Interaction of Gsalpha with the Cytosolic Domains of Mammalian Adenylyl Cyclase*

(Received for publication, April 15, 1997, and in revised form, June 20, 1997)

Roger K. Sunahara , Carmen W. Dessauer , Richard E. Whisnant , Christiane Kleuss Dagger and Alfred G. Gilman

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Forskolin- and Gsalpha -stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ~25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native Mr ~ 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 µmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S)-Gsalpha is responsible for the observed enzymatic activity and can be isolated. In addition, GTPgamma S-Gsalpha interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsalpha and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsalpha can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsalpha , indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.


INTRODUCTION

Eleven distinct isoforms of mammalian adenylyl cyclase have been identified to date, and the regulatory properties of several of these proteins have been characterized extensively (1, 2). Although there is remarkable variation in the responsiveness of these enzymes to inhibitory effects of the Gialpha 1 proteins and to such agents as G protein beta gamma subunits and Ca2+, the catalytic activity of all of the known isoforms is stimulated by the alpha  subunit of Gs and, presumably nonphysiologically, by the diterpene forskolin. The adenylyl cyclases share a unique structure for an enzyme, resembling transporters such as the P-glycoproteins topographically. They are intrinsic membrane proteins by virtue of their two large hydrophobic domains, each of which is hypothesized to contain six membrane-spanning helices. The first of these hydrophobic regions follows a short amino-terminal sequence and precedes a roughly 40-kDa cytoplasmic domain (C1). The second hydrophobic region separates C1 from a second cytosolic domain (C2) of comparable size. Each of the two cytosolic domains includes a sequence of 200-250 amino acid residues that is typically 50% similar to its consort, 50-90% similar to the corresponding domains of other adenylyl cyclase isoforms, and 20-25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl cyclases.

Detailed biochemical characterization of adenylyl cyclase is impaired by the insolubility, instability, and sparsity of the native enzyme, as well as our incapacity to express necessary amounts of the protein in heterologous systems. To overcome these hurdles we have synthesized (in Escherichia coli) portions of the two cytosolic domains of adenylyl cyclase in the absence of the remainder of the protein, first as a 55-kDa chimeric fusion protein containing the C1a domain of type I adenylyl cyclase linked to the C2 domain of the type II enzyme (3, 4). The specific activity of this engineered enzyme is remarkably high, it is soluble in the absence of detergent, and it is adequately stable. Importantly, it is activated synergistically by Gsalpha and forskolin and inhibited by so-called P-site inhibitors and the G protein beta gamma subunit complex, providing ample justification to pursue investigation of this and similar artificial entities. To overcome the remaining hurdle of a relatively low level of accumulation of the chimera in bacterial cytosol, we and others prepared the two cytosolic domains of adenylyl cyclase as distinct entities and found that enzymatic activity with similar regulatory properties can be reconstituted by simple mixture of the two roughly 25-kDa proteins (5, 6). Large amounts (50-100 mg or more) of the C2 domain of type II adenylyl cyclase can be prepared readily, but similar results are difficult to achieve with the C1a domain of the type I enzyme. We have now extended this approach by utilizing a fragment of the C1 domain of type V adenylyl cyclase, which can be expressed in reasonable (but not exuberant) quantities, and we have characterized the interactions of the two cyclase fragments with each other and with Gsalpha .


