The Regulation of Type 7 Adenylyl Cyclase by Its C1b Region and Escherichia coli Peptidylprolyl Isomerase, SlyD*

Shui-Zhong YanDagger , Jeff A. BeelerDagger , Yibang Chen§, Robyn K. SheltonDagger , and Wei-Jen TangDagger

From the Dagger  Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637 and the § Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, November 15, 2000, and in revised form, December 5, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2. The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit alpha  (Galpha s). In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-trans peptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD protein can inhibit the Galpha s- and/or forskolin-activated activity of both soluble and membrane-bound AC7. Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli slyD-deficient strain. This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6. This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.



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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Changes in intracellular cAMP concentration regulate a variety of physiological responses, such as sugar and lipid metabolism, olfaction, and cell growth and differentiation. Cyclic AMP directly activates diverse molecules including cAMP-dependent protein kinase, cyclic nucleotide-gated Na+/Ca2+ and Na+/K+ channels, and cAMP-regulated guanine nucleotide exchangers of Rap-1 (1-3). Intracellular cAMP concentration is controlled primarily through regulation of adenylyl cyclase. To date, there are nine isoforms of membrane-bound adenylyl cyclase and one soluble adenylyl cyclase found in mammals (4-6). Each isoform of adenylyl cyclase has its own pattern of tissue distribution and unique regulation to fit its physiological roles.

All nine isoforms of mammalian membrane-bound adenylyl cyclases share a common structure including two ~40-kDa cytoplasmic domains (C1 and C2), each following an ~20-kDa hydrophobic domain (M1 and M2) (see Fig. 1) (4, 5). When expressed separately and then mixed in vitro, the conserved portions of the C1 and C2 domains, C1a and C2a, form a soluble enzyme that exhibits most of the regulatory properties of the membrane-bound enzyme (7-22). This soluble enzyme can be activated by subunit alpha  of G protein (Galpha s)1 and by the diterpene, forskolin, and can be inhibited by subunit alpha  of Gi and by adenosine analogs termed P-site inhibitors.

Such soluble enzyme models have yielded high resolution structures of the C2a homodimer of AC2 (2C2a) and the 5C1a/2C2a heterodimer (23, 24). These structures suggest a catalytic mechanism and basic mode of regulation. The catalytic site of adenylyl cyclase is formed in one of two small pockets at the interface of C1a and C2a. Forskolin binds the other pocket to shift the alignment of C1a and C2a. The activation of extracellular signals, such as hormones and neurotransmitters, triggers the exchange of guanine nucleotide in Galpha s, resulting in conformational changes in the switch regions of Galpha s. The switch 2 region of Galpha s interacts with both C2a (alpha 2 and alpha 3/beta 4 regions) and C1a (N-terminal loop of beta 1) to promote the proper alignment between C1a and C2a (9, 24). Amino acid residues from both C1a and C2a domains contribute to the "two-metal"- mediated catalysis, a catalytic mechanism also used in many DNA polymerases and nucleotidyltransferases (25, 26). Proper alignment of C1a and C2a induced by forskolin and Galpha s facilitates catalysis.

The C1b region links C1a to the M2 region of mammalian adenylyl cyclase and is far less conserved than the C1a and C2a regions among the nine isoforms of membrane-bound adenylyl cyclase (Fig. 1). Although C1b is not required for catalysis, it is essential in several regulatory functions, such as calmodulin activation of AC1 and cAMP-dependent protein kinase modulation of AC6 (27-30). In this paper, we use the soluble adenylyl cyclase from the C1a and C2a domains of AC7 and its membrane-bound counterpart to address the regulatory role of AC7 C1b.



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Fig. 1.   Model of mammalian adenylyl cyclases (left) and sequence comparison of the conserved portion of C1b regions among nine isoforms of mammalian adenylyl cyclase (right). The sequences of three peptides, ISLL, FGSI, and FLLT, derived from the C1b region of rat AC2, human AC7, and rat AC6, respectively, are compared with the C1b of six other isoforms of adenylyl cyclase. The conserved sequences are highlighted in bold. The asterisk symbol marks the highly conserved glutamic acid at residue 582 of human AC7.



