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J. Biol. Chem., Vol. 275, Issue 49, 38626-38632, December 8, 2000

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Isolation and Characterization of Constitutively Active Mutants of Mammalian Adenylyl Cyclase^*

Mark E. Hatley, Benjamin K. Benton^§¶, Jun Xu^§, John P. Manfredi^§**, Alfred G. Gilman, and Roger K. Sunahara

From the  Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041 and ^§ Cadus Pharmaceutical Corporation, New York, New York 10153

Received for publication, August 7, 2000, and in revised form, September 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G_s. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C₁ and C₂) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the 4-5 loop of the C₂ domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C₁ domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C₂ domain of adenylyl cyclase for the wild type C₁ domain was exploited to isolate a complex containing VC₁, IIC₂, and G_s-guanosine 5'-3-O-(thio)triphosphate (GTPS) in the absence of forskolin and a complex of VC₁, IIC₂, forskolin, and P-site inhibitor in the absence of G_s-GTPS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G_s.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian adenylyl cyclases are membrane-bound enzymes that catalyze the synthesis of the intracellular second messenger cyclic AMP from ATP. Nine isoforms of the enzyme have been detected, and they display characteristic regulatory properties and patterns of cellular distribution (1, 2). Cellular rates of cyclic AMP synthesis are controlled by a variety of extracellular ligands that interact with heptahelical receptors in the plasma membrane. Relevant receptors can either stimulate cyclic AMP synthesis, usually via the intermediacy of a G protein¹ (G_s) that activates adenylyl cyclase, or inhibit cyclic AMP synthesis, often by interaction with an inhibitory G protein, G_i. Mammalian adenylyl cyclases can also be activated by the diterpene forskolin (3) and inhibited by certain adenosine analogs and adenine nucleotides called P-site inhibitors (4). Certain adenylyl cyclases are also regulated by Ca²⁺, Ca²⁺-calmodulin, and phosphorylation (5).

Mammalian adenylyl cyclases are integral membrane proteins that appear to contain two sets of six membrane-spanning helices that are separated by a large (~40 kDa) cytoplasmic loop and followed by a similarly sized carboxyl-terminal cytosolic domain (6). The cytosolic domains, termed C₁ and C₂, have been extensively studied; they are responsible for catalytic activity and most of the regulatory properties of the enzymes (7). The first 200-250 amino acids of each cytosolic domain, designated C_1a and C_2a, are the most highly conserved regions among adenylyl cyclases. Strikingly, the C_1a and C_2a domains are approximately 50% similar and 25% identical to each other within a single isoform of adenylyl cyclase, and they are 20-25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl cyclases.

The C_1a and C_2a domains of adenylyl cyclases can be expressed separately and purified as recombinant proteins. When mixed together, they display the characteristics of membrane-bound adenylyl cyclase with respect to regulation by G_s, G_i, forskolin, and P-site inhibitors (8-10). The crystal structures of the soluble catalytic core of adenylyl cyclase bound to G_s and forskolin (11) and of this complex bound with the competitive substrates -L-2',3'-ddATP and ATPS (12) have provided detailed insights into mechanisms of catalysis of cyclic AMP synthesis and regulation of the activity of the enzyme.