MATERIALS AND METHODS

Plasmid Construction

DNA containing nucleotides 961-2010 (amino acid residues 321-670) of canine type V adenylyl cyclase (7) was generated with the polymerase chain reaction using oligonucleotides A: 5'-CCATGGCTGAGGTCTCCCAG-3'; and B: 5'-TTGCGGCCGCGGATCCGGTCAGGCTCCCTGAAGG-3', as primers. Note that oligonucleotide A is 5' to a unique NcoI site in the cDNA for canine type V adenylyl cyclase and includes the codon for Met364. Further truncations of this fragment were generated by taking advantage of unique restriction sites for AvrII (at nucleotide 1815), BstBI (at nucleotide 1858), and Bsu36AI (at nucleotide 1900) to create VC1(364-606), VC1(364-620), and VC1(364-635), respectively. VC1(364-567), VC1(364-591), and VC1(364-591)Flag were generated with the polymerase chain reaction using the following pairs of oligonucleotide primers: A and 5'-TTCTCCGGATCCAAGCTTGCAGCGCAGGA-3', A and 5'-GATTCGAAGCTTGTGCCCAATGGAG-3', and A and 5'-GCTAATTAAGCTTGTCATCGTCGTCCTTGTAGTCGTGCCCAATGGAGTTG-3'. The last construct contains a carboxyl-terminal Flag epitope (Kodak), DYKDDDDK, as well as a cleavage site for enterokinase. All of the VC1 constructs were ligated into pQE60-H6 (8) with NcoI and HindIII. Thus, each of the encoded proteins contains a hexa-histidine tag at its amino terminus, followed by the residue corresponding to Met364 of canine type V adenylyl cyclase, and terminates at the stop codon contained in pQE60-H6. We will subsequently designate these proteins by reference to their carboxyl-terminal residue.

Expression and Purification of Proteins

E. coli strain BL21 was co-transformed with pREP4 and pQE60-H6-VC1 (various) and grown overnight at 30 °C in 200 ml of LB medium containing ampicillin (50 µg/ml) and kanamycin (50 µg/ml). This culture was used to inoculate 10 liters of T7 medium (8), which was incubated at 30 °C until OD600 reached 1.3. Synthesis of VC1 was then induced with 30 µM isopropyl thiogalactoside, and incubation was continued for 4 h at room temperature. Cells were harvested by centrifugation, frozen in liquid N2, and stored at -70 °C. Frozen cells were pulverized and suspended in 750 ml of buffer A (50 mM Tris-HCl, pH 8, 120 mM NaCl, 1 mM beta -mercaptoethanol, and a mixture of protease inhibitors (4)) prior to incubation with 0.25 µg/ml lysozyme in buffer A for 30 min at 4 °C and subsequently 8 µg/ml DNase and 1 mM MgCl2 in buffer A for an additional 30 min. Cellular debris was removed by centrifugation at 100,000 × g for 40 min at 4 °C.

All purification steps were performed at 4 °C. The supernatant (750 ml) was applied to a 5-ml metal chelate column (Talon, CLONTECH) equilibrated in buffer A, and the column was washed with 20 volumes of buffer A containing 500 mM NaCl and 10 volumes of buffer B (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM beta -mercaptoethanol, and protease inhibitors). H6-VC1 was eluted from the column in 2-ml fractions with buffer B containing 100 mM imidazole. Peak fractions were pooled, diluted with 2 volumes of buffer C (20 mM NaHepes, pH 8.0, 20 mM NaCl, 2 mM dithiothreitol, and protease inhibitors) and applied to a Mono Q 5/5 FPLC column (Pharmacia). This column was washed with 5 ml of buffer C and protein was eluted with a 25-ml linear gradient of NaCl (20-300 mM in buffer C). Peak fractions (about 150 mM NaCl) were pooled and concentrated to 3 mg/ml unless noted otherwise.

The C2 domain of type II adenylyl cyclase (IIC2) was expressed in E. coli and purified as described previously (5), as was bovine Gsalpha (short form) (8). Gsalpha was activated with GTPgamma S by incubation with 50 mM NaHepes, pH 8.0, 10 mM MgSO4, 1 mM EDTA, 2 mM dithiothreitol, and 800 µM GTPgamma S for 30 min at 30 °C.

Gel Filtration

Proteins (Gsalpha and/or the adenylyl cyclase domains) were applied (200-µl sample volumes) to a Superdex 200 column (HR10/30; Pharmacia) or to Superdex 75 (HR10/30) and 200 columns in tandem. Samples were eluted with 20 mM NaHepes, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 2 mM dithiothreitol, 100 mM NaCl, and (where indicated) 50 µM forskolin. The flow rate was 0.3 ml/min and fractions were 400 µl unless otherwise indicated.