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ABSTRACT
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EXPERIMENTAL PROCEDURES
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Materials-- Forskolin was from Calbiochem. Restriction enzymes and Vent DNA polymerase were from New England Biolabs (Beverly, MA). The Bradford reagent was from Bio-Rad. Renaissance Western blot Chemiluminescence Reagent Plus and [alpha -32P]ATP were from PerkinElmer Life Sciences. Ni-NTA resin was from Qiagen (Chatsworth, CA). Quick-Change kit for site-directed mutagenesis was from Stratagene (La Jolla, CA). Big-dye kit for automatic DNA sequencing was from PerkinElmer Life Sciences. Reagents for yeast two-hybrid analysis were from CLONTECH (Palo Alto, CA).

Plasmids-- The coding sequence for AC7 C1b was amplified by 18 cycles of polymerase chain reaction using the human AC7 as the template, Vent DNA polymerase, and primers 5'-GGTCGAATTCCCCGGAGCCAGCAGCCACCCCCGCCCAGCC-3' and 5'-CGCAAAGCTTCTAGTGGCGGGCCCGGGGGATGGGTGCCAG-3' (7C1b from amino acids 483 to 596). The resulting DNAs were digested with EcoRI and HindIII and ligated into pProEx-HAH6, which was digested with the same enzymes, resulting in pProEx-HAH6-7C1b. Plasmid used to express 7C1b(E582A) was constructed by Quick-change site-directed mutagenesis using pProEx-HAH6-7C1b as the template. Mutagenic oligonucleotides contained 25-30 complementary nucleotides flanking each side of the codon(s) targeted for mutation. Mutations were confirmed by DNA sequencing.

The yeast two-hybrid vectors, pLexA-W1 and pB42AD-W1, were constructed by Quick-Change site-directed mutagenesis using pLexA and pB42AD as the templates (CLONTECH) so that the coding of the DNA binding domain of LexA and activating domain of B42 can fuse in-frame with the multiple cloning site of the pProEx-HAH6 vector. To construct pLexA-W1-7C1a and pLexA-W1-7C2a, the HindIII-blunted EcoRI fragments of 7C1a and 7C2a coding sequences were excised from pProEx-HAH6-7C1a and pProEx-HAH6-7C2a and ligated into pLexA-W1, which was digested with EcoRI and PvuII. A similar strategy was used for constructing pB42AD-W1-7C1b except that the filled in XhoI site of pB42AD-W1 was used.

Expression and Purification of 7C1b-SlyD Protein-- To express 7C1b and 7C1b mutants, pProEx-HAH6-7C1b and pProEx-HAH6-7C1b(E582A) were transformed into Escherichia coli strain BL21(DE3), and the cells were grown in T7 medium containing 50 µg/ml ampicillin at 30 °C. When A600 reached 0.4, isopropyl-1-thio-beta -D-galactopyranoside was added to 30 µM final concentration. After 4 h, the induced cells were collected and lysed. The recombinant proteins were purified using Ni-NTA and Q-Sepharose columns similar to the purification of 1C1a and 2C2a as described (7). E. coli SlyD was copurified with 7C1b. 7C1b-SlyD and 7C1b(E582A)-SlyD complexes were further purified using Amersham Pharmacia Biotech Superdex 200 column and identified based on molecular weight and immunoblot. The concentration of proteins was determined using the Bradford reagent with bovine serum albumin as standard (32).