Less information is available about the biochemical and structural properties of low activity states of adenylyl cyclase. Crystallization of the C₁ and C₂ domains in the absence of G_s and/or forskolin would permit structural comparison of high and low activity states of the enzyme. We have approached this goal with a combination of genetic and biochemical techniques. Full-length mammalian adenylyl cyclase was introduced into a mutant strain of Saccharomyces cerevisiae that does not express the endogenous enzyme. Co-expression of the mammalian protein together with G_s relieves dependence of cyclic AMP for growth at non-permissive temperatures. We have utilized a genetic screen to isolate constitutively active mutants of adenylyl cyclase within this strain. We describe the isolation of several partially active mutants, as well as the biochemical consequences of these mutations in the context of the soluble, recombinant adenylyl cyclase system.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genetic Screen-- To achieve high level constitutive expression of type II adenylyl cyclase (ACII) in yeast, a 0.94-kb fragment containing the S. cerevisiae PGK1 promoter and a 3.3-kb fragment encoding rat ACII (PCR-amplified from a template plasmid provided by Randall Reed, The John Hopkins University) were subcloned into pRS425 (13). The resulting expression plasmid, Cp1512 (genotype LEU2 PGK1p-ACII 2µ-ori REP3 bla), was used as the template for error-prone PCR amplification of two separate fragments as follows: nucleotides 5514-6384, which encode the C_1a domain, and nucleotides 7033-8264, which encode the C_2a domain (primer sequence available upon request). The mutagenized fragments encoding the C_1a domain were then used to transform yeast strain TC41F2-1 (genotype MATa cyr1::ura3 cam1 cam2 cam3 leu2-3 leu2-112 his3-532 his4 ura3; provided by Warren Heideman, University of Wisconsin) along with a "gapped" vector prepared as follows. Cp1512 was digested with ApaI (which cuts at nucleotide 5682 of ACII) and DraIII (which cuts at nucleotide 6254), and the 10.1-kb fragment, which lacks the C_1a coding sequence, was isolated. Similarly, TC41F2-1 was co-transformed with mutagenized fragments encompassing the C_2a domain and a gapped vector prepared as follows. Cp1512 was digested with PvuII (which cuts at nucleotide 7111) and BamHI (which cuts at nucleotide 8168), and the 9.7-kb fragment, which lacks the C_2a coding region, was isolated. For both transformations, cells were plated on SC-Leu (14) without supplemental cyclic AMP and incubated at room temperature. Colonies from these plates were replica-plated to SC-Leu plates and incubated at 34 °C. Colonies that grew at 34 °C were expected to harbor stably replicating episomes that result from in situ recombination between the gapped vector and a PCR-amplified fragment and that, as a result of this recombination, express an adenylyl cyclase that has constitutive (i.e. G_s-independent) activity.

In all cases, the plasmid dependence of growth under selective conditions (on SC-Leu at 34 °C) was further tested by allowing spontaneous loss of plasmid under non-selective conditions (on YEPD with supplemental cyclic AMP at 30 °C), followed by re-testing under selective conditions. Plasmids were rescued from those colonies whose growth under selective conditions was dependent on plasmid; the plasmids were then reintroduced into naive host strain TC41F2-1, and the transformants were tested for growth on SC-Leu at 34 and at 37 °C. All plasmids reported here confer plasmid-dependent growth at 34 °C when introduced into TC41F2-1.

Mutagenesis of Adenylyl Cyclase Domains-- The mutants that appeared to confer the highest level of constitutive activity in yeast were analyzed using the soluble mammalian adenylyl cyclase system. The vectors pQE60-H6-VC₁(364-591)FLAG (10), pQE60-IIC₂-H6 (8), and pQE60-ArgC-IIC₂ (11) served as templates for site-directed mutagenesis (QuikChange^TM, Stratagene). The mutations in the C_1a domain were made in the conserved residues in the C_1a domain from canine type V adenylyl cyclase corresponding to the mutations in the rat type IIC₁ domain obtained from the yeast screen. The mutations in the C₂ domain from the yeast screen were made in the C₂ domain of rat type II adenylyl cyclase using the vector pQE60-IIC₂-H6. The vector pQE60-ArgC-IIC₂ was used to generate a non-tagged IIC₂-K1014N. Sequences of synthetic mutagenic sense and antisense primers are available upon request.

Expression and Purification of Proteins-- Wild type and mutant H6-VC₁(591)FLAG and IIC₂-H6 were expressed in Escherichia coli and purified as described previously (8, 10). Non-tagged ArgC-IIC₂ wild type and mutant K1014N were expressed and purified as described (11). Bovine G_s (short form) was purified and activated by GTPS as described (15).

Adenylyl Cyclase Assays-- Adenylyl cyclase activity was measured as described by Smigel (16). All assays were performed in a volume of 100 µl for 10 min at 30 °C. The final concentration of MgCl₂ was 10 mM. The concentration of ATP was 1 mM unless otherwise stated. Kinetic constants were determined by varying MgATP from 20 µM to 5 mM with a fixed excess of Mg²⁺. Unless otherwise stated, assays contained a limiting concentration of VC₁ and an excess of IIC₂ for both wild type and mutant assays. All specific activities reported are with respect to the concentration of the limiting domain. Each experiment was repeated two to three times.

