The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase*

(Received for publication, November 27, 1996, and in revised form, February 14, 1997)

Shui-Zhong Yan , Zhi-Hui Huang , Robin S. Shaw and Wei-Jen Tang Dagger

From the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgements
REFERENCES


ABSTRACT

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C2 domain is catalytically active and the C1 domain is not; the role of the C1 domain is to promote the catalytic activity of the C2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C2 domains, but not among the C1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30-100-fold reduction in Kcat of adenylyl cyclase. However, the same mutations do not affect the Km for ATP, the half-maximal concentration (EC50) for the C2 domain of type II adenylyl cyclase to associate with the C1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without Gsalpha . This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in Kcat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.


INTRODUCTION

The activity of mammalian adenylyl cyclase, the enzyme that converts ATP to cAMP, is the key step in modulating intracellular cAMP concentration in response to extracellular stimulation by hormones, neurotransmitters, and odorants. Nine isoforms of mammalian adenylyl cyclases have been cloned to date, and they belong to a rapid expanding cyclase family that includes class III adenylyl cyclases1 and guanylyl cyclases (1). All nine isoforms have a similar structure, including two intensely hydrophobic domains (M1 and M2) and two 40-kDa cytoplasmic domains (C1 and C2). The two cytoplasmic domains contain sequences (C1a and C2a) that are homologous to each other and to class III adenylyl cyclases and guanylyl cyclases. Each isoform of adenylyl cyclase not only has distinct patterns of tissue expression but also has unique responses to extracellular and intracellular stimuli (1-3). In common, each adenylyl cyclase can be activated by the G protein2 alpha  subunit, designated as Gsalpha , and each can be inhibited by certain adenosine analogues (P-site inhibitors). However, there are several subtype-specific regulators, including forskolin, G protein beta gamma subunits, the alpha  subunit of Gi, Go, and Gz, Ca2+ ion, Ca2+-calmodulin, Ca2+-calcineurin, cAMP-dependent protein kinase, and PKC (for review, see Refs. 1-3). Certain ones of these regulators (e.g. beta gamma ) can be either stimulatory or inhibitory, depending on the subtype of adenylyl cyclase (4). When acting concurrently, the effects of regulators can be independent or interdependent (antagonistic or synergistic). The binding sites of adenylyl cyclases for Ca2+-calmodulin and G protein beta gamma subunits are in the C1 and C2 domains, respectively (5-8). The sites for other regulators and the mechanisms by which they modulate activity of adenylyl cyclase remain to be determined.

One key step in studying the mechanisms for the complicated regulation of adenylyl cyclase is to understand the molecular basis of catalysis. The Gsalpha - and forskolin-stimulated soluble adenylyl cyclases can be formed either by mixing IC1 protein (C1 domain of type I adenylyl cyclase) and IIC2 protein (C2 domain of type II adenylyl cyclase) in vitro or by covalent linkage of IC1 and IIC2 proteins (IC1IIC2) (9-12). From the genetic and biochemical analysis of this soluble adenylyl cyclase and mutational analysis of type I enzyme, we have shown that both C1 and C2 domains are required for the high enzymatic activity (13). Furthermore, the data suggests that the C2 domain is the catalytic domain while the C1 domain enhances the activity of the C2 domain. In this paper, genetic and biochemical analyses are used to identify two amino acid residues, asparagine and arginine that are crucial for catalysis and might play an important role in inactivating the enzymatic activity of the C1 domain.


EXPERIMENTAL PROCEDURES

Materials

Forskolin was from Calbiochem (La Jolla, CA); 2'-d-3'-AMP was from Sigma; restriction enzymes and Vent DNA polymerase were from New England Biolabs (Beverly, MA); Bradford reagent was from Bio-Rad; ECL system was from Amersham Corp.; Ni-NTA resin was from Qiagen (Chatsworth, CA); and Escherichia coli XL1-red cells were from Stratagene (La Jolla, CA). GTPgamma S was purchased from Pharmacia Biotech Inc. and purified by FPLC Mono Q column chromatography.

Isolation of Loss of Function of IC1IIC2 Mutants and the Second Site Suppressor of IC1IIC2-N1025S

E. coli strain, WJT-1 (Delta cya AraD- endoA crp/crp3 malT/malT3) was constructed so that cAMP synthesis by the cloned cyclase replaced such synthesis by the bacteria gene and that made it possible to enrich for mutations that decreased synthesis of cAMP. To construct E. coli WJT-1 cells, E. coli TK2313 (F- thi rha lacZ nagA Delta (kdpAE81) trk1 trkA405 endoA) was transduced with P1 lysate of a thr34::Tn10 strain, and a tetracycline-resistant colony was purified. This strain was then transduced to Thr+, araD139 with a lysate of strain MC4100 (14). One araD139 derivative of TK2313 was purified and transduced to ilv500::Tn10 with a lysate of strain MRi134 (pertinent marker is ilv500::Tn10), and a tetracycline-resistent colony was purified. The resulting strain was transduced to ilv+, Delta cya with a lysate of strain TK2648 (pertinent marker Delta (cya)8306, a deletion originally obtained from J. Beckwith (Harvard Medical School) in strain CA8306), and a Delta cya transductant was purified. Finally, the F141 plasmid that carried the crp malT TrkA genes was introduced by selection for growth on medium containing 0.1 mM K+ (15).

Two conditions were used to select the loss of function IC1IIC2 mutants in which expression of cAMP-dependent genes was growth inhibitory or lethal. The first was the sensitivity of E. coli to infection and killing by lambda  phage, and the second was its inhibition of growth by a metabolic intermediate of arabinose catabolism. A library of plasmids that had mutations at the coding region of IC1IIC2 was constructed by propagating plasmid pProEx-HAH6-IC1IIC2 in E. coli mutator strain, XL-1 red (mutD5 mutS mutT). Plasmid DNAs were isolated and transformed into WJT-1 cells that harbored pBB131-Gsalpha Q227L [WJT-1 (Gsalpha Q227L)]. The transformants were grown in Luria broth (LB) that contained 0.2% maltose and supplement A (2 mM KCl, 5 mM MgSO4, 100 µM IPTG, 25 µM ampicillin, and 50 µM kanamycin) overnight. The transformants (107 cells) were then infected with lambda  phage, lambda vir (1010 plaque-forming units) in LB medium that contained 1% maltose and supplement A for 1 h, and then plated on LB plates that contained 1% maltose, 1% arabinose, and supplement A. Plasmid DNAs from the resulting colonies were isolated and transformed into WJT-1 (Gsalpha Q227L). The resulting cells were tested on MacConkey plates (MacConkey agar base, Difco) that contained 1% maltose and supplement A. The coding sequences for IC1IIC2 of the mutated DNAs from colonies that were negative on maltose-MacConkey plates were then excised by EcoRI and HindIII from plasmids encoding the mutants and cloned into pProEx-HAH6 that had been digested with the same enzymes. The DNAs that were transformed into strain WJT-1 cells and were again negative on maltose-MacConkey plates were analyzed further for expression and enzymatic activity of IC1IIC2. Double-stranded DNA sequencing of the coding region of IC1IIC2 mutants was performed to identify the mutations.