Purification of 7C1b Protein from E. coli slyD - Strain-- To obtain 7C1b free of SlyD, pProEx-HAH6-7C1b was transformed into E. coli RY-3041, a BL21(DE3) slyD- strain, and grown to A600 = 0.45. For induction, isopropyl-1-thio-beta -D-galactopyranoside was added to 30 µM, and the incubation temperature was lowered to 22 °C. Cells were harvested 4 h after induction. Cells were lysed in buffer containing 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 0.025% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 µM pepstatin A, 0.5 µg/ml aprotinin, 0.1 mM benzamidine, and 0.1 mg/ml lysozyme, followed by a 4-min sonication (1 s on, 3 s off) and centrifugation at 100,000 × g for 30 min. Lysate was loaded on a Ni-NTA affinity column (10 ml of resin) pre-equilibrated to the same buffer as used for lysis (without lysozyme). The column was washed with 100 ml of low imidazole (same buffer with 15 mM imidazole) and then eluted with a 15-200 mM imidazole gradient (3-min fractions and 2 ml/min flow rate). 7C1b was detected by Coomassie Blue staining of SDS-PAGE, and fractions containing relatively pure 7C1b were concentrated to 1 ml by Amicon positive pressure filtration with Millipore filter YM10 membrane. A combination of membrane dialysis (Spectra/Por 16 mm, molecular weight cut off 10,000) and Amicon filtration were used to bring NaCl and imidazole concentrations to less than 2 and 0.2 mM, respectively.

Purification of SlyD and SlyD Mutants-- To express SlyD and its mutant proteins, the plasmids for the expression of wild type SlyD and SlyD mutants (L11R and Delta 107-111) in plasmid pJF118EF were transformed into E. coli BL21(DE3) wild type and slyD-(MCX) strains, respectively. The resulting cells were grown in modified T7 media with 50 µg/ml ampicillin at 30 °C to an A600 = 0.4. Protein expression was then induced with 100 µM isopropyl-1-thio-beta -D-galactopyranoside. 19 h after infection, 0.1 µM phenylmethylsulfonyl fluoride was added. The cells were then spun down, and the cell pellet was frozen at -80 °C. SlyD and its mutant proteins were purified by Ni-NTA and Q-Sepharose column in the manner similar to that of 7C1b. The yields of SlyD and its mutant proteins were about 10-20 mg with greater than 95% purity from each liter of E. coli culture.

Adenylyl Cyclase Assay-- Adenylyl cyclase assays were performed in the presence of 10 mM MgCl2 and 0.5 mM ATP at 30 °C for 20 min as described previously (7). To express wild type and mutated forms of Galpha s, pQE60 that carried wild type or mutant forms of Galpha s were transformed to BL21(DE3) that carried pUBS520, and the induction and purification of recombinant Galpha s were performed as described previously (33). Recombinant Galpha s was activated by 50 µM AlCl3 and 10 mM NaF. Expression of soluble and membrane-bound AC7 and purification of 7C1a and 7C2a were performed as described (34). Adenylyl cyclase activity of Sf9 cell membranes was completed as described previously (35).

Peptide Synthesis-- Peptides were synthesized with an Applied Biosystems model 430A peptide synthesizer. Identity of the peptides was verified by mass spectrometry. Peptides were dissolved in water and used at final concentration as indicated.

beta -Galactosidase Assay-- Yeast EGY48 strain that carried pSH18-34 was transformed with pLexA-W1-7C1a or pLexA-W1-7C2a. The resulting transformants were then transformed with pB42AD-W1-7C1b. The yeast clones that carried all three plasmids were grown in synthetic defined media lacking uridine, histidine, and tryptophan and containing 2% galactose and 1% raffinose at 30 °C until the cells reached a density of A600 = 1. beta -Galactosidase assay was performed with 1 ml of cells as described previously (36).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Characterization of 7C1a and 7C2a-- We made C1a (amino acids 263-476) and C2a (amino acids 864-1080) from human AC7 that effectively could be expressed and purified (Fig. 2A) (34). To compare soluble adenylyl cyclase with its native enzyme, a recombinant baculovirus was constructed to express membrane-bound AC7 (34). Both membrane-bound AC7 and soluble 7C1a-7C2a complex were substantially stimulated by Galpha s and forskolin, consistent with the observation that a coexpressed Gs-coupled receptor could raise cAMP formation over 20-fold in mammalian cells overexpressing AC7 (34, 37). Significant synergy was observed in both membrane-bound AC7 and soluble 7C1a-7C2a complex when Galpha s and forskolin were coapplied (Fig. 3).