Sedimentation Equilibrium

Sedimentation equilibrium analysis was performed by centrifugation of samples (100 µl) for 60 h at 4 °C in a Beckman TL100 centrifuge as described previously (9). The buffer contained 50 mM NaHepes, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 20 mM beta -mercaptoethanol, 50 mM NaCl, 50 mM 6-O-(3'-(piperidino)propionyl)forskolin, and 5 mg/ml dextran T40 to provide density stabilization. Immediately after centrifugation, 7-µl fractions were collected with a Brandel microfractionator. The concentration of [35S]GTPgamma S-Gsalpha in each fraction was determined by liquid scintillation spectrometry. The concentration of adenylyl cyclase in each aliquot was determined by quantification of catalytic activity under linear conditions with addition of either 1 µM VC1(591) or IIC2 in the presence of 50 µM forskolin and 0.2 µM GTPgamma S-Gsalpha . The addition of exogenous GTPgamma S-Gsalpha and either the C1 or C2 domain of adenylyl cyclase was necessary to assure linearity of activity throughout the range of concentrations found in the gradient. Molecular weights were calculated from the slope of a plot of ln C/Co versus r2, where C is the protein concentration in a given fraction, Co is the initial concentration, and r is the radial distance in the centrifuge (cm). Similar results were obtained for all conditions at two different rotational velocities.

Chemical Cross-linking

Cross-linking studies were performed utilizing the bifunctional amine-coupling reagent disuccinimidyl suberate (Pierce). Purified proteins were diluted into a buffer containing 20 mM NaHepes (pH 8.0), 1 mM EDTA, and 2 mM MgCl2. Cross-linking was initiated by addition of freshly prepared disuccinimidyl suberate (100 µM final concentration) and allowed to progress for 15 min at room temperature. Reactions were terminated with SDS-PAGE sample buffer containing 10 mM glycine and 20 mM dithiothreitol, and proteins were resolved by SDS-PAGE. Proteins were visualized by immunoblotting using antibodies specific for H6-VC1(364-591)Flag (anti-Flag), IIC2-H6 (10), or Gsalpha (11).

Adenylyl Cyclase Assays

Adenylyl cyclase activity was measured as described by Smigel (12). All assays were performed for 10 min at 30 °C in a volume of 100 µl. In reconstitutive assays the final concentration of IIC2 was at least 1-5 µM to maintain linearity with variable concentrations of VC1. The final concentration of ATP was 1 mM unless stated otherwise, and an ATP regenerating system was used only when crude preparations of enzyme were assayed.


RESULTS

Expression and Purification of Proteins Containing the VC1a Domain

The C1 domain of type V adenylyl cyclase was truncated at its carboxyl terminus in attempts to obtain a pure, stable protein with high specific enzymatic activity when reconstituted with IIC2. Bacterial lysates containing constructs as large as VC1(670), for example, supported high forskolin-stimulated adenylyl cyclase activity (>50 nmol/min/mg in the lysate), but immunoblot analysis of SDS-PAGE gels indicated contamination with several proteolytic products (data not shown). Extracts containing truncations VC1(635), VC1(621), or VC1(606) displayed progressively less such contamination, but susceptibility to degradation was again high with VC1(567); the latter protein is analogous to the C1 domain of type I adenylyl cyclase described previously (5). However, extracts of bacteria expressing VC1(591) or its carboxyl-terminally Flag-tagged counterpart, VC1(591)Flag, contained only a single significant proteolytic product (molecular mass ~ 25 kDa; mass of VC1(591) ~ 28 kDa), and its accumulation was minimized by shortening the length of time for expression to 4 h. The specific activities of such lysates for reconstitution of adenylyl cyclase activity with IIC2 were typically 50-100 nmol/min/mg extract protein. These values are roughly 1% of those observed with purified native adenylyl cyclases (12-14).

Metal chelate column chromatography (Talon resin) of extracts containing VC1(591)Flag resulted in 100-fold or more purification of the protein with loss of 60% of the total activity (Table I). The remaining activity was not found in the flow-through or column washes. Subsequent Mono Q column chromatography resulted in 2-3-fold purification and removal of the remaining major contaminants. This final product appeared essentially homogeneous when analyzed electrophoretically (Fig. 1) and had a reconstitutive specific activity of 30 µmol/min/mg when assayed with maximally effective concentrations of purified IIC2 and 50 µM forskolin as the sole activator. This purified protein will subsequently be designated VC1.