The second site suppressors from IC1IIC2-N1025S mutants were obtained from E. coli that grew faster than the control (E. coli that express both IC1IIC2-N1025S and Gsalpha Q227L) on maltose-minimal medium. To increase the sensitivity of cAMP detection, E. coli WJT-3 cells (Delta cya cpdB-) were constructed by transducing E. coli Crookes strain (cpdB-) to Delta cya by the protocol used to put this mutation into WJT-1 cells (16). The procedures in analyzing the second site suppressors were similar to that in analyzing the loss of function mutants of IC1IIC2 proteins.

Plasmids

To construct pProEx-HAH6-IIC2-M16 or pProEx-HAH6-IIC2-N1025S(TCG), 0.8 kbp of DNA was amplified by 15 cycles of polymerase chain reaction using pProEx-HAH6-IC1IIC2-M16 or pProEx-HAH6-IC1IIC2-N1025S(TCG) as the template, respectively, Vent DNA polymerase, and two oligonucleotides (5'-CGAGGAATTCTGGAGAACGTGCTTCCTGCACAC and 5'-TGCGTTCTGATTTAATCTGTATCAGGCTGA) as the primers.4 The resulting DNAs were digested with EcoRI and HindIII and ligated into pProEx-HAH6 that had been digested with the same enzymes. The resulting constructs were confirmed by dideoxy nucleotide sequencing. Kunkle's method (17) was used to incorporate the desired point mutations into the phagemid vectors, pProEx-HAH6-IIC2 and pProEx-HAH6-IIC2-M16 (N1025S). Oligonucleotides (23-mer) used for mutagenesis contained 10 complementary nucleotides flanking each side of the target codon, which was replaced with the appropriate codon for the mutants that had single point mutation (N1025S, N1025A, N1025D, N1025K, N1025Q, N1025T, and R1029A) or double point mutations (IIC2-P1015S, N1025S; P1015A,N1025S; P1015D,N1025S; P1015E,N1025S; P1015N,N1025S; and P1015Q,N1025S). Mutations were confirmed by dideoxy nucleotide sequencing of the plasmid DNA.

To construct pRC/CMV-ACII N1025K, the 0.7-kbp DNA was isolated from pProEx-HAH6-IIC2-N1025K that was digested with restriction enzymes, BspEI and XbaI, and cloned into pSK-ACII that had been digested with the same enzymes. The resulting plasmid was then digested with HindIII and XbaI, and the 3.3-kbp DNA, which encoded type II adenylyl cyclase, was excised and ligated to pRC/CMV that had been digested with HindIII and XbaI. The same approach was used to construct pRC/CMV-ACII for expression of type II adenylyl cyclase.

Expression and Purification of Wild Type and Mutant Forms of IIC2

The plasmids that encoded wild type or mutant forms of IIC2 were transformed into E. coli BL21(DE3) cells. E. coli that harbored the desired plasmid were cultured in LB medium containing 50 mg/ml ampicillin at 30 °C. When A600 reached 0.4, IPTG (100 µM) was added. After 3-4 h, the induced cells were then collected and lysed, and IIC2 proteins were purified using the Ni-NTA column and FPLC Q-Sepharose column as described (9). The Coomassie Blue staining of SDS-polyacrylamide gel electrophoresis was used to determine the protein peak in the fractions from the Q-Sepharose column. The concentration of proteins was determined using Bradford reagent and bovine serum albumin as standard (18).

Expression of ACII-N1025K in HEK 293 (ATCC CRL1573) and Mouse Adrenal Y1

Mouse adrenal Y1-t4 cells (kindly provided by Dr. E. Simpson, Univ. of Texas Southwestern Medical School) were selected for their high sensitivity to morphological changes upon cAMP stimulation. HEK 293 and mouse adrenal Y1-t4 cells (106/60-mm dish) were transfected with pRC/CMV, PRC/CMV-ACII, or pRC/CMV-ACII-N1025K by lipofection (Life Technologies, Inc). Geneticin (0.5 mg/ml) was used to select HEK 293 and mouse adrenal Y1-t4 cells that had stably incorporated the expression constructs, and cell colonies surviving antibiotic selection from a single transfection were pooled into a polyclonal population for subsequent analysis.

Adenylyl Cyclase Assay

Activities of the soluble adenylyl cyclases were assayed for 20 min at 30 °C in the presence of 10 mM MgCl2 (19). Wild-type IIC2 and IIC2 mutant proteins were premixed with IC1 on ice for 10 min before the assay. Recombinant Gsalpha was purified and activated by GTPgamma S as described (20, 21). Plasma membranes from the pools of HEK 293 and mouse adrenal Y1-t4 stable transformants were isolated as described (22). Enzyme activity of the membrane fractions was assayed for 30 min at 30 °C in the presence of 30 µM AlCl3, 10 mM MgCl2, and 10 mM NaF (AMF) or 100 µM forskolin (Fsk).


RESULTS

Isolation and Analysis of Loss of Function IC1IIC2 Mutants

We have shown that coexpression of IC1IIC2 and constitutively active Gsalpha , Gsalpha Q227L can complement the genetic defects of E. coli Delta cya, which has a deletion of the adenylyl cyclase gene and thus fails to synthesize cAMP (10). To select IC1IIC2 mutants that have reduced adenylyl cyclase activity (loss of function mutants), we have constructed an E. coli strain, WJT-1 (Delta cya AraD- endoA crp/crp3 malT/malT3). Two conditions were used to select the loss of function IC1IIC2 mutants in which expression of cAMP-dependent genes was growth inhibitory or lethal. The first was the sensitivity of E. coli to infection by lambda  phage. A virulent mutant of bacteriophage lambda (lambda vir) kills E. coli. Absorption of lambda  to the cell begins with binding to the LamB protein. Expression of LamB protein is induced when E. coli is grown with medium containing maltose, and expression of the LamB is dependent on cAMP. Thus, strains that express the maltose genes are killed by lambda vir. Mutants that do not express a functional LamB protein do not absorb lambda  phage and thus survive. Therefore, selection for resistance to lambda vir enriches for mutants unable to make cAMP. As expected, most of WJT-1 cells (>99.9%) that expressed both IC1IIC2 and Gsalpha Q227L were sensitive to infection by lambda vir (plaques were opaque), while those that expressed IC1IIC2 or Gsalpha Q227L alone were insensitive to infection (data not shown).