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Fig. 2.   Purified soluble AC7 domains and SlyD. Purified 7C1a (HAH6-7C1a) and 7C2a (H6-7C2) (A), 7C1b-SlyD complex (B), and SlyD and its mutants (C) (2 µg each) were run onto SDS-PAGE and stained by Coomassie Blue.



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Fig. 3.   Comparison of soluble 7C1a-7C2a and membrane-bound AC7 activated by Galpha s and forskolin. Adenylyl cyclase activities of soluble 7C1a-7C2a (A) and membrane-bound AC7 (mAC7) (B) are shown. Adenylyl cyclase activity of 7C1a-7C2a (0.4 µM each) and membrane-bound AC7 (80 µg) were activated by Galpha s and forskolin. The concentration of Galpha s in the assay is 240 nM. Sum (Fsk + Galpha s) is the sum of adenylyl cyclase activity observed in the presence of forskolin or Galpha s alone (open circle ), Fsk + Galpha s is adenylyl cyclase activity observed in the presence of both forskolin and Galpha s (). The means ± S.E. are representative of three experiments. Fsk, forskolin.

Purification and Characterization of 7C1b-SlyD-- The soluble 7C1a-7C2a complex is used to address the role of C1b because it does not contain C1b. We constructed a vector to express a polypeptide chain from amino acids 457 to 596 of human AC7 named 7C1b. Adding lysates of E. coli containing 7C1b, we observed 30% inhibition in Galpha s and forskolin-stimulated 7C1a-7C2a activity (data not shown). However, no detectable 7C1b protein was found by immunoblot, suggesting that 7C1b may undergo proteolysis at its N terminus. To test this hypothesis, we constructed and expressed four N-terminal truncation mutants of 7C1b. Lysates of E. coli containing one of four mutants, 7C1b-Delta 2 (amino acids 483-596 of human AC7), exhibited 30% inhibition in 7C1a-7C2a activity stimulated by Galpha s and forskolin and had the highest immunoreactivity. Thus, 7C1b-Delta 2 was used in the subsequent study and renamed as 7C1b.

C1b contains a short stretch of sequence that is relatively conserved among nine isoforms of adenylyl cyclase (Fig. 1). We made 7C1b mutants with single point mutations in this region and screened for one that expressed similar to wild type 7C1b but lost its ability to inhibit 7C1a-7C2a activity. Upon assaying E. coli lysates containing 7C1b mutants, we found that 7C1b(E582A), a mutant with a conserved glutamic acid at residue 582 that changed to alanine, fit these criteria (Fig. 1, data not shown). We characterized both wild type 7C1b and 7C1b(E582A) in our subsequent study.

7C1b and 7C1b(E582A) proteins were purified through three chromatographic steps. After purification through Ni-NTA, Q-Sepharose, and Superdex 200 columns, the expected 16-kDa 7C1b and 7C1b(E584A) were obtained with the yield of 2.2 mg from 1 liter of E. coli culture (Fig. 2B). A 25-kDa protein was copurified with 7C1b at about 4-fold molar excess after purification through the Ni-NTA column. A stoichiometric quantity of 25 kDa and 7C1b was obtained after purification through the Q-Sepharose and Superdex 200 gel permeation columns. Based on the gel permeation column, the molecular size of 7C1b is approximately 40 kDa, which is not a high molecular mass aggregate. This suggests that 7C1b forms a complex with the 25-kDa protein in a 1:1 stoichiometric ratio. The N-terminal protein sequence analysis of the 25-kDa protein resulted in XXVAXDLLVVLAYQ that matched the N-terminal sequence of SlyD. SlyD, a 25-kDa protein with a histidine-rich C-terminal region, can bind the Ni-NTA column effectively (38, 39). The predicted pI of SlyD and 7C1b are 4.8 and 8.5, respectively, which may lead to the charge-charge interaction of these two proteins. In the subsequent study, we refer to our purified 7C1b and 7C1b(E582A) preparations as 7C1b-SlyD or 7C1b(E582A)- SlyD.