Table I. Purification of H6-VC1(591)Flag from E. coli

Fractions containing H6-VC1(591)Flag from a 10-liter bacterial culture were mixed with purified IIC2 (2 µM) and assayed for adenylyl cyclase activity as described under "Materials and Methods." Forskolin (100 µM) was included in all assays.

Purification step Volume Protein Specific activity Total activity Recovery Purification

ml mg µmol/mg/min µmol/min % -fold
Lysates 1000 7100 0.05 355 100
Talon pool 10 12 12 144 41 240
Mono Q pool 4.5 4 30 120 34 600


Fig. 1. Purification of H6-VC1(364-591)Flag. H6-VC1(364-591)Flag was expressed in E. coli as described under "Materials and Methods." Aliquots from various stages in the purification were resolved by SDS-PAGE on a 15% polyacrylamide gel, and proteins were visualized by staining with Coomassie Blue. Lane A, 5 µg of E. coli lysate; lane B, 1 µg of Talon column eluate; lane C, 1 µg of Mono Q pool.
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Adenylyl Cyclase Activity Reconstituted from VC1 and IIC2

The adenylyl cyclase activity observed in mixtures of VC1 and IIC2 resembles that seen previously with mixtures of IC1 and IIC2 (5, 6); many of the regulatory features that characterize native membrane-bound adenylyl cyclases are retained. Activities shown in Fig. 2, A, B, and D, were measured with low concentrations of IIC2 and either near saturating (2 µM, Fig. 2, A and B) or variable (Fig. 2D) concentrations of VC1. Activities shown in Fig. 2C were measured with low concentrations of VC1 and near saturating (2 µM) concentrations of IIC2. The reconstituted enzyme is activated by either forskolin (EC50 > 10 µM) or Gsalpha (EC50 ~ 400 nM) (Fig. 2, A and B). Unlike the mixture of IC1 and IIC2, GTPgamma S-Gsalpha could not activate a mixture of VC1 and IIC2 maximally. The effects of forskolin and GTPgamma S-Gsalpha are synergistic; the presence of one activator increasing the apparent affinity (EC50) of the enzyme for the other by 40-fold or more. In the absence of an activator, the rate of cyclic AMP synthesis is low and the two domains of the enzyme have an apparent affinity for each other of greater than 5 µM (Fig. 2D, inset). This value is lowered in the presence of activators; the apparent affinity of VC1 and IIC2 is 0.66 µM with GTPgamma S-Gsalpha , 1.2 µM with forskolin, and 150 nM when both activators are present.


Fig. 2. Reconstitution of adenylyl cyclase activity. VC1 was reconstituted with IIC2 and assayed with the indicated activators. A, VC1 (2 µM) and IIC2 (1 nM) were activated with increasing concentrations of forskolin in the absence (open circle ), or presence of either 50 nM GTPgamma S-Gsalpha (square ) or 400 nM GTPgamma S-Gsalpha (black-square). B, VC1 (2 µM) and IIC2 (1 nM) were activated with increasing concentrations of either GDP-bound Gsalpha (open circle , square ) or GTPgamma S-bound Gsalpha (bullet , black-square) in the absence (open circle , bullet ) or presence (square , black-square) of 100 µM forskolin. C, IIC2 (2 µM) and VC1 (1 nM) were activated with increasing concentrations of forskolin in the absence (open circle ) or presence (black-square) of 400 nM GTPgamma S-Gsalpha . D, IIC2 (1 nM) was assayed with increasing concentrations of VC1 in the absence (inset) or presence of 100 µM forskolin (bullet ), 400 nM GTPgamma S-Gsalpha (square ), or 400 nM GTPgamma S-Gsalpha and 100 µM forskolin (black-square).
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Activation of adenylyl cyclase activity by Gsalpha is dependent on the nature of the bound nucleotide, but not to the extent usually assumed. GDP-Gsalpha is only 10-fold less potent than GTPgamma S-Gsalpha and is equally efficacious (in the presence or absence of forskolin (Fig. 2B)). Identical results were obtained with limiting concentrations of either VC1 or IIC2 (data not shown).