The second condition for selection of loss of function mutants was the growth inhibition of E. coli by a metabolic intermediate of arabinose catabolism. Arabinose induces the enzymes specific to early steps of arabinose catabolism. When ribulose-1-phosphate epimerase is defective, the product produced by the araD gene, ribulose-1-phosphate, accumulates in the cell. This sugar phosphate, like many others, is growth inhibitory when accumulated to high levels. Since expression of the genes for arabinose catabolism is also dependent on cAMP, araD mutants that make cAMP are inhibited by arabinose, while those that do not make cAMP are not. E. coli WJT-1 strain was AraD-, and thus 90% of WJT-1 cells that expressed IC1IIC2 and Gsalpha Q227L were growth-arrested when arabinose was added (both in minimal and complex media). As a control, arabinose had no effect on the growth of WJT-1 cells that expressed either IC1IIC2 or Gsalpha Q227L alone. The combination of selection by lambda vir and arabinose provided 104-fold enrichment of WTJ-1 cells that failed to produce high concentrations of intracellular cAMP (Fig. 1A).


Fig. 1. Characterization of IC1IIC2 mutants, M8 and M16. Phenotypic characterization and adenylyl cyclase activity (A) and expression of the IC1IIC2 mutants (B). AC activity = nmol·min-1·mg-1. Phenotypic characterization was performed using WJT-1 cells that expressed Gsalpha Q227L. MacConkey (hrs) shows the required time (hours) to be positive on maltose-MacConkey medium for WJT-1 cells that expressed no protein (-), wild-type IC1IIC2 (WT), IC1IIC2-M8 (M8), and IC1IIC2-M16 (M16). Survival after lambda vir infection (%) shows the relative ability of WJT-1 cells that expressed IC1IIC2 (WT) and the mutants (M8, M16) to form colonies on LB plate with 1% arabinose after infection with lambda vir (1:1,000 ratio of cell to lambda vir). Adenylyl cyclase assays were performed using lysates from E. coli BL21(DE3) cells that expressed no protein (-), IC1IIC2 (WT), and the mutants (M8, M16) without activator (Basal), with 100 µM forskolin (Fsk), with 1 µM GTPgamma S-Gsalpha (Gsalpha ), and with 100 µM forskolin + 1 µM GTPgamma S-Gsalpha (Gsalpha +Fsk). Immunoblot of lysates (25 µg) from E. coli BL21(DE3) cells that expressed no protein (-), IC1IIC2 (WT) and the mutants (M8, M16) was performed using a monoclonal antibody, 12CA5.
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The first step in selecting IC1IIC2 mutants that failed to be activated by Gsalpha Q227L in vivo was random mutagenesis of the plasmid containing the coding region of IC1IIC2 by growth in mutator strain XL-1 red (mutT mutD mutS). This strain can introduce a variety of mutations, including transitions, transversions, and frameshifts. The cells that failed to produce cAMP were selected based on their insensitivity to infection by lambda vir and to growth inhibition by arabinose. Approximately 20-fold higher numbers of colonies of WJT-1 cells that carried the mutagenized plasmids (pProEx-HAH6-IC1IIC2) were observed than those that carried the unmutagenized pProEx-HAH6-IC1IIC2, indicating that this selection did enrich for the loss of function mutants.

Thirty clones were selected for analysis. The coding regions for IC1IIC2 of 30 colonies were introduced back to unmutated pProEx-HAH6, and they were analyzed genetically (phenotype on maltose-MacConkey medium) and biochemically (immunoblot and adenylyl cyclase activity). Two IC1IIC2 mutants (M8 and M16) were of interest. WJT-1 cells that carried either IC1IIC2-M8 or M16 were insensitive to infection by lambda vir and growth arrest by arabinose and were delayed in being positive on maltose-MacConkey medium (Fig. 1A). Lysates from BL21(DE3) cells that expressed IC1IIC2-M8 and M16 mutants had normal expression of 60 kDa IC1IIC2, but they had a significant reduction (4- and 95-fold, respectively) in Gsalpha - and forskolin-stimulated adenylyl cyclase activity (Fig. 1, A and B). IC1IIC2-M8 had a relatively conserved leucine to phenylalanine change at aa 362 of type I adenylyl cyclase (CTCright-arrowTTC) (Fig. 2). IC1IIC2-M16 has two transitions, resulting in the change of a highly conserved asparagine to serine at aa 1025 (AACright-arrowAGC) and a silent mutation at aa 832 (TTGright-arrowTTA) of type II adenylyl cyclase (Fig. 2).