SlyD Inhibits the Activity of Type 7 Adenylyl Cyclase-- SlyD protein, one of several gene products originally identified as suppressor of phage lysis, is a member of the FK506-binding protein family and exhibits cis-trans peptidylprolyl isomerase (PPIase) activity (38, 39). We tested whether SlyD protein could affect the activity of the 7C1a-7C2a complex using purified SlyD protein (Figs. 2C and 4). In the presence of Galpha s, forskolin, or the combination of Galpha s and forskolin, we observed up to 80% inhibition of adenylyl cyclase activity by SlyD in a dose-dependent manner. To address whether PPIase activity is required for this inhibition, we purified and tested two SlyD mutant proteins, SlyD(L11R), which has no detectable PPIase activity, and SlyD(Delta 107-111), which has only 20% PPIase activity (Fig. 2C) (47). SlyD(L11R) showed no inhibition to 7C1a-7C2a, and SlyD(Delta 107-111) had reduced potency, suggesting that PPIase activity plays a role in SlyD inhibition of adenylyl cyclase (Fig. 4).



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Fig. 4.   Regulation of 7C1a-7C2a activity by SlyD. 7C1a-7C2a (0.4 µM each) was stimulated by 2.2 µM Galpha s (Gsalpha ) (A), 100 µM forskolin (Fsk) (B), and 0.2 µM Galpha s with 100 µM forskolin (Gsalpha /Fsk) (C) in the presence of the indicated SlyD concentration. The specific activities of 7C1a-7C2a, stimulated by 2.2 µM Galpha s, 100 µM forskolin, and 0.2 µM Galpha s with 100 µM forskolin, were 130, 18, and 660 nmol·min-1·mg-1, respectively. The means ± S.E. are representative of four experiments.

We then tested whether SlyD could modulate the activity of membrane-bound adenylyl cyclases. Using the membrane preparations from Sf9 cells that expressed individual isoforms of adenylyl cyclase, we observed that SlyD protein could inhibit the activity of both AC2 and AC7 while having little effect on the activity of AC1. Interestingly, SlyD could increase the activity of AC5 and AC6 (Fig. 5A). These effects could not be observed with the PPIase-defective mutant, SlyD(L11R) (Fig. 5B). Thus, we conclude that the effect mediated by SlyD is both isoform-specific and involves PPIase activity.



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Fig. 5.   Regulation of membrane-bound adenylyl cyclase by SlyD. The membranes of Sf9 cells containing the indicated isoform of adenylyl cyclase (80 µg of each) were assayed for their adenylyl cyclase activity in the presence of 100 µM forskolin, and 0.24 µM Galpha s was assayed in the presence of 20 µg of SlyD (A) and 20 µg of SlyD(L11R) (A and B). The data shown represent adenylyl cyclase activities after subtracting out the activity of membranes of Sf9 cells infected with control virus. The specific activities of the control membranes in the absence and presence of SlyD were 0.9 and 1.5 nmol·min-1·mg-1, respectively. The means ± S.E. are representative of at least two experiments.

Inhibition in the Activity of Soluble 7C1a-7C2a by 7C1b-SlyD and 7C1b-- To address the role of 7C1b in regulating AC7, we used 7C1b-SlyD and 7C1b(E582A)-SlyD. As a control, we used an equal quantity of purified SlyD compared with that in our 7C1b-SlyD preparations and based on protein concentration and SDS-PAGE. In the concentration range of 0.1-0.6 µM Galpha s, 7C1b-SlyD exhibited 40-50% more inhibition than SlyD alone (Fig. 6A). The inhibition is dose-dependent (Fig. 6B). Such inhibition in excess of the SlyD control was not observed with 7C1b(E582A)-SlyD (Fig. 6). At a high concentration of Galpha s (2.2 µM) or in the presence of both Galpha s and forskolin, no C1b-mediated inhibition of 7C1a-7C2a was observed (Fig. 6A, data not shown). When the activity of membrane-bound AC7 was analyzed, we observed no additional effect of 7C1b-SlyD that was not accountable by SlyD protein (data not shown).