Gel Filtration and Sedimentation Equilibrium Centrifugation

We have examined the interactions of VC1 and IIC2 with each other and with Gsalpha by gel filtration, sedimentation equilibrium centrifugation, and (see below) chemical cross-linking. Gel filtration of VC1 or IIC2 on tandem columns of Superdex 75 and 200 suggests that each of these proteins exists in solution as roughly 50-kDa homodimers (Fig. 3, A and B); each individual protein has a molecular mass of approximately 28,000. Similarly, sedimentation equilibrium analysis of IIC2 revealed a molecular weight of 46,000 (Fig. 6D). Gel filtration of a mixture of VC1 and IIC2 in the presence (Fig. 3B) or absence (not shown) of forskolin revealed a single peak of protein with an apparent molecular weight of 50,000, while sedimentation equilibrium analysis of the mixture indicates a molecular weight of 52,000 (Fig. 6B). Because of the similarity of the molecular masses of VC1 and IIC2, we do not know if these values represent homodimers, heterodimers, or a mixture of the two.


Fig. 3. Gel filtration of VC1 and IIC2. A, VC1 (500 µg; approximately 100 µM) was gel filtered on a Superdex 200 (HR 10/30) column. B, VC1 (100 µM) was gel filtered on a Superdex 200 (HR 10/30) column by itself, with IIC2 (100 µM), or in the presence of IIC2 and GTPgamma S-Gsalpha (100 µM). IIC2 was also gel filtered by itself. Forskolin (50 µM) was present in the samples and the elution buffer. The positions of elution of molecular weight markers are shown.
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Fig. 6. Determination of the molecular weights of Gsalpha , adenylyl cyclase, and their complex by sedimentation equilibrium. A, [35S]GTPgamma S-Gsalpha (3 µM) was subjected to sedimentation equilibrium ultracentrifugation at 11,000 rpm at 4 °C in the absence (bullet ) or presence (black-square) of 15 µM VC1 and 15 µM IIC2. The concentration of [35S]GTPgamma S-Gsalpha was determined by scintillation counting. B, VC1 and IIC2 (4 µM each) were centrifuged at 11,000 rpm in the absence (bullet ) or presence (black-square) of 12 µM GTPgamma S-Gsalpha . Adenylyl cyclase concentrations were determined by measuring adenylyl cyclase activity in the presence of 1 µM IIC2, 50 µM forskolin, and 0.2 µM GTPgamma S-Gsalpha . Data (A and B) are representative of four experiments at 3 different rotational velocities. C, [35S]GTPgamma S-Gsalpha (5 µM) was subjected to sedimentation equilibrium ultracentrifugation at 15,000 rpm in the absence (bullet ) of additional proteins or in the presence of 100 µM IIC2 (black-square), 200 µM IIC2 (black-triangle), or 100 µM VC1 (black-down-triangle ); the concentration of [35S]GTPgamma S-Gsalpha was measured. D, IIC2 (3 µM) was subjected to centrifugation at 14,000 rpm in the absence (bullet ) or presence (black-square) of 25 µM GTPgamma S-Gsalpha . The concentration of IIC2 was assayed by measurement of adenylyl cyclase activity in the presence of 1 µM VC1, 50 µM forskolin, and 0.2 µM GTPgamma S-Gsalpha . Data (C and D) are representative of two experiments at two different rotational velocities.
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Mixture of VC1, IIC2, and GTPgamma S-Gsalpha (in the presence of forskolin) results in formation of a high-affinity complex. This is detected by gel filtration with the appearance of a new peak of optical density, migrating with an apparent molecular weight of roughly 100,000 (Fig. 3B). SDS-PAGE analysis of fractions obtained by gel filtration of such a mixture containing an excess of IIC2 indicates that this high molecular weight peak contains all three proteins, with an apparent stoichiometry of 1:1:1 (by scanning densitometry) (Fig. 4). (Note that essentially all of the VC1 is found in this high molecular weight peak, presumably indicating that most of the protein is active.) Similarly, equilibrium sedimentation of such mixtures reveals a 104-107 kDa complex when either Gsalpha or adenylyl cyclase activity is monitored (Fig. 6, A and B). The anticipated molecular weight of a 1:1:1 complex of Gsalpha , VC1, and IIC2 is 102,000. 