Fig. 2. Sequence comparison of adenylyl cyclases and guanylyl cyclases at the two highly conserved regions, A and B. Diagram (top) shows the relationship of regions A and B, which are connected by a highly variable region (dotted line). The consensus sequences (bottom) were obtained using the cyclase domains from closely related family of cyclases (three to ten types of cyclases), and those consensus sequences were then used to compare among the distally related families of cyclases. Sequences in bold are absolutely conserved within the family of the indicated groups of cyclases, and sequences underlined are conserved among nine out of ten adenylyl cyclases. The mutations at the IC1IIC2 mutants, M8 and M16, and IIC2 mutants, R1029A and P1015S,N1025S are shown. The letters at the tops of the sequences mean absolutely conserved glycine (a), Asn-1025 and Arg-1029 that are conserved among all adenylyl and guanylyl cyclases except those of the C1 domain of adenylyl cyclases with 12 transmembrane helices and the alpha  subunits of soluble guanylyl cyclases (b), aspartate that is conserved among all cyclases except those of the C2 domain of adenylyl cyclases with 12 transmembrane helices and is crucial for enzyme activity (c) (13), mutation at these residues results increase of Km for ATP (d) (13), and mutation at the fly learning mutant, D. rutabaga (e) (42). The protein sequences analyzed include (with GenBankTM accession number in parentheses) adenylyl cyclases from mammalian and D. melanogaster (mammal + fly, AC-C1 (M25579[GenBank]), AC-C22 (M80550[GenBank]), AC3 (M55075[GenBank]), AC4 (M80633[GenBank]), AC5 (M88649[GenBank]), AC6 (M94968[GenBank]), AC7 (U12919[GenBank]), AC8 (L26986[GenBank]), AC9 (Z50190[GenBank]), and rutabaga (M81887[GenBank])); from Dictyostelium (ACA (M87279[GenBank]) and ACG (M87278[GenBank])); from yeast (yeast AC; S. cerevisiae (M12057[GenBank]), S. pombe (M24942[GenBank]), and S. klyuyveri (X56042[GenBank])); from Trypanosoma blucei (Tb ESAG (X52118[GenBank], X52120[GenBank], and X52121[GenBank])); from Leishmania donovani (Ld RAC-A; (U17042[GenBank])); from Brevibacterium liquefaciens (BlAC (X57541[GenBank])); and from Rhizobium meliloti (RmAC (M35096[GenBank])). The protein sequences analyzed also include guanylyl cyclases as membrane-bound forms (memb GC; GC-A (J05677[GenBank]), GC-B (M26896[GenBank]), GC-C (M55636[GenBank]), GC-D (L37203[GenBank]), GC-E (L36029[GenBank]), and GC-F (L36030[GenBank]), D. melanogaster (X72800[GenBank], L35598[GenBank]), and sea urchin (M22444[GenBank])); and those as the soluble heterodimeric enzymes, alpha  (alpha 1 (M57405[GenBank]), alpha 2 (X63282[GenBank]), and D. melanogaster (U27117[GenBank])); and beta  (beta 1 (M22562[GenBank]), beta 2 (M57507[GenBank]), and D. melanogaster (U27123[GenBank])). The sequence alignment was performed using DNA* MegAlign Clustal method with structural residue weight table.
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Three IC1IIC2 mutants had 2-5-fold reduction in expression of 60 kDa proteins that were correlated well with reduced adenylyl cyclase activity (data not shown). Immunoblot showed that all three mutants had increased amounts of smaller molecular weight immunoreactive bands, suggesting that the reduced protein expression was associated with increased proteolysis (not shown). Sequence analysis of one of these mutants (M25) showed that IC1IIC2-M25 had a glutamine to lysine change at aa 848 of type II adenylyl cyclase (GAGright-arrowAAG). We have shown that the region from aa 821 to aa 855 of type II adenylyl cyclase is not required for catalysis (9). Therefore, the mutation at aa 848 is likely to cause a structural change that renders IC1IIC2 highly susceptible to proteolysis. Although WJT-1 cells that carried the plasmids for the remaining 25 IC1IIC2 mutants did take longer to be positive on maltose-MacConkey, E. coli lysates of these mutants did not have a significant reduction in enzyme activity. Thus, they were not analyzed further.

Characterization of the IIC2-N1025 and R1029A Mutants

Genetic and mutational analyses suggest that the C2 domain of mammalian adenylyl cyclase is the catalytic domain, and the C1 domain is not (10, 13). This begs the questions as to which amino acids are crucial for catalytic activity of the C2 domains, and are they absent at the C1 domains? More than 30 different adenylyl cyclases and guanylyl cyclases share the common homologous cytoplasmic domains, designated as the cyclase domains (1). For the functional enzyme, the cyclase domains can work as the homo-oligomer, such as adenylyl cyclases from yeast, Trypanosome, Dictyostelium (ACG), Leishmania, Brevibacterium, and Rhizobium (23-30). The cyclase domains can also exist as an obligatory heterodimer for enzymatic activity such as the C1 and C2 domains from type I to type IX adenylyl cyclases, Drosophila rutabaga, and Dictyostelium ACA, and the alpha  and beta  subunits of soluble guanylyl cyclases (22, 23, 31-46). The amino acid residues that are crucial for catalysis should be conserved among all the cyclase domains that function by themselves and among one of the two cyclase domains among the obligatory hetero-dimers. Two amino acid residues (Asn-1025 and Arg-1029 of type II adenylyl cyclase) fits this criterion (Fig. 2, letter b). They are conserved among all of the cyclase domains that function by themselves. They are also conserved among the catalytic C2 domain of adenylyl cyclases and the beta  subunits of guanylyl cyclases but are not conserved in the C1 domain of adenylyl cyclases or the alpha  subunit of soluble guanylyl cyclases. We thus examined the effects of mutations at Asn-1025 and Arg-1029 on enzyme activity of IC1+IIC2 complex.

To evaluate the effects of mutations of Asn-1025 of type II adenylyl cyclase, we constructed and purified IIC2 mutant proteins that had the Asn-1025 changed to Thr, Ser, Ala, Gln, Asp, and Lys (IIC2-N1025T, N1025S, N1025A, N1025Q, N1025D, and N1025K). All six mutants expressed normal amounts of enzyme compared with wild-type IIC2 based on immunoblot of E. coli BL21(DE3) lysates (Fig. 3). They also could be purified readily (Fig. 3). The biochemical properties of these IIC2-N1025 mutants are summarized in Table I and Fig. 4. All the enzyme assays were performed in the presence of a fixed amount of IC1. Mutations of Asn-1025 caused a range of reduction (10-5000-fold) in forskolin- and Gsalpha -activated adenylyl cyclase activity (Kcat) with the order of N1025T, N1025S, N1025A, N1025Q, N1025D, and N1025K from the highest enzyme activity to the lowest. IIC2-N1025T and IIC2-N1025S had similar values of Km for ATP and of half-maximal concentration (EC50) for forskolin stimulation in the presence of GTPgamma S-Gsalpha compared with wild-type IIC2. In addition, similar values were observed in EC50 for wild-type IIC2 and IIC2 mutants (N1025T and N1025S) to form complexes with IC1 and reach maximal enzyme activity that was stimulated by either forskolin alone or forskolin plus GTPgamma S-Gsalpha . Although the enzyme activity of mutants IIC2-N1025A, N1025Q, N1025D, and N1025K was too low to determine Km values of ATP and EC50 values for forskolin stimulation and for IIC2 requirement, all four mutant proteins could effectively compete with wild-type IIC2 to form a complex with IC1 and block wild-type IIC2 enzyme activity (Fig. 4D). The concentrations of IIC2 mutants required to achieve half-maximal inhibition (IC50) were close to the concentration of wild-type IIC2 protein in the assays (0.26 µM). This result indicated that the mutation of Asn-1025 caused reduction in catalysis without gross structural alteration. Both IIC2 mutants (N1025T and N1025S) had reduced sensitivity to 2'-d-3'-AMP, an inhibitor of adenylyl cyclase activity, when both forskolin and GTPgamma S-Gsalpha were present (Fig. 3C and Table I).