Gel Filtration-- Proteins (G_s and adenylyl cyclase domains) were separated by fast protein liquid chromatography with tandemly arranged Superdex 75 and 200 (HR10/30) columns. Proteins were eluted in a buffer containing 20 mM NaHepes, pH 8.0, 2 mM MgCl₂, 1 mM EDTA, 2 mM dithiothreitol, and 50 mM NaCl. Flow rates were 0.2 ml/min, and 300-µl fractions were collected. An aliquot of each fraction (15 µl) was analyzed by SDS-PAGE on a 15% acrylamide gel and stained with Coomassie Blue.

Talon Column Binding Studies-- H6-VC₁(591)FLAG (15 µM) and ArgC-IIC₂ wild type or K1014N (75 µM) were incubated on ice for 15 min in 150 µl of buffer A (20 mM NaHepes, pH 8.0, 2 mM MgCl₂, 1 mM EDTA, and 50 mM NaCl) in the presence or absence of 100 µM forskolin, 100 µM 2'd,3'-AMP and 100 µM PP_i. The mixture was applied to a 25-µl metal chelate column (Talon^TM, CLONTECH) equilibrated with buffer A. The column was washed twice with 100 µl of buffer B (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM MgCl₂, 100 µM forskolin, 100 µM 2'd,3'-AMP, and 100 µM PP_i). The column was then eluted with 100 µl of buffer B containing 100 mM imidazole. An aliquot (15 µl) of the eluate was resolved by SDS-PAGE on a 15% acrylamide gel and stained with Coomassie Blue.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genetic Screen-- We isolated 24 plasmids that conferred cyclic AMP-independent growth at 34 °C when introduced into strain TC41F2-1; this phenotype is presumed to reflect the expression of G_s-independent adenylyl cyclase activity. The mutagenized regions of these plasmids were sequenced, and their mutations are listed in Table I. Cp4465, which encodes ACII I259V/Y402C, was used to generate the two plasmids ACII I259V and ACII Y402C; similarly, Cp4522 was used to generate two plasmids encoding ACII M253V and ACII C305R. In this way it was found that ACII C305R and ACII Y402C are constitutively active mutants. In several cases mutations that were observed in genetically selected plasmids were engineered by site-directed mutagenesis into wild type ACII to test their effects independently of coincident mutations. Table II lists the mutations and the growth characteristics of all constitutively active ACII single mutants.

View this table: [in this window] [in a new window]   Table I Growth of yeast harboring plasmids encoding mutant adenylyl cyclases that confer independence of Gs and cAMP

It is possible that plasmid-dependent variations in copy number could account for the phenotypes conferred by the ACII mutants. We addressed that possibility by subcloning six of the genetically selected missense mutants into centromeric vectors whose copy number is stably maintained at 1-3 per cell (17). In each case the phenotype conferred by the centromeric plasmid was the same as that imparted by its high copy equivalent (data not shown). We thus suggest that all 14 mutants listed in Table II have elevated specific activities.

Adenylyl Cyclase Assay-- The mutations identified in the yeast screen were generated in the soluble mammalian adenylyl cyclase system to facilitate biochemical characterization. The mutations were made in the C_1a domain of canine type V adenylyl cyclase at the residues that correspond to those mutated in the C_1a domain of rat type II adenylyl cyclase. The mutations F298Y, C305R, N315S, K334R, and V377I from rat ACIIC_1a were made to the following residues in canine ACVC_1a: F400Y, C407R, N417S, K436R, and V479I. A similar screen in yeast using rat type IV adenylyl cyclase² generated mutations Y265H, V388I, G968S, and K998N; these mutations were made in VC₁ and IIC₂, respectively, as Y383H, V506I, G970S, and K1014N. No conversion was necessary for the remainder of the mutations since they were already in rat ACIIC₂. All mutant proteins, with the exception of C407R, K436R, and V506I, were expressed and purified to degrees comparable to levels described previously for the wild type proteins. The mutants were assayed for activity at limiting concentrations of the VC₁ domain and increasing concentrations of the IIC₂ domain in the absence of activators or in the presence of forskolin. The apparent affinity between the two soluble domains of adenylyl cyclase was expressed as the EC₅₀, and the apparent V_max was extrapolated from the asymptote of the curve. The apparent EC₅₀ and V_max values for the mutant proteins are summarized in Table III.