Fig. 4. Purification of VC1 associated with IIC2 and GTPgamma S-Gsalpha . VC1 (100 µM), IIC2 (300 µM), and GTPgamma S-Gsalpha (100 µM) were incubated for 30 min on ice in gel filtration buffer containing 50 µM forskolin. The mixture was applied to a Superdex 75 (HR 10/30) gel filtration column in tandem with a Superdex 200 (HR 10/30) column. The column was eluted with gel filtration buffer containing 50 µM forskolin. Fractions 5-17 (5 µl of each 400-µl fraction) 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.
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Interaction between Gsalpha and IIC2 alone can also be detected by gel filtration and equilibrium sedimentation. Incubation of 50 µM [35S]GTPgamma S-Gsalpha with 5, 35, or 250 µM IIC2 causes elution of radioactivity at a position consistent with progressively higher molecular weights (Fig. 5C). At the highest concentration of IIC2, the complex is consistent with molecular mass ~70 kDa. No such interactions were detected between VC1 and GTPgamma S-Gsalpha (Fig. 5A) or IIC2 and GDP-Gsalpha (Fig. 5B). With sedimentation equilibrium, the apparent molecular weight of Gsalpha was increased by 24,000 following mixture with IIC2 (but not with VC1) (Fig. 6C) and analysis of adenylyl cyclase activity revealed a complex between GTPgamma S-Gsalpha and IIC2 with mass = 64 kDa. (Note: observation of enzymatic activity in this experiment required addition of VC1; the complex of GTPgamma S-Gsalpha with IIC2 does not have detectable adenylyl cyclase activity.) All of these observations indicate formation of a relatively low affinity 1:1 complex between IIC2 and GTPgamma S-Gsalpha , and, thus, that interaction of the adenylyl cyclase domains with the activated G protein alpha  subunit disrupts the homodimeric interactions characteristic of VC1 and IIC2.


Fig. 5. Gel filtration of VC1 or IIC2 with Gsalpha . [35S]GTPgamma S-Gsalpha or [32P]GDP-Gsalpha was chromatographed on a Superdex 75 (HR 10/30) gel filtration column in the absence or presence of either VC1 or IIC2. The chromatogram demonstrates elution of bound radioactive nucleotide. A, [35S]GTPgamma S-Gsalpha (~50 µM) was chromatographed in the absence (bullet ) or presence (triangle ) of 40 µM VC1. B, [35S]GTPgamma S-Gsalpha (50 µM) (bullet ) was gel filtered alone. [32P]GDP-Gsalpha (50 µM) (Delta , black-triangle) was chromatographed in the absence (Delta ) or presence (black-triangle) of 250 µM IIC2. C, [35S]GTPgamma S-Gsalpha (50 µM) was gel filtered in the absence (bullet ) or presence of 5 µM (open circle ), 35 µM (square ), or 250 µM (black-square) IIC2.
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Chemical Cross-linking

Although direct interactions between VC1 and Gsalpha were not detected by gel filtration or sedimentation equilibrium, chemical cross-linking studies do suggest that the molecules are at most 11 Å away from each other when tightly complexed with IIC2. Fig. 7A illustrates the forskolin (and IIC2-, not shown)-dependent appearance of a 70-kDa species representing covalent coupling of Gsalpha and VC1 by the 11-Å cross-linker, disuccinimidyl suberate. Analysis of the same fractions with an anti-Gsalpha antibody also reveals a 70-kDa species that appears in a forskolin-dependent manner. It is unclear in this panel if this species represents Gsalpha -VC1, Gsalpha -IIC2, or both, since the cyclase domains have similar molecular weights. This was also complicated by the inability of available IIC2 antibodies to detect IIC2-Gsalpha complexes by immunoblotting. Cross-linking studies performed at higher protein concentrations (2 µM VC1, IIC2, and Gsalpha ) and analyzed with an anti-Gsalpha antibody reveal a IIC2-Gsalpha cross-linked species in the absence of VC1 (Fig. 7E, lane 4). Moreover, a large species (Mr ~ 95,000) appears in the presence of all three proteins and 100 µM forskolin; we presume this to be the VC1-IIC2-Gsalpha heterotrimer (Fig. 7E, lane 7). Observation of both the cross-linked heterotrimer and the IIC2-Gsalpha heterodimer is dependent on the presence of GTPgamma S-activated Gsalpha (Fig. 7E, lane 3 versus and lane 6 versus 7).