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Fig. 6.   Adenylyl cyclase activity of 7C1a-7C1a regulated by SlyD, 7C1b-SlyD, and 7C1b(E582A)-SlyD. A, the effects of SlyD, 7C1b-SlyD, and 7C1b(E582A)-SlyD (5.3 µM each) on the activities of 7C1a-7C1a (0.4 µM each) in the indicated Galpha s concentration. Inset shows the effect of SlyD (solid bar), 7C1b-SlyD (open bar), and 7C1b(E582A)-SlyD (hatched bar) in the indicated Galpha s concentration. Differences are statistically significant. *, p = 0.002; +, p = 0.0005. B, the effect on the activities of 7C1a-7C2a (0.4 µM each) in the indicated concentrations of SlyD, 7C1b-SlyD, and 7C1b(E582A)-SlyD. Adenylyl cyclase assays were performed in the presence of 0.6 µM Galpha s. The means ± S.E. are representative of six experiments.

To demonstrate 7C1b inhibition in the absence of SlyD, we expressed and purified 7C1b in RY3041, an E. coli BL21(DE3) strain that is defective in the expression of SlyD. Expression of 7C1b in the slyD- strain was comparable with the expression in the BL21(DE3) strain based on the immunoblot of the lysate. Following the Ni-NTA column, we obtained 7C1b protein as seen on a Coomassie Blue-stained SDS-PAGE gel and immunoblot. However, the purified 7C1b could no longer be detected after only a few hours in the cold room, making further purification steps impossible. After testing a panel of stabilizing agents, we determined that 0.025% Triton X-100 in combination with 0.5 M NaCl stabilized the protein sufficiently to conduct further concentration and buffer exchange steps. We further determined that the salt concentration could be reduced to < 2 mM if the protein was frozen in dry ice quickly after salt removal and stored at -80 °C. This finding allowed us to obtain 7C1b that is ~50% pure but free of SlyD (Fig. 7A). The partially purified 7C1b inhibited the activity of 7C1a-7C2a (Fig. 7B). As a control for the detergent in the buffer, we boiled our 7C1b preparation (100 °C for 3 min), which abolished the inhibition (data not shown). This further supports an inhibitory role for 7C1b.



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Fig. 7.   Inhibitory effect of SlyD-free 7C1b on adenylyl cyclase activity of soluble 7C1a-7C2. A, purified 7C1b (1 µg) protein was run onto SDS-PAGE and stained by Coomassie Blue. B, inhibition of adenylyl cyclase activity of 7C1a-7C2a by purified 7C1b (1 µg). Adenylyl cyclase assay was performed in the presence of 0.75 µM Galpha s. The means ± S.E. are representative of five experiments.

Inhibition of Membrane-bound Adenylyl Cyclases by Peptides Derived from C1b-- To address the role of 7C1b further, we made 28-mer peptides derived from the most conserved region of C1b across three adenylyl cyclases (Fig. 1). FGSI and ISLL peptides (named by their first four amino acid sequences) were derived from two closely related adenylyl cyclases AC7 and AC2, respectively. FGSI peptide inhibited the adenylyl cyclase activity of 7C1a-7C2a activated by forskolin and/or Galpha s (Fig. 8A). Furthermore, the addition of FGSI and ISLL resulted in the inhibition of adenylyl cyclase activity of soluble and membrane-bound AC7 in a dose-dependent manner (Figs. 8B and 9A). FLLT peptide was derived from the region corresponding to FGSI in a distantly related isoform, AC6, and had little effect on the enzymatic activity of soluble and membrane-bound AC7 (Figs. 8B and 9A). FLLT is active because it inhibited the activity of membrane-bound AC6 as described previously (Fig. 9B) (29). Interestingly, FGSI but not ISLL was a potent inhibitor of membrane-bound AC6 (Fig. 9B). Taken together, these data indicate that C1b is capable of inhibiting the activity of adenylyl cyclase, and peptides from a particular region of C1b may confirm isoform specificity.