Surprisingly, most mutant proteins displayed only modest differences in the apparent affinity or V_max when compared with the corresponding wild type domain. Mutants C407R and K436R yielded very little protein with no measurable adenylyl cyclase activity. The V506I mutation in VC₁ caused an increase in basal enzymatic activity and a 3-fold decrease in EC₅₀ in the presence of forskolin. However, the low level of expression of this protein precluded detailed characterization. The mutations I1010M, K1014N, and P1015Q, all located in the 4-5 loop of IIC₂, caused an increase in V_max in both the basal and the forskolin-stimulated conditions.

The K1014N mutation appeared to cause the largest degree of constitutive activation; IIC₂-K1014N was thus purified to homogeneity by anion-exchange chromatography and characterized biochemically. The apparent affinity of IIC₂-K1014N for wild type VC₁ was determined in various activation states. The activities shown in Fig. 1 were determined with a limiting concentration of VC₁ and either variable concentrations (Fig. 1, A-C) or a saturating concentration (Fig. 1D) of IIC₂. The apparent affinity for VC₁ and the maximal catalytic activity were both 3-fold greater for the K1014N mutant compared with wild type IIC₂ in the absence of activators (Fig. 1A). Similarly, the apparent affinity of IIC₂-K1014N for VC₁ was 3-fold higher in the presence of forskolin (Fig. 1B) and 10-fold higher in the presence of G_s-GTPS (Fig. 1C) compared with its wild type counterpart. No significant changes were observed in V_max under these conditions. There was no difference in either the EC₅₀ or the V_max in the presence of both forskolin and Gs-GTPS, demonstrating that the mutation does not create a hyperactive enzyme. Similar results were observed using limiting concentrations of IIC₂ and varying concentrations of VC₁ (data not shown). The apparent affinity of G_s-GTPS for adenylyl cyclase is shown in Fig. 1D. The EC₅₀ for G_s-GTPS was 0.05 µM for K1014N compared with 0.4 µM for the wild type IIC₂. These values were 6 and 25 nM, respectively, in the presence of forskolin.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   The apparent affinity of IIC2-K1014N for VC1 and Gs-GTPs. VC1 was mixed with wild type IIC2 or IIC2-K1014N and assayed for adenylyl cyclase activity in the presence of the indicated activators. A, adenylyl cyclase activity was assayed with VC1 (30 nM) and increasing concentrations of IIC2 () or IIC2-K1014N () in the absence of activators. B, adenylyl cyclase was assayed with VC1 (2 nM) and increasing concentrations of IIC2 () or IIC2-K1014N () in the presence of 100 µM forskolin. C, adenylyl cyclase was assayed with VC1 (2 nM) and increasing concentrations of IIC2 () or IIC2-K1014N () in the presence of 400 nM Gs-GTPS with (filled symbols) and without (open symbols) 100 µM forskolin. D, adenylyl cyclase was assayed with 2 nM VC1 and 2 µM IIC2 () or IIC2-K1014N () with increasing concentrations of Gs-GTPS in the presence (filled symbols) and absence (open symbols) of 100 µM forskolin.

Determination of Kinetic Constants-- The kinetic constants for substrate were also determined under various conditions (Fig. 2 and Table IV). Reconstituted adenylyl cyclase containing IIC₂-K1014N exhibited a K_m value for ATP that was 6-fold less than that observed with the wild type protein under basal conditions. As noted above, V_max is increased under this condition. Assays performed with activated G_s revealed a 6-fold decrease in the K_m value for ATP when the mutation was present. No changes were obvious in the presence of forskolin, with or without G_s-GTPS.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Substrate kinetics with IIC2-K1014N. The kinetic constants for ATP were determined from the Michaelis-Menten plots generated by measuring adenylyl cyclase activity with a limiting concentration of VC1 and an excess of IIC2 while varying the ATP concentration from 20 µM to 5 mM. A, 30 nM VC1 and 10 µM IIC2 () or IIC2-K1014N () were assayed in the absence of activators. B-D, 2 nM VC1 and 3 µM IIC2 () or IIC2-K1014N () were assayed in the presence of 100 µM forskolin (B), 400 nM Gs-GTPS (C), or 100 µM forskolin and 400 nM Gs-GTPS (D).