Fig. 3. Purified IIC2 mutant proteins. IIC2 mutant proteins were electrophoresed on 11% SDS-polyacrylamide gel electrophoresis and stained by Coomassie Blue (top). Lysates of E. coli BL21(DE3) (10 µl) that expressed wild type and mutant forms of IIC2 were prepared 3 h after IPTG induction. Immunoblot was performed with a monoclonal antibody, 12CA5 (bottom).
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Table I. Biochemical properties of wild type IIC2 and IIC2 mutant proteins with a single or double point mutation


Adenylyl cyclase activitya
Kmb Kcat/Km EC50
IC50e 2'd3'-AMP
Gsalpha Fsk Gsalpha  + Fsk IIC2c
Fskd
Gsalpha  + Fsk Fsk

mM min-1/mM µM
WT 9.3  ± 0.1 4400  ± 300 3900  ± 200 6800  ± 400 0.8  ± 0.1 534 0.06  ± 0.01 0.9  ± 0.0 0.5  ± 0.1 11.5  ± 0.6
N1025T 2.9  ± 0.1 76  ± 4 51  ± 6 725  ± 9 1.3  ± 0.2 34 0.05  ± 0.00 0.5  ± 0.1 0.9  ± 0.3 2600  ± 500
N1025S 0.7  ± 0.0 23  ± 8 26  ± 2 220  ± 40 1.0  ± 0.2 14 0.08  ± 0.02 0.6  ± 0.0 1.1  ± 0.9 1300  ± 100
N1025A 0.8  ± 0.1 6.8  ± 0.1 6  ± 1 126  ± 0.0 NDf ND ND ND ND ND
N1025Q 0.2  ± 0.0 4.6  ± 0.6 3.3  ± 0.4 29  ± 6 ND f ND ND ND ND ND
N1025D 0.5  ± 0.1 2.9  ± 0.4 2.5  ± 04 10  ± 2 ND f ND ND ND ND ND
N1025K 0.5  ± 0.01 0.8  ± 0.1 0.7  ± 0.1 1.2  ± 0.1 ND f ND ND ND ND ND
R1029A 0.4  ± 0.02 45  ± 2 43  ± 3 216  ± 9 0.9  ± 0.0 14 0.04  ± 0.01 0.6  ± 0.1 1.1  ± 0.1 2000  ± 200
N1025S,R1029A 0.7  ± 0.1 0.7  ± 0.1 2.1  ± 0.5 2.9  ± 0.3 ND f ND ND ND ND ND

a Adenylyl cyclase activity = nmol·min-1·mg-1. Adenylyl cyclase assays contained 0.1 µM purified IC1, 0.26 µM of purified wild type or mutant IIC2, 100 µM forskolin and/or 0.2 µM Gsalpha and were performed at 30 °C for 20 min. The means ± S.E. are the averages of at least two experiments.
b The Km values were determined using ATP concentrations ranging from 0.06 to 2 mM with 2-fold dilution and calculated from a least square fit of the enzyme activity to a simple rectangular hyperbola. The means ± S.E. are the averages of at least two experiments.
c Purified IC1 (0.06 µM) was mixed with different quantities of IIC2 or mutant proteins in the presence of either forskolin (100 µM; Fsk) or Gsalpha plus forskolin (0.2 µM and 100 µM, respectively; Gsalpha  + Fsk). The means ± S.E. are the averages of at least two experiments.
d Purified IC1 (0.1 µM) and IIC2 proteins (wild type or mutants, 0.26 µM) were assayed for adenylyl cyclase activity in the presence of 0.2 µM Gsalpha and different amounts of forskolin. The means ± S.E. are the averages of at least two experiments.
e Purified IC1 (0.1 µM) and IIC2 proteins (wild type or mutants, 0.26 µM) were assayed for adenylyl cyclase activity in the presence of 100 µM forskolin. The means ± S.E. are the averages of at least two experiments.
f ND, not determined.


Fig. 4. Biochemical analysis of IIC2 mutant proteins that have a mutation at aa 1025 (asparagine) or aa 1029 (arginine). A, forskolin activation of wild type and mutant IIC2 (N1025S, N1025T, and R1029A) mixed with IC1. B, complementation of IC1 activity by wild type or mutant IIC2. C, inhibition of IC1 protein complexed with wild type or mutant IIC2 proteins by the P-site inhibitor (2'-D-3'-AMP). D, inhibition of enzyme activity of the mixed wild-type IC1 + IIC2 proteins by IIC2 mutant proteins. Purified IC1 (0.1 µM) plus wild type or mutant IIC2 proteins (0.26 µM) were mixed with the indicated quantities of forskolin (A), of P-site inhibitor (C), or of mutant proteins (D). Purified IC1 (60 nM) was mixed with the indicated quantities of wild type or mutant IIC2 proteins (B). Adenylyl cyclase assays were performed in the presence of 0.2 µM GTPgamma S-Gsalpha (A), 100 µM forskolin and 0.2 µM Gsalpha (B and C), or 100 µM forskolin (D) at 30 °C for 20 min. The means ± S.E. are representative of at least two experiments.
[View Larger Version of this Image (43K GIF file)]

We then examined the effects of the mutation from arginine to alanine at aa 1029 (IIC2-R1029A). IIC2-R1029A could be expressed normally and was readily purified to homogeneity (Fig. 3). The biochemical properties of IIC2-R1029A are similar to IIC2-N1025S (Table I and Fig. 4). When mixed with IC1, IIC2-R1029A had 30-fold reduction in catalysis compared with wild-type IIC2. Similar values to that of wild-type IIC2 were observed in Km of ATP, EC50 for forskolin to maximally activate the complex of IC1 and IIC2-R1029A, and EC50 for IIC2-R1029A to form a complex with IC1 for reaching maximal activation (Table I and Fig. 4). The IC50 value of 2'-d-3'-AMP for IIC2-R1029A was about 200-fold higher than that of wild-type IIC2. A similar mutation in recombinant type I adenylyl cyclase was analyzed and this mutation also had reduced catalysis without altered ability of the mutant protein to interact with GTPgamma S-Gsalpha (13).