Fig. 7. Chemical cross-linking of Gsalpha , VC1, and IIC2. VC1 (200 nM) and IIC2 (2 µM) were incubated with 400 nM GTPgamma S-Gsalpha and increasing concentrations of 6-O-[3'-(piperidino)propionyl]forskolin at room temperature for 5 min prior to cross-linking with disuccinimidyl suberate. Cross-linked samples were resolved by SDS-PAGE in an 11% gel, transferred to nitrocellulose, and probed with anti-Flag (i.e. anti-VC1) antibody (panel A) or antibody 584 (anti-Gsalpha ) (11) (panel B). C, VC1 (2 µM) and IIC2 (200 nM) were incubated with 400 nM GTPgamma S-Gsalpha and increasing concentrations of 6-O-[3'-(piperidino)propionyl]forskolin at room temperature for 5 min. Samples were cross-linked, resolved on a 7.5% gel, transferred to nitrocellulose, and analyzed with anti-IIC2 antibody. D, VC1 (200 nM) and IIC2 (2 µM) were incubated with 400 nM GTPgamma S-Gsalpha and increasing concentrations of 6-O-[3'-(piperidino)propionyl]forskolin at room temperature for 5 min. Samples were cross-linked, resolved on a 7.5% gel, transferred to nitrocellulose, and analyzed with anti-Flag (i.e. anti-VC1) antibody. E, GDP- or GTPgamma S-bound Gsalpha (2 µM) was incubated with 2 µM IIC2 in the absence or presence of either 2 µM VC1, with or without 100 µM 6-O-[3'-(piperidino)propionyl]forskolin, as indicated. Samples were cross-linked, resolved on an 11% gel, and transferred to nitrocellulose. The blot was analyzed with an anti-Gsalpha antibody.
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Chemical cross-linking also revealed forskolin-dependent formation of heterodimers between VC1 and IIC2 (Fig. 7, C and D). Although each domain exists as a homodimer under nonactivating conditions, formation of heterodimers occurs in a forskolin (and Gsalpha -)-dependent manner.


DISCUSSION

We have expressed and purified a fragment of the C1 domain of type V adenylyl cyclase, consisting of amino acid residues 364-591 and including hexa-histidine and Flag tags at the amino and carboxyl termini, respectively. Although this protein itself has no adenylyl cyclase activity, catalysis of cyclic AMP synthesis is restored by simple mixture of VC1 with an appropriate fragment from the second cytosolic domain of the enzyme, such as IIC2. This interaction is similar to that described previously between IC1 and IIC2 (5, 6); the major advantages are the yield of VC1, which exceeds that of IC1 by 20-fold, and the apparent homogeneity of the product. The adenylyl cyclase activity of the VC1-IIC2 mixture is stimulated markedly by either Gsalpha or forskolin, and these two regulators activate the enzyme synergistically when present simultaneously. These are characteristics of native type II and type V adenylyl cyclases.

A notable difference between this reconstituted adenylyl cyclase and the native enzymes is the maximal stimulated activity, which typically exceeds 100 µmol/min/mg in the case of the mixture of VC1 and IIC2; values of 10 µmol/min/mg typify purified preparations of native enzymes (12-14). Although the source of this discrepancy is not known, we suspect that the values observed with the VC1/IIC2 mixture may indeed approximate a true Vmax for mammalian adenylyl cyclase. Several factors may cause underestimation of maximal activity when dealing with a native adenylyl cyclase. Overexpression of these enzymes in Sf9 cells is plagued by production of nonfunctional protein; detergents are necessary to maintain solubility of the native proteins but may alter estimates of specific activity; lengthy purification schemes may cause denaturation of these labile entities. Alternatively, it is possible that inhibitory domains have been removed from the soluble constructs described above or that the membrane spanning domains of the enzymes do not permit optimal orientation of the interacting cytosolic segments. Another notable difference between native adenylyl cyclases and the engineered soluble enzymes (whether or not the cytosolic domains are linked covalently) is the relatively low (reduced 20-50-fold) apparent affinities of the soluble enzymes for Gsalpha . It is possible that the transmembrane spans, the loops that connect them, and/or residues immediately surrounding the remnants of the C1 and C2 domains in the constructs utilized may contribute to the binding site for Gsalpha . Nevertheless, the essential features of activation of adenylyl cyclase by the G protein alpha  subunit are retained.