Gel Filtration of the VC₁·IIC₂-K1014N·G_s Complex-- Purified H6-VC₁ and wild type or IIC₂-K1014N were combined with G_s-GTPS and gel-filtered using tandem Superdex 75 and 200 columns. Fractions were analyzed by SDS-PAGE (Fig. 3). In the absence of forskolin, there is no evidence for formation of a complex between VC₁, wild type IIC₂, and G_s (Fig. 3B). The largest apparent species (78 kDa) is likely a heterodimer consisting of IIC₂ and G_s; similar results have been reported previously (10). In contrast, protein in the mixture of VC₁, IIC₂-K1014N, and G_s eluted as two major peaks with the largest species representing a 100-kDa complex. Analysis by SDS-PAGE indicates a complex of VC₁, IIC₂-K1014N, and G_s with an apparent stoichiometry of 1:1:1 (Fig. 3A). Similar results were observed with wild type IIC₂ only when forskolin was present (10).

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Gel filtration of VC1, IIC2-K1014N, and Gs in the absence of forskolin. A mixture of VC1 (100 µM), IIC2-K1014N (50 µM) (A) or wild type IIC2 (50 µM) (B) and Gs-GTPS (50 µM) was applied to a Superdex 75 (HR10/30) gel filtration column in tandem with a Superdex 200 (HR10/30). Fractions 18-40 (15 µl of 300-µl fractions) were resolved by SDS-PAGE on a 15% polyacrylamide gel and stained with Coomassie Blue. The positions of elution of two gel filtration standards are indicated.

Isolation of a Complex of VC₁·IIC₂·Forskolin·2'd,3'-AMP· PP_i-- Purified H6-VC₁ and non-tagged wild type or IIC₂-K1014N were combined and applied to a metal chelate chromatographic column (Talon^TM, CLONTECH). Samples were eluted with imidazole and analyzed by SDS-PAGE (Fig. 4). No complex was detected by SDS-PAGE when H6-VC₁ and IIC₂ or IIC₂-K1014N were incubated with forskolin. Some IIC₂-K1014N was retained on the column in the presence of the P-site inhibitor 2'd,3'-AMP·PP_i; wild type IIC₂ was not. When both forskolin and 2'd,3'-AMP·PP_i were present, a complex of H6-VC₁ and either IIC₂ or K1014N was isolated. These complexes had apparent stoichiometries of 1:1 (determined by scanning densitometry). Similar results were obtained when Mn²⁺ replaced Mg²⁺ (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Complexes of VC2 and IIC2. IIC2 or IIC2-K1014N (75 µM) were incubated for 15 min on ice in the presence or absence of H6-VC1 (15 µM) with 100 µM forskolin and/or 100 µM 2'd,3'-AMP·PPi as indicated. The mixtures were applied to 25-µl Talon columns, which were washed and then eluted with imidazole. Aliquots of the eluates were resolved by SDS-PAGE on a 15% polyacrylamide gel and visualized by staining with Coomassie Blue.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The crystal structure of the cytosolic portions of adenylyl cyclase demonstrates that the C₁ and C₂ domains are arranged as a pseudo-2-fold symmetrical dimer (see inset in Fig. 5) (11). The contributions of several residues within each domain to substrate and Mg²⁺ binding, as well as catalysis, have been investigated in previous studies. Adenylyl cyclases, and presumably guanylyl cyclases, contain palm domains. These domains were defined previously in DNA polymerases, enzymes that catalyze very similar reactions (18). Crystal structures of the cytosolic domains of adenylyl cyclase have revealed significant conformational changes upon substrate binding (11). The 1 and 2 helices and the 3 and 4 helix/strand of C₁ and the 7-8 loop of C₂ collapse around the nucleotide and align the nucleotide and two metal ions for catalysis. Located within the active site is the 2-3 loop of C₁, containing aspartate residues 396 and 440 that coordinate two Mg²⁺ ions. These divalent cations participate in deprotonation of the 3' hydroxyl of the ribose moiety (a critical step in the synthesis of cyclic AMP) and stabilize the pentavalent transition state. The conserved aspartate residues are also found among DNA polymerases and guanylyl cyclases (11, 12, 19-22).

