©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Cytoplasmic Domains of Mammalian Adenylyl Cyclase Form a G- and Forskolin-activated Enzyme in Vitro(*)

(Received for publication, February 2, 1996; and in revised form, March 14, 1996)

Shui-Zhong Yan David Hahn Zhi-Hui Huang Wei-Jen Tang (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C(1) and C(2)). The first cytoplasmic domain of type I enzyme (IC(1)) and the second cytoplasmic domain of type II enzyme (IIC(2)-Delta3, a construct in which 36 N-terminal amino acids of the C(2) region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in G- and forskolin-activated enzyme activity. The turnover number for G- and forskolin-stimulated enzyme activity of the complex between IC(1) and IIC(2)-Delta3 was 8.2 s. The concentration of IIC(2)-Delta3 to achieve half-maximal activation of IC(1) was 0.8 and 1.3 µM when stimulated by forskolin and G, respectively. The concentration of IIC(2)-Delta3 needed to complex with IC(1) was reduced 10-fold (0.08 µM) when the enzyme was activated by both forskolin and G, suggesting that G and forskolin increased the affinity of the two cytoplasmic domains for each other.


INTRODUCTION

The enzymatic activity of adenylyl cyclase is the key step in regulating the intracellular cAMP concentration upon stimulation of a variety of hormones, neurotransmitters, and other regulatory molecules. There are at least nine distinct mammalian adenylyl cyclases which have a similar structure (Fig. 1A)(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) . This includes two intensely hydrophobic domains (M(1) and M(2)) and two 40-kDa cytoplasmic domains (C(1) and C(2)). The C(1) and C(2) domains contain sequences (C and C) that are similar to each other and to other adenylyl and guanylyl cyclases(12, 13) . Each isoform of adenylyl cyclase has its own distinct tissue distribution and unique regulatory properties, providing modes for different cells to respond diversely to similar stimuli(12, 14) .


Figure 1: Properties of soluble type I-type II adenylyl cyclase constructs expressed in E. coli. A, the top shows a model of mammalian adenylyl cyclase with each of its regions labeled. ACI = type I enzyme; ACII = type II enzyme. Below are shown the constructs used in this work. The first four include, at their amino termini, a hexa-histidine and a HA1 epitope tag with a short linker to create the EcoRI and NcoI restriction sites. B, adenylyl cyclase activities and protein expression of the IC(1)IIC(2) construct in protease-deficient E. coli strains, BL21DE3 and SG22094. Samples (10 µl) were taken for enzyme assay and immunoblot assay with either 12CA5 or C2-1077 antibodies at the indicated times after IPTG induction. C, adenylyl cyclase activities for a mixture of 10 µl of a bacterial lysate (from BL21DE3 cells) containing the components at the top that were obtained after the indicated hours of induction with IPTG and 10 µl of an lysate (from BL21-DE3 cells) containing either IIC(2) or IC(1). The immunoblot shows the amount of the component at the top after the indicated hours of induction by IPTG. p.i. = post-IPTG induction. Adenylyl cyclase activity is shown as nmolbulletminbulletmg. Data are representative of two experiments.



Membrane-bound adenylyl cyclases are expressed in small quantities, and the enzyme is labile and difficult to manipulate in detergent-containing solutions. To facilitate biochemical and structural analysis, a soluble adenylyl cyclase has been constructed by linking the C and C domains of type I and type II adenylyl cyclases, respectively(15) . The resulting protein is sensitive to activation by G(^1)and forskolin and to inhibition by P-site inhibitors, indicating the essential roles of C and C domains for catalysis and regulation. In this paper, we describe the expression and purification of the C and C domains of type I and type II adenylyl cyclase, respectively. Alone, each has no adenylyl cyclase activity; however, mixing of the two domains in vitro results in G- and forskolin-activated enzyme activity.