We also examined the effects of combined mutations at both arginine 1029 to alanine and asparagine 1025 to serine (IIC2-N1025S,R1029A). IIC2-N1025S,R1029A could be expressed and purified similar to wild-type IIC2 (Fig. 3). It had 3,000-fold reduction in activity compared with wild-type IIC2 (Table I). When mixed with wild-type IC1 and IIC2 proteins, IIC2-N1025S,R1029A was a potent inhibitor for enzyme activity, indicating that it interacted with IC1 normally. Together, this result indicates that both Asn-1025 and Arg-1029 are crucial for catalysis, and the effect of mutations on catalysis by these two residues is additive.

Effect of Asparagine Mutation at aa 1025 on Type II Adenylyl Cyclase

To evaluate the effect of Asn-1025 on full-length type II adenylyl cyclase, we constructed expression vectors with the cytomegalovirus (CMV) promoter that carried cDNA for type II adenylyl cyclase with or without the N1025K mutation. HEK 293 and mouse adrenal Y1-t4 cells were transfected with the expression vectors, and 50-200 colonies of stable transformants were pooled together and analyzed for their adenylyl cyclase activity (Fig. 5). As expected, plasma membranes from HEK 293 and mouse adrenal Y1-t4 cells that had stably integrated the vector with type II adenylyl cyclase had 2-8-fold increases in adenylyl cyclase activity over cells that had integrated the control vector. When stimulated by forskolin, plasma membranes from HEK 293 and mouse adrenal Y1-t4 cells that had integrated the vector with type II adenylyl cyclase containing the N1025K mutation not only did not have increased enzyme activity but had a 25-50% reduction in enzyme activity compared with that from the control cells. This indicates that type II adenylyl cyclase containing the N1025K mutation is catalytically inactive and can dominantly block the activation of endogenous adenylyl cyclases. The mechanism for such blocking is currently under investigation.


Fig. 5. Adenylyl cyclase activity of HEK 293 and adrenal Y1 cells that expressed either type II adenylyl cyclase (AC-II) or the mutant form of type II adenylyl cyclase, AC-II N1025K. HEK 293 and adrenal Y1 cells were transfected with pRC/CMV (Control), pRC/CMV-ACII, or pRC/CMV-ACII-N1025K, and the neomycin-resistant clones were pooled for the assay. Adenylyl cyclase assays were performed without activators (Basal); with 30 µM AlCl3, 10 mM MgCl2, and 10 mM NaF (AMF); or with 100 µM forskolin (Fsk). The means ± S.E. are representative of four experiments.
[View Larger Version of this Image (27K GIF file)]

Selection and Analysis of the Second Site Suppressor of the IC1IIC2-N1025S

Cyclic nucleotide phosphodiesterase hydrolyzes cAMP, and thus a defect in the cpdB gene enhances accumulation of intracellular cAMP. Therefore, an E. coli strain, WJT-3 (Delta cya CpdB-), was constructed to increase the sensitivity of our genetic screen. As expected, E. coli WJT-3 cells can grow on maltose-minimal medium with 10-fold less cAMP than is required by E. coli Delta cya, CpdB+. When harboring plasmids for expression of Gsalpha Q227L and IC1IIC2, E. coli WJT-3 cells can grow about twice as fast (2 days) on maltose-minimal medium than E. coli Delta cya, CpdB+ (4 days).

We employed the second site suppressor approach to search for amino acid residues that can suppress the catalytic defect of IC1IIC2-N1025S. A library of randomly mutated plasmids that encoded IC1IIC2-M16 was generated by passing through E. coli XL-1 red cells and then transformed to E. coli WJT-3 cells that expressed Gsalpha -Q227L. More than 200 colonies that carried mutated IC1IIC2-M16 and Gsalpha -Q227L were grown within 3 days, similar to those that carried wild-type IC1IIC2 and Gsalpha -Q227L. Eight independent clones that had different colony sizes were selected to further analyze. All eight IC1IIC2 revertants had similar adenylyl cyclase activity and time required for being positive on maltose-MacConkey, as did wild-type IC1IIC2. Sequence analysis revealed that all of them had a transition to revert serine back to asparagine at aa 1025. This result indicates again that asparagine at aa 1025 is crucial for the enzyme activity of adenylyl cyclase.

To avoid the true revertants in the genetic screen, we constructed pProExHAH6-IC1IIC2-N1025S(TCG) which had the TCG as codon for serine at aa 1025. In this plasmid, three mutational events are required to revert the TCG codon to the codon for asparagine (AAC/T). A library of randomly mutated pProEx-HAH6-IC1IIC2(TCG) plasmids was generated by passage through E. coli XL1 red cells and was used to select the revertants as described above. As expected, E. coli WJT-3 cells that expressed IC1IIC2-N1025S and Gsalpha Q227L were negative on maltose-minimal medium and were not positive on maltose-MacConkey plates (Fig. 6). When coexpressed with Gsalpha -Q227L in E. coli WJT-3 cells, two independent IC1IIC2 revertants allowed E. coli to grow in 4 days on maltose-minimal medium and were positive within 44 h on maltose-MacConkey plates, significantly better than the IC1IIC2 mutant but not as well as wild-type IC1IIC2 (Fig. 6A). Lysates from E. coli BL21(DE3) cells that expressed IC1IIC2 revertants had normal expression of 60 kDa IC1IIC2. Consistent with the results from the genetic analysis, lysates from E. coli BL21(DE3) cells that expressed IC1IIC2 revertants had 17-fold higher adenylyl cyclase activity than IC1IIC2 mutants but 10-fold lower than wild-type IC1IIC2 (Fig. 6A). DNA sequencing analysis revealed that both revertants retained the TCG codon at aa 1025 but had acquired a proline to serine at aa 1015 (CCAright-arrowTCA) of type II adenylyl cyclase.