View larger version (60K): [in this window] [in a new window]   Fig. 5.   Catalytic core of adenylyl cyclase. Inset, the heterodimeric complex formed by VC1 (khaki) and IIC2 (mauve) viewed along its pseudo-2-fold axis toward the hypothesized cytoplasmic face. Forskolin (Fsk) and ATP bind between VC1 and IIC2 and are shown as stick models. The 4-5 loop of IIC2 containing I1010M, K1014N, and P1015Q is highlighted in green. Bottom right, interactions of 4-5 loop of IIC2. The side chains of the 4-5 loop (green) of IIC2 (mauve) and their interactions with VC1 (khaki) are shown. Dashed gray lines show side chain-side chain and side chain-main chain hydrogen bonds. Carbon atoms are gray, nitrogen atoms blue, and oxygen atoms red.

The 4-5 loop of C₂ supports the 2-3 loop of C₁ (Fig. 5). Perturbations in either the contact regions or the loop-fold could have dramatic effects on C₁:C₂ structure and hence activity. Several residues in both the 2-3 loop and the 2 helix of C₁ and the 4-5 loop of C₂ have been investigated by site-directed mutagenesis and have various effects on adenylyl cyclase activity. Structural evidence strongly suggests that Asp-424 in the 2 helix and Arg-434 in the 2 sheet of C₁ engage in extensive hydrogen bonding with the 4-5 loop of C₂ (Fig. 5). Asp-424 forms a salt bridge with Arg-434 and a hydrogen bond with the backbone nitrogens of Ala-1012 and Gln-1013. The side chain of Arg-434 forms a hydrogen bond with the side chain of Gln-1016; the backbone carbonyl of Arg-434 forms a hydrogen bond with the side chain of Gln-1013. Mutations of either Asp-424 or Arg-434 have previously been shown to have detrimental effects on adenylyl cyclase activity. Mutations of these residues have broad effects on cyclase activity without affecting the affinity of the enzyme for G_s, as follows: R434A increases the IC₅₀ value for P-site inhibitors (23); R434S increases the K_m value for MgATP, the K_i value for ATPS, and the EC₅₀ value for Mg²⁺ (24); D424A and D424N decrease forskolin- and G_s-stimulated enzymatic activity (23, 25).

Mutations of residues in the 4-5 loop of C₂ have also been shown to affect adenylyl cyclase activity. The mutations Y1017A and D1018A (Y999A and D1000A in type I adenylyl cyclase) obliterate activity without eliminating G_s binding (23). Asp-1018 coordinates substrate binding through the purine ring and is responsible for dictating nucleotide specificity (11, 26). Alteration in neighboring side chains that perturb the conformation of the amino acid chain backbone would likely perturb activity. Residues Ile-1010, Lys-1014, and Pro-1015, investigated in this study, are all located in the 4-5 loop of C₂ and therefore are intimately involved in the arrangement of the 2-3 loop of C₁. The mutations I1010M, K1014N, and P1015Q are all within bonding distance of the 2-3 loop of C₁. The P1015Q mutation, which displays slightly elevated affinity between C₁ and C₂, was previously identified as a second site suppresser of a catalytically inactive mutant (N1025S) but unfortunately was not characterized alone (27). Prediction of the positions of the side chains of the 4-5 loop of C₂ is difficult because of its flexibility; however, some explanations of the activating mutations can be extracted from the crystal structure. We expect K1014 upon mutation to asparagine to pack between adjacent glutamines in the 4-5 loop of C₂ and form a stabilizing hydrogen bond with Arg-434 in the 2-3 loop of C₁. P1015Q could rearrange the 4-5 loop, causing a more active conformation. I1010M fills space in the hydrophobic pocket with a larger hydrophobic residue. The introduction of new side chain interactions contributed by mutations I1010M or K1014N or the removal of main chain constraints with mutation P1015Q may alter the C₁ 2-3 loop and enhance activity. Taken together, these mutations suggest that proper formation of a competent active site is inhibited by decreased and promoted by increased interactions between the 4-5 loop of C₂ and the 2-3 loop of C₁.

The 4-5 loop of the C₁ domain has a congruous interaction with the 2-3 loop of C₂ because of the pseudosymmetrical structure of adenylyl cyclase. Of the clones obtained from the genetic screen, only V506I displayed significant enhancement of activity when tested in the soluble adenylyl cyclase system. V506I adds a methyl group that may form a primary contact with forskolin and increase the hydrophobicity of the forskolin-binding pocket. Another possible explanation is that substitution of the isoleucine may enhance C₁-C₂ interactions by altering van der Waals contacts with neighboring residues in the 4-5 loop of C₁. The structural effect of this minor change at the base of the loop may be amplified along the length of the loop and thus alter interactions with the 2-3 loop of C₂.