EXPERIMENTAL PROCEDURES

Plasmids

For construction of the expression plasmid vector pProEx-HAH6, the NcoI and EcoRI 4.9-kb fragment of pProEx-1 (Life Technologies, Inc.) was ligated with the phosphorylated linkers (5`-CATGCATCACCATCACCATCACGCGGCCGCCTACCCGTATGATGTCCCGGATTACGCCGGAATTCCCATGGC and 5`-AATTGCCATGGGAATTCCGGCGTAATCCGGGACATCATACGGGTAGGCGGCCGCGTGTGGTGATGGTGATG). Proper insertion of cDNA at the NcoI site of pProEx-HAH6 vector would result in the expression of a fusion protein that contained both HA1 and hexo-histidine tags at the N terminus (Fig. 1A). The BspHI and HindIII fragments were excised from pTrc-IC(1) and pTrc-IC(1)IIC(2)-L(3)(15) . The resulting fragments were ligated with NcoI- and HindIII-digested pProEx-HAH6 to construct vectors, pProEx-HAH6-IC(1) and pPro-HAH6-IC(1)IIC(2). To construct pProEx-HAH6-IIC(2), the BspHI-blunted EcoRI fragment was excised from pUC-IIC(2) and ligated into pProEx-HAH6 that was digested with NcoI and SmaI(15) . To construct pProEx-HAH6-IIC(2)-Delta3, an 0.8-kb DNA fragment encoding IIC(2) was amplified by 15 cycles of polymerase chain reaction using pProEx-HAH6-IIC(2) as the template, Vent DNA polymerase, and two oligonucleotides (5`-CGAGGAATTCTGGAGAACGTGCTTCCTGCACAC and 5`-TGCGTTCTGATTTAATCTGTATCAGGCTGA) as the primers. The resulting DNA was digested with EcoRI and HindIII and ligated into pProEx-HAH6 that was digested with the same enzymes. Plasmids pProEx-HAH6-IC(1), -IIC(2), -IIC(2)-Delta3, -IC(1)IIC(2) were used to express IC(1), IIC(2), IIC(2)-Delta3, and IC(1)IIC(2), respectively.

Expression of Soluble Adenylyl Cyclase

Escherichia coli cells with a plasmid were grown in Luria's broth containing ampicillin (50 µg/ml) at 30 °C and IPTG to 0.1 mM was added when the culture reached an A of 0.4. Cells were harvested at suitable times, centrifuged at 6,000 times g at 4 °C, and frozen. Frozen cells was thawed in 1/10 culture volume of Tbeta(5)P (20 mM Tris HCl, pH 8.0, 5 mM beta-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride), lysozyme to 0.1 mg/ml was added, the cells were sonicated briefly, and the supernatant after centrifugation (150,000 times g for 30 min, 4 °C) was saved. The concentration of proteins was determined using Bradford reagent (Bio-Rad) and bovine serum albumin as standard(16) . The proteins were separated by electrophoresis on 11% SDS-PAGE and immunoblot was performed using the ECL system (Amersham). Ascites fluid of hybridoma 12CA5 was raised and collected as described(17) .

Purification of Soluble Adenylyl Cyclase

All steps of the purification were performed at 4 °C in a cold room. Supernatant of E. coli lysate (from 4 liters, harvested 4 h after IPTG induction) was applied directly to a 20-ml Ni-NTA column (Qiagen) that was equilibrated with Tbeta(5)P containing 100 mM NaCl. The Ni-NTA column was washed with 100 ml of Tbeta(5)P containing 500 mM NaCl and 100 ml of Tbeta(5)PN containing 20 mM imidazole (pH 7.0). The column was then eluted with 100 ml of Tbeta(5)PN containing 150 mM imidazole (pH 7.0). The eluate was concentrated by ultrafiltration (Amicon, positive pressure ultrafiltration device, PM 10 membrane) and then diluted 2-fold with TE(1)D(1) (20 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM DTT), these steps being repeated three times to reduce NaCl concentration to below 15 mM. The resulting concentrate was applied to a Pharmacia Mono Q HR 10/10 fast protein liquid chromatography (FPLC) column that had been equilibrated with TE(1)D(1). The column was washed with 1 volume of TE(1)D(1), and absorbed proteins were eluted at 1 ml/min with the 240-ml linear gradient of NaCl (200-500 mM and 100-300 mM NaCl for purification of IC(1) and IIC(2)-Delta3, respectively) in TE(1)D(1); 4-ml fractions were collected. The protein peak of IC(1) was determined by its adenylyl cyclase activity when the fractions were mixed with E. coli lysates that contained IIC(2); the peak of IIC(2)-Delta3 was determined using the lysates containing IC(1). Recombinant protein, IC(1) was eluted at about 350 mM NaCl and IIC(2)-Delta3 at about 200 mM NaCl. The peak fractions of IC(1) and IIC(2)-Delta3 were then separated by electrophoresis on 11% SDS-PAGE and analyzed for purity by Coomassie Blue staining. The purest fractions of IC(1) were then applied to a Pharmacia Superdex 200 HR10/30 gel filtration column that had been equilibrated with TE(1)D(1) and peak activity was collected. Purified IC(1) and IIC(2)-Delta3 were concentrated in a Centricon 10 microconcentrator (Amicon) and stored at -80 °C (protein concentration > 1 mg/ml).