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Fig. 8.   Effects of ISLL, FGSI, and FLLT peptides derived from the C1b region of AC7, AC2, and AC6 on adenylyl cyclase activity of 7C1a-7C2a. The adenylyl cyclase activity of 7C1a-7C2a (0.4 µM each) activated by 100 µM forskolin, 2.2 µM Galpha s, or 100 µM forskolin with 2.2 µM Galpha s, in the presence of FGSI peptide (A) and 100 µM forskolin with 0.24 µM Galpha s (B), was performed in the indicated peptide concentration. The specific activities of 7C1a-7C2a were 310 (2.2 µM Galpha s), 850 (2.2 µM Galpha s and 100 µM forskolin), 22 (100 µM forskolin), and 660 (100 µM forskolin and 0.24 µM Galpha s) nmol·min-1·mg-1. The means ± S.E. are representative of at least three experiments.



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Fig. 9.   Effects of ISLL, FGSI, and FLLT peptides derived from the C1b region of AC7, AC2, and AC6 on adenylyl cyclase activity of membrane-bound AC7 (mAC7) and membrane-bound AC6 (mAC6). The adenylyl cyclase activity of mAC7 (A) and mAC6 (B) (80 µg each) activated by 100 µM forskolin and 0.24 µM Galpha s was performed in the indicated peptide concentration. The specific activities of mAC7 and mAC6 were 17 and 8 nmol·min-1·mg-1, respectively. The means ± S.E. are representative of at least three experiments.

Interaction of 7C1b with 7C1a and 7C2a-- To initially assess interaction of 7C1b to 7C1a and/or 7C2a, we used the yeast two-hybrid system where transactivation of the lacZ reporter indicates potential interaction (40). We observed light blue colonies on medium containing 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (X-gal) for yeast strain EGY48(pSH18-34) that contained 7C1a or 7C2a fused to the DNA binding domain of LexA (LexA-DB-7C1a or LexA-DB-7C2a, respectively) (data not shown). When the 7C1b-B42AD fusion was coexpressed in yeast strains containing LexA-DB-7C1a or LexA-DB-7C2a, a >2-fold increase was observed in the beta -galactosidase activity (standard beta -galactosidase unit multiplied by 1000: 7C1a with control plasmid = 36 ± 1 while 7C1a with 7C1b = 84.6 ± 0.4 and 7C2a with control = 40 ± 2 while 7C2a + 7C1b = 85.8 ± 0.5). This finding suggests that 7C1b can interact with both 7C1a and 7C2a.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The catalytic core of mammalian adenylyl cyclase consists of two cytoplasmic domains, C1a and C2a (22). The catalytic site is located at the interface of C1a and C2a and includes residues from both domains (24, 26). The activation of adenylyl cyclase does not seem to involve a major conformational change in either C1a or C2a upon the binding of Galpha s. Instead, catalysis is activated by an induced juxtaposition of these two domains to properly form the catalytic cleft at their interface. Interestingly, the structure of 2C2 homodimer and 5C1a/2C2a heterodimer reveals significant contact between C1a and C2a (23, 26). Because C1a and C2a are tethered by the transmembrane domains (M1 and M2) in their native membrane-bound configuration, the question arises of how C1a and C2a are kept in an inactive conformation in the resting state of the enzyme.

The C1b region joins C1a to C2a by connecting C1a to the second transmembrane domain (M2), which then connects to C2a (Fig. 1). Thus, C1b is in an ideal location to dynamically regulate the interaction between C1a and C2a and thus modulate the catalytic rate. Using both C1b protein and peptides derived from C1b, our data show that AC7 C1b can inhibit AC7. We also observed that both C1b and peptides derived from C1b inhibit soluble 7C1a-7C2a more potently than they inhibit membrane-bound AC7. Such a difference is probably due to the presence of endogenous C1b in the membrane-bound AC7.

