, M. K., Cambier, J. C., Prasad, K. N., Masserano, J. M., Schlegel, W., and Cooper, D. M. F. (1988) Anal. Biochem. 175, 177-190[Medline] [Order article via Infotrieve]
  32. Nakahashi, Y., Nelson, E., Fagan, K., Gonzales, E., Guillou, J.-L., and Cooper, D. M. F. (1997) J. Biol. Chem. 272, 18093-18097[Abstract/Free Full Text]
  33. Cali, J. J., Parekh, R. S., and Krupinski, J. (1996) J. Biol. Chem. 271, 1089-1095[Abstract/Free Full Text]
  34. Boyajian, C. L., Garritsen, A., and Cooper, D. M. F. (1991) J. Biol. Chem. 266, 4995-5003[Abstract/Free Full Text]
  35. Tang, W.-J., Stanzel, M., and Gilman, A. G. (1995) Biochemistry 34, 14563-14572[Medline] [Order article via Infotrieve]
  36. Yan, S.-Z., Huang, Z.-H., Shaw, R. S., and Tang, W.-J. (1997) J. Biol. Chem. 272, 12342-12349[Abstract/Free Full Text]
  37. Yan, S.-Z., Huang, Z.-H., Rao, V. D., Hurley, J. H., and Tang, W.-J. (1997) J. Biol. Chem. 272, 18849-18854[Abstract/Free Full Text]
  38. Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305[Abstract/Free Full Text]
  39. Cooper, D. M. F., Yoshimura, M., Zhang, Y., Chiono, M., and Mahey, R. (1994) Biochem. J. 297, 437-440[CrossRef][Medline] [Order article via Infotrieve]
  40. Cech, S. Y., Broaddus, W. C., and Maguire, M. E. (1980) Mol. Cell. Biochem. 33, 67-92[Medline] [Order article via Infotrieve]
  41. Cooper, D. M. F. (1994) Methods Enzymol. 238, 71-81[Medline] [Order article via Infotrieve]
  42. Caldwell, K. K., Boyajian, C. L., and Cooper, D. M. F. (1992) Cell Calcium 13, 107-121[Medline] [Order article via Infotrieve]
  43. Goldhammer, A., and Wolff, J. (1982) Anal. Biochem. 124, 45-52[Medline] [Order article via Infotrieve]
  44. Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777[Abstract/Free Full Text]
  45. Zhi, G., Abdullah, S. M., and Stull, J. T. (1998) J. Biol. Chem. 273, 8951-8957[Abstract/Free Full Text]
  46. Tokumitsu, H., Wayman, G. A., Muramatsu, M., and Soderling, T. R. (1997) Biochemistry 36, 12823-12827[CrossRef][Medline] [Order article via Infotrieve]
  47. Yokokura, H., Picciotto, M. R., Nairn, A. C., and Hidaka, H. (1995) J. Biol. Chem. 270, 23851-23859[Abstract/Free Full Text]
  48. Wakabayashi, S., Ikeda, T., Iwamoto, T., Pouyssegur, J., and Shigekawa, M. (1997) Biochemistry 36, 12854-12861[CrossRef][Medline] [Order article via Infotrieve]
  49. Hashimoto, Y., Perrino, B. A., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1924-1927[Abstract/Free Full Text]
  50. Cooper, D. M. F., Karpen, J. W., Fagan, K. A., and Mons, N. E. (1998) Adv. Second Messengers Phosphoprotein Res. 32, 23-51[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.