Fig. 6. Characterization of the second site suppressor of IC1IIC2-N1025S. A, phenotypic characterization, adenylyl cyclase activity, and the expression of second site suppressor of IC1IIC2-N1025S. Phenotypic characterization was performed using E. coli WJT-3 cells that expressed Gsalpha Q227L. MacConkey (hrs) showed the required time (hours) to be positive on maltose-MacConkey medium of WJT-3 cells that expressed wild-type IC1IIC2 (WT), IC1IIC2-N1025S mutant (N1025S), and second site suppressor of IC1IIC2 (P1015S,N1025S). Lysates from E. coli BL21(DE3) that expressed wild-type IC1IIC2 (WT), IC1IIC2-N1025S (N1025S), and IC1IIC2-P1015S,N1025S (P1015S, N1025S) were used for adenylyl cyclase assay in the presence of 100 µM forskolin and for immunoblots using a monoclonal antibody, 12CA5. B, purified IIC2-P1015,N1025S mutant proteins. IIC2 mutant proteins were analyzed in the same manner as described in Fig. 3.
[View Larger Version of this Image (32K GIF file)]

Characterization of IIC2-P1015,N1025S) Mutations

To evaluate the effect of proline mutation at aa 1015 of type II enzyme, we constructed six IIC2 mutants, all having serine at aa 1025 and a mutation from Pro 1015 to Ser, Ala, Asp, Glu, Asn, or Gln (IIC2-P1015S,N1025S; P1015A,N1025S; P1015D,1025S; P1015E,N1025S; P1015N,N1025S; and P1015Q,N1025S). Based on immunoblot analysis of lysates from E. coli BL21(DE3) cells, IIC2-P1015S,N1025S, IIC2-P1015A,N1025S, and IIC2-P1015E,N1025S were expressed normally, whereas IIC2-P1015D,N1025S, IIC2-P1015N, N1025S and IIC2-P1015Q,N1025S had 50-90% reductions in expression as the soluble proteins. To characterize these proteins, all were purified to near homogeneity (Fig. 6B). The biochemical properties of these mutants are summarized in Table II. All six mutants had 3-10-fold higher Gsalpha - or forskolinstimulated enzyme activities compared with IIC2-N1025S, while their enzyme activities were still significantly lower than wild-type IIC2. The IC50 values for 2'-d-3'-AMP to inhibit the enzyme activity were slightly reduced compared with IIC2-N1025S (particularly IIC2-P1015D,N1025S and IIC2-P1015N,N1025S) but were still significantly higher than that of wild-type IIC2. Their Km values for ATP and EC50 values for IIC2 mutants to achieve the maximal forskolin-stimulated enzyme activity were similar to that of wild-type IIC2. Interestingly, the EC50 values for forskolin to maximally activate the complex of IC1 and IIC2 mutants were altered in some mutants IIC2-P1015S,N1025S; IIC2-P1015A,N1025S; IIC2-P1015N, N1025S; and IIC2-P1015Q,N1025S).

Table II. Biochemical properties of wild type IIC2 and IIC2 mutant proteins with the double point mutations


Adenylyl cyclase activitya
Km EC50
IC50 2'-d3'-AMP
Gsalpha Fsk Gsalpha  + Fsk IIC2 Fsk

mM µM
WT 6  ± 2 3400  ± 300 4400  ± 300 5200  ± 700 0.8  ± 0.2 0.7  ± 0.2 0.6  ± 0.1 11  ± 2
N1025S 0.8  ± 0.1 25  ± 3 30  ± 6 310  ± 30 0.9  ± 0.3 0.6  ± 0.1 1.1  ± 0.2 1500  ± 200
P1015S,N1025S 4  ± 1 141  ± 6 80  ± 3 800  ± 100 0.8  ± 0.2 1.1  ± 0.5 6  ± 1 800  ± 200
P1015A,N1025S 0.4  ± 0.0 136  ± 4 100  ± 10 620  ± 80 0.5  ± 0.01 1.2  ± 0.3 8  ± 1 700  ± 200
P1015D,N1025S 1.5  ± 0.5 240  ± 30 80  ± 7 800  ± 90 1.1  ± 0.1 0.7  ± 0.1 1.7  ± 0.8 400  ± 100
P1015E,N1025S 4  ± 1 260  ± 40 160  ± 20 1400  ± 100 0.6  ± 0.3 1.3  ± 0.2 1.9  ± 0.2 600  ± 100
P1015N,N1025S 0.5  ± 0.0 250  ± 30 64  ± 3 570  ± 40 0.7  ± 0.2 0.7  ± 0.2 7  ± 2 400  ± 200
P1015Q,N1025S 0.4  ± 0.1 100  ± 10 48  ± 9 120  ± 0.2 0.5  ± 0.1 0.5  ± 0.1 9  ± 4 1600  ± 300

a Adenylyl cyclase activity = nmol·min-1·mg-1. Adenylyl cyclase assays were performed the same as in Table I except 0.07 µM GTPgamma S-Gsalpha was used. The means ± S.E. are the averages of at least two experiments. The means ± S.E. are the averages of at least two experiments.


DISCUSSION

Mammalian adenylyl cyclase has a complex molecular design, presumably to integrate and respond to various extracellular and intracellular stimuli. Genetic and biochemical analyses of type I and soluble adenylyl cyclases indicate that both the C1 and C2 domains are essential for the high enzyme activity and only the C2 domain is catalytic.5 In this paper, we have shown that the conserved asparagine and arginine residues are crucial for catalysis, and they might be two of the key residues that prevent the C1 domain from being catalytically active.6 The residue in the C1 domain that corresponds to Asn-1025 of the C2 domain is commonly a threonine. Interestingly, this replacement of Asn-1025 in the C2 domain results in the retention of the highest activity of the replacements examined (Fig. 2). The corresponding residue of the conserved C2 arginine in the C1 domain is either histidine or lysine (Fig. 2). We have shown that the arginine to lysine substitution in type I adenylyl cyclase did not alter the enzyme activity, highlighting the role of the substitution at the corresponding site of the C2 asparagine in inactivating the enzyme activity of the C1 domain (13). The role of the conserved asparagine and arginine in catalysis remains to be determined. One possible role is to coordinate and stabilize the PO5 transition state similar to the way in which the Gln and Arg are involved in the hydrolysis of GTP in the G protein alpha  subunit (47, 48). Structural determination of the cyclase domain with its substrate should help to elucidate the roles of these two residues in catalysis.

The changes in free energy (Delta Delta G) have been used to determine whether the two mutations have an additive effect on catalysis (49). If the free energy change due to the first mutation is denoted by Delta Delta Gx and that due to the second mutation is Delta Delta Gy, the change in free energy observed in the double mutant, Delta Delta Gx,y, can be expressed as Delta Delta Gx,y = Delta Delta Gx + Delta Delta Gy + Delta Gi, where Delta Gi represents an interaction energy between sites. When Delta Gi is near 0, the two sites behave independently, and the double mutant displays "simple additivity." The change in transition-state stabilization energy (Delta Delta GT) is equal to -RT*ln(Kcat/Km)mutant/(Kcat/Km)wild-type. Thus, both Delta Delta GT for both mutant IIC2-N1025S and IIC2-R1029A are 2.2 kcal/mol. We could not determine Km of double mutant IIC2-N1025S,R1029A due to its extremely low enzyme activity. However, it might be reasonable to assume that the Km of the double mutants is not altered significantly because we observed no change in Km for either IIC2-N1025S or IIC2-R1029A mutants. If this is true, Delta Delta GT for double mutant IIC2-N1025S,R1029A would be 4.8 kcal/mol, close to the sum of Delta Delta GT (4.4 kcal/mol) for mutant IIC2-N1025S and IIC2-R1029A. Thus, it appears that the conserved Asn-1025 and Arg-1029 act independently in the catalytic activity of adenylyl cyclase.