It is difficult to determine why other mutations displayed strong phenotypes in yeast but failed to produce substantial changes in the soluble adenylyl cyclase assays. As demonstrated in this study, mutations that increase favorable interactions between the 2-3 loop of C₁ and the 4-5 loop of C₂ likely account for the increased activity that was observed. Alternatively, increased activity may be related to the method of protein expression, since the soluble C₁ and C₂ domains remain as homodimers when expressed and purified individually. Mutations in the interface region may alter homodimerization. Mutations that impair homodimerization may favor heterodimerization and hence increase adenylyl cyclase activity. More likely, the lack of increased basal activity in the in vitro assays of many of the mutants could be explained by the inherent sensitivity to small changes in cyclic AMP concentrations of the yeast screen. The lack of change in activity in vitro could also be explained by the ablation of the membrane domains and the putative regulatory C_1b domain in the soluble constructs. For example, the F400Y mutation has been shown to increase both basal activity and sensitivity to the activators G_s and forskolin and to abrogate inhibition by G_i (28). However, when assayed in the soluble system, this mutation caused no increase in basal activity or sensitivity to forskolin compared with the wild type enzyme. The contribution of the C_1b domain to activity are not known, nor is there any structural information on this domain.

As mentioned previously, all complexes of adenylyl cyclase whose structures have been determined to date contain both G_s and forskolin. Endogenous forskolin-like substances have yet to be discovered, begging the questions of the physiological significance of the C₁·C₂·forskolin·G_s-GTPS structure and the degree to which it resembles the structure of C₁:C₂:G_s-GTPS. Or rather, what is the mechanism of activation of adenylyl cyclase by forskolin? This is a particularly interesting question, since some forms of adenylyl cyclase (types II, IV, and V-VII) are activated synergistically by forskolin and G_s-GTPS, whereas others (2) are activated only additively (types I, III, and VIII). The K1014N mutation facilitated the isolation of a C₁·C₂·G_s-GTPS complex in the absence of forskolin. Determination of the structure of this complex would further our understanding of activation of adenylyl cyclase by the diterpene.

The crystal structure of a homodimer of the C₂ domain bound with two molecules of forskolin has also been determined (29). The structure also contains a 2-fold symmetrical arrangement of the domains and has been used as a model for the basal, nonactivated form of adenylyl cyclase. However, the presence of two forskolin molecules in a complex without an active site inherently precludes this structure as a precise model of nonactivated adenylyl cyclase. It is our hope that the structure of the constitutively active mutants described herein, particularly that of VC₁ associated with IIC₂-K1014N, may represent a closer approximation of the low activity basal state.

	ACKNOWLEDGEMENTS

We thank Julie Collins for technical assistance. We also thank J. J. G. Tesmer for critically reading the manuscript, helpful discussions of the adenylyl cyclase structure, and constructing Fig. 5.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM34497, the Welch Foundation Grant I-1271, and the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology.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.

^¶ Present address: OSI Pharmaceuticals, Inc., 777 Old Saw Mill River Rd., Tarrytown, NY 10591.

Present address: Wyeth-Ayerst Research, 401 North Middleton Rd., Pearl River, NY 10965.

^** Present address: Myriad Pharmaceuticals, Inc., 320 Wakara Way, Salt Lake City, UT 84108.

To whom correspondence should be addressed: Dept. Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-2370; Fax: 214-648-8812; E-mail: alfred.gilman@email.swmed.edu.

Published, JBC Papers in Press, September 11, 2000, DOI 10.1074/jbc.M007148200

² S. Haney, J. Xu, S.-Y. Lee, C.-L. Ma, E. Duzic, J. Broach, and J. Manfredi, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; G_s, the  subunit of the G protein that stimulates adenylyl cyclase; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5'-3-O-(thio)triphosphate; ATPS, adenosine 5'-(-thio)-triphosphate; ACII, type II adenylyl cyclase; kb, kilobase pair; PCR, polymerase chain reaction.

	REFERENCES

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