We demonstrate herein that the C1 and C2 domains of adenylyl cyclase interact to form a catalytically active adenylyl cyclase and that the apparent affinity of C1 for C2 is enhanced in the presence of Gsalpha and/or forskolin. Gel filtration, equilibrium sedimentation, and cross-linking analyses all demonstrate that Gsalpha interacts with the C2 domain of adenylyl cyclase in a GTP-enhanced manner and that this interaction is further stabilized by C1. Direct interactions between VC1 and Gsalpha were not detected by gel filtration or sedimentation equilibrium. However, these two proteins could be cross-linked by disuccinimidyl suberate in a IIC2-dependent manner. We further demonstrate that Gsalpha , C1, and C2 form a relatively high-affinity complex with a 1:1:1 stoichiometry. Although this is not surprising, the similarities in the primary sequence of the C1 and C2 domains of adenylyl cyclase raised the possibility of binding sites for two molecules of Gsalpha . Several adenylyl cyclase isoforms are inhibited by Gialpha . Although high concentrations of Gialpha can apparently compete for the Gsalpha -binding site, inhibition of adenylyl cyclase activity by Gialpha is not dependent on Gsalpha and is not competitive with the stimulatory alpha  subunit (15). We presume that there is a distinct binding site for Gialpha on certain adenylyl cyclases; Gialpha may well be found to interact predominantly with C1.

Homodimerization of C1 and C2 is difficult to interpret. The phenomenon may clearly be an artifact of protein engineering and have no significance with regard to the membrane-bound native enzyme. However, we have previously detected oligomers of near native adenylyl cyclases in detergent solution, suggesting the possibility of relevance of homodimerization of the cytosolic domains (16).

The relative capacities of GTP- and GDP-bound Galpha proteins to interact with their effectors has been long debated and may be dependent on the system in question. However, no such interaction has probably been examined previously in a system containing such highly purified proteins. The Gsalpha , VC1, and IIC2 proteins utilized herein are all expressed abundantly in bacteria, greatly facilitating their purification to a high degree. Significant levels of contaminating nucleotide kinases are extremely unlikely, obviating such concerns as conversion of GDP to GTP in the presence of ATP. Nevertheless, the apparent affinity of GDP-Gsalpha for adenylyl cyclase is only about 10-fold less than that of GTPgamma S-Gsalpha . The GTPase activity of a G protein alpha  subunit thus facilitates deactivation of the system by reducing the affinity of Galpha for its effector. However, the affinity of the G protein beta gamma subunit complex for Galpha is more highly dependent on the nature of the bound nucleotide. GTP hydrolysis thus greatly favors association of alpha  with beta gamma , and it is this interaction that prevents access of either alpha  or beta gamma to effectors.

More critical and microscopic analyses of the interactions of the two cytosolic domains of adenylyl cyclase with each other and with their regulators are necessary. Although this need has been recognized for a long time, progress has been greatly limited by the availability and purity of reagents. Milligram quantities of G protein-regulated adenylyl cyclase domains may now be prepared readily, as can a high-affinity complex of Gsalpha with the crucial components of the cyclase. We hope that all relevant tools can now be utilized to understand the complex regulatory features of these interesting enzymes.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant GM34497, a postdoctoral fellowship from The Medical Research Council of Canada (to R. K. S.), National Institutes of Health National Research Service Award GM16905 (to C. W. D.), 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.
Dagger    Present address: Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany.
1   The abbreviations used are: Gsalpha , the alpha  subunit of the G protein that stimulates adenylyl cyclase; Gialpha , the alpha  subunit of the G protein that inhibits adenylyl cyclase; G protein, heterotrimeric guanine nucleotide-binding protein; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We thank Julie Collins and Jeff Laidlaw for superb technical assistance and Alex Duncan for generously providing purified wild type Gsalpha .


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