How does a proline to serine mutation at aa 1015 increase 10-15-fold enzyme activity of IIC2-N1025S? Proline can exist as either cis- or trans-peptide bonds. However, a charged lysine residue, not Phe, Tyr, or Leu, immediately proceeds proline at aa 1015, making it less likely to be cis-Pro (50) (Fig. 2). The entropy effect of proline contributes positively in protein stability; thus, mutation at a proline residue might introduce an increased flexibility (50, 51). We hypothesize that the open-close conformational switch is essential for the catalytic activity of adenylyl cyclase and that the increased flexibility of the protein by the proline to serine mutation might facilitate such a switch (1, 13). Further experiments are required to test this hypothesis. Proline is involved in numerous gain of function mutants in other proteins. Proline to leucine has been shown to render G protein-coupled alpha -factor receptor to be constitutively active and second site suppressor mutations from other amino acids to proline enhance the functioning of several mutated proteins, i.e. triosephosphate isomerase, F1F0 ATP synthase, and lactose permease (52-55).

Three genetic model systems have been set up to study catalysis and regulation of adenylyl cyclases that have 12-transmembrane helices (10, 56-58). In both E. coli and Saccharomyces cerevisiae, the paradigm of cAMP-dependent cell growth under certain growth conditions are utilized, while in D. discoideum, that of cAMP-mediated cell aggregation is applied. Conditions that allow selection of mutants that gain or lose cAMP accumulation have been established both in E. coli and in D. discoideum. Mutations that inactivate or increase the basal enzyme activity have been found in ACA and in soluble adenylyl cyclase (56, 57, see above). These systems should permit the genetic analysis of interactions between adenylyl cyclases and its regulators. In addition, these systems should help us to identify the pharmacological reagents that could modulate the enzyme activity of adenylyl cyclase with isoform specificity.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM53459, the Cancer Research Foundation, and the Brain Research Foundation.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 Pharmacological and Physiological Sciences, University of Chicago, 947 E. 58th St. Chicago, IL 60637.
1   Three classes of adenylyl cyclases have been cloned based on the conservation of their catalytic domains: class I, adenylyl cyclases from Enterobacteria including E. coli; class II, toxin-adenylyl cyclases including calmodulin activated toxins from Bordetella pertussis and Bacillus anthracis; and class III, adenylyl cyclases homologous from bacteria to human (1). Class III adenylyl cyclases have four distinct molecular designs. First are the integral membrane proteins that contain two hydrophobic stretches (~20 kDa each), each consisting of six putative transmembrane helices and two homologous cytoplasmic domains (C1 and C2 domains, 30-40 kDa each). This design includes nine mammalian adenylyl cyclases (type I to type IX), fruit fly rutabaga (Dm rutabaga), and slime mold (Dd ACA) (22, 23, 31-42). Second are the integral membrane proteins that have one transmembrane helix. This class is found in the parasites, Trypanosoma brucei (Tb ESAG, expression site-associated gene), and Leishmania donovani (Ld-RAC, receptor-adenylyl cyclase), and in the slime mold (Dictyostelium discoideum, Dd ACG) (23-25). Third are the peripheral membrane proteins from yeast (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Saccharomyces klyuyveri) (26-28). Fourth are the soluble enzymes from the bacteria, Brevibacterium liquefaciens (Bl AC) and Rhizobium meliloti (Rm AC) (29, 30).
2   The abbreviations used are: G protein, guanine nucleotide binding regulatory protein; Gsalpha , the alpha  subunit of the G protein that stimulates adenylyl cyclase; Fsk, forskolin; IC1 protein, C1 domain of type I adenylyl cyclase; IIC2 protein, C2 domain of type II adenylyl cyclase; GTPgamma S, guanosine-5'-[gamma -thio]triphosphate; aa, amino acid(s); 2'-D-3'-AMP, 2'deoxyadenosine 3'-monophosphate; kbp, kilobase pair; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; LB, Luria broth; CMV, cytomegalovirus; IBMX, isobutylmethylxanthine.
3   crp/crp and malT/malT mean that the strain is diploid for two genes due to presence of the F141 plasmid (15). Mutations in E. coli to resistance to lambda  phage infection can arise from chromosomal mutations, most frequently in one of three genes, malT, lamB, or crp. To reduce the frequency of two of such undesired mutations, strain WJT-1 was made diploid for the malT and the crp genes. Since strains diploid for both matT and crp genes had to have mutations on both copies of either of these genes to survive infection by lambda vir, the frequency of resistance was much lower in diploids than in strains with only one copy of the gene.
4   IIC2 here is the same as IIC2-Delta 3 described previously (9).
5   We have previously shown that IIC2 protein had no detectable enzyme activity in the presence of IBMX, phosphodiesterase inhibitor (9). However, using a high quantity of IIC2 (100 µg) in the absence of IBMX, low but reproducible Gsalpha - and forskolin-stimulated enzyme activity was detected (9.1 nmol·min-1·mg-1; 1000-fold less than the IC1 + IIC2 complex) when only IIC2 protein was present. IBMX may have inhibited the low activity of IIC2 protein.
6   We have constructed IC1 mutants that have single or double mutations from Thr-426 and Val-430 (corresponding residues of C2 Asn-1025 and Arg-1029) to Asn and Arg, respectively. When mixed with wild-type IIC2 protein, the forskolin-stimulated activities of E. coli lysates containing IC1 mutants were reduced 10-50-fold compared with that of wild-type IC1. When mixed with mutant forms of IIC2 proteins (IIC2-N1025S and IIC2-R1029A), the enzyme activity of E. coli lysates containing IC1 mutants were similar to or less than that of wild-type IC1. Thus, IC1 protein cannot be converted to be catalytically active by changing either Thr-426 to Asn or Val-430 to Arg or by changing both residues simultaneously.

Acknowledgements

We thank David Hahn for superb technical help; W. Epstein for helpful suggestions in constructing E. coli WJT-1 and WJT-3 cells and the genetic screening; and W. Epstein, H. Fozzard, and C. Skoczylas for critical reading of the manuscript.


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