How does C1b inhibit the activity of AC7? Our yeast two-hybrid analysis suggests that AC7 C1b can bind 7C1a and 7C2a. Using 7C1b-SlyD, our coimmunoprecipitation experiment shows that 7C1b can bind both 7C1a and 7C2a, which is in agreement with our yeast two-hybrid analysis.2 Using antibody that can immunoprecipitate 7C2a, the active 7C1a-7C2a-Galpha s complex can be coimmunoprecipitated.2 Interestingly, 7C1b-SlyD can compete with Galpha s to bind the 7C1a-7C2a complex, suggesting that one possible mechanism for 7C1b-mediated inhibition is blocking the binding of Galpha s to 7C1a-7C2a complex.2 However, the presence of SlyD in our 7C1b preparation precludes drawing any definitive conclusion. It would be interesting to determine whether 7C1b directly interacts with 7C1a and 7C2a and whether the interaction of 7C1b to 7C1a and 7C2a is involved in 7C1b-mediated inhibition. It is worth noting that Scholich et al. (20) have shown that AC5 C1b can interact with 5C2a by yeast two-hybrid analysis. Unfortunately, they did not test the interaction of AC5 C1b with its C1a domain.

C1b is one of the key regions for isoform-specific regulation. An amphipathic region that is only present in AC1 C1b is involved in the binding and activation of Ca2+/calmodulin (27, 28, 30). Splicing variants of AC8 that have different C1b sequences are differentially sensitive to calmodulin (41). AC6 C1b has a cAMP-dependent protein kinase phosphorylation site that confers feedback inhibition by cAMP (29). The C1b region may also be involved in inhibition by calcium and Galpha i in AC5 and regulation by calcineurin in AC9 (17, 19, 42). We show here that peptides from the C1b region of different isoforms of adenylyl cyclase can inhibit the activity of a subset of adenylyl cyclase isoforms. This isoform-specific inhibition by peptides may provide a tool with which to address the physiological functions of each isoform of adenylyl cyclase in the intact cells (43).

We were surprised to find that the catalytic activity of certain isoforms of mammalian adenylyl cyclase can be stimulated or inhibited by SlyD. Using mutant forms of SlyD, we find that this isoform-specific regulation may be due to PPIase activity of SlyD. This raises the possibility that mammalian PPIases may regulate the activity of selective isoforms of mammalian adenylyl cyclase directly. There are three gene families of eukaryotic PPIases: FK506-binding protein, cyclophilins, and parvulins (44). PPIase is known as a folding helper enzyme; however, growing evidence suggests that PPIases may serve to regulate biological activity after the proper folding of proteins (31, 45, 46). Thus, an understanding of how SlyD regulates mammalian adenylyl cyclases may not only provide a novel molecular tool for modulating adenylyl cyclase activity in an isoform-specific manner but may also lead to a new area of research in the regulation of adenylyl cyclase activity by PPIases.


    ACKNOWLEDGEMENTS

We are grateful for the help in oligonucleotide synthesis and DNA sequencing from P. Gardner (Howard Hughes Medical Institute, University of Chicago), protein sequencing from Andrew Bohm (Boston Biomedical Research Institute), plasmids for the expression of SlyD and its mutants from Bill Roof (Texas A&M University), and insightful suggestions from R. Iyengar (Mount Sinai School of Medicine) and Ryland F. Young (Texas A&M University).


    FOOTNOTES

* This research was supported by National Institute of Health Grant GM53459 and American Heart Association Established Investigator Award (to W. -J. T.), National Institute of Health Grant DK38761 (to R. Iyengar), and Fellowship from American Heart Association Chicago Affiliate and University of Chicago Committee on Cancer Biology (to S. -Z. Y.).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.

To whom correspondence should be addressed: Dept. of Neurobiology, Pharmacology, and Physiology, The University of Chicago, 947 E. 58th St., MC0926, Chicago, IL 60637. Tel.: 773-702-4331; Fax: 773-702-3774; E-mail: wtang@uchicago.edu.

Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M010361200

2 S.-Z. Yan, J. Beeler, and W.-J. Tang, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: Galpha s, subunit alpha  of the G protein that stimulates adenylyl cyclase; AC, adenylyl cyclase; PPIase, cis-trans peptidylprolyl isomerase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NI-NTA, nickel-nitrilotriacetic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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