Gel Filtration

Purified IC(1), IIC(2)-Delta3, or mixed IC(1) and IIC(2)-Delta3 (200 µl) were applied to a Pharmacia Superdex 200 HR 10/30 gel filtration column; the flow rate was 0.3 ml/min and 0.3-ml fractions were collected. For Fig. 5A, TE(1)D(1)N was used in sample dilution and in equilibrating and running the column. For Fig. 5B (to mimic assay condition), samples were incubated in 200 µl of TD(1) with 10 mM MgCl(2), 1 mM ATP, and 100 µM forskolin at 30 °C for 2 min; forskolin (100 µM, 200 µl) was applied to a Pharmacia Superdex 200 HR 10/30 gel filtration column that had been equilibrated with TD(1)N containing 10 mM MgCl(2) and 1 mM ATP, and samples were applied immediately after application of forskolin. Adenylyl cyclase activity was measured in the presence of 100 µM forskolin. As a control, a lysate of E. coli containing IC(1)IIC(2) (300 µg in Tbeta(5)PN) was applied under the same conditions.


Figure 5: Superdex 200 gel filtration chromatography of purified IC(1), IIC(2)-Delta3, mixed IC(1) and IIC(2)-Delta3, and an extract containing IC(1)IIC(2) using TE(1)D(1)N (A) and TD(1)N with 10 mM MgCl(2), 1 mM ATP, and forskolin (B). Molecular size markers (Bio-Rad) are thyroglobin (670 kDa), -globulin (158 kDa), chicken ovalbumin (44 kDa), and horse myoglobin (17 kDa). A, total activity values of the purified IC(1) (5 µg), IIC(2)-Delta3 (5 µg), mixed IC(1) (0.5 µg) and IIC(2)-Delta3 (5 µg), and an extract containing IC(1)IIC(2) (300 µg) were 80, 183, 47, and 9.9 nmolbulletmin, respectively. B, total activity values of the purified IC(1) (5 µg), IIC(2)-Delta3 (5 µg), mixed IC(1) (1 µg) and IIC(2)-Delta3 (50 µg), and an extract containing IC(1)IIC(2) (300 µg) were 82.5, 66.2, 115.8, and 6.37 nmolbulletmin, respectively. Data are representative of two experiments.



Adenylyl Cyclase Assay

Activity was assayed in a 100-µl final volume for 10-20 min at 30 °C in the presence of 10 mM MgCl(2)(18) . Recombinant IC(1), IIC(2), and IIC(2)-Delta3 were premixed on ice for 10 min before assay. Recombinant G was purified and activated as described(19, 20) . Membranes of Sf9 cells that expressed IM(1)C(1)-(1-570), IM(1)C(1)-(1-484), IM(2)C(2), and IIM(2)C(2) were prepared as described(21) .


RESULTS AND DISCUSSION

Expression of IC(1)IIC(2), IC(1), and IIC(2)

A soluble adenylyl cyclase was constructed by linking the conserved cytoplasmic domains from type I (C(1)) and type II (C(2)) adenylyl cyclases(15) . The resulting protein, IC(1)IIC(2), could be activated by G and forskolin, and the activated enzyme could be inhibited by P-site inhibitors. IC(1)IIC(2) was tagged with hexa-histidine and the HA1 epitope at the N terminus to facilitate the detection and purification (Fig. 1A). Hexo-histidine allowed affinity purification using immobilized metal affinity chromatography (e.g. using Ni-NTA resin), and the HA1 epitope permitted the detection of recombinant protein using monoclonal antibody made by hybridoma, 12CA5(20, 22) . High speed supernatant of lysates from both E. coli BL21DE3 and SG22094 cells that expressed IC(1)IIC(2) had increased forskolin-stimulated adenylyl cyclase activities, and the enzyme activities were higher (4-120-fold) in lysates of cells that were harvested after 8-19 h of IPTG induction than after only 2-4 h of IPTG induction (Fig. 1B)(23) . Using monoclonal antibody 12CA5 and anti-peptide antiserum C2-1077 (detecting the N and C terminus of IC(1)IIC(2), respectively), the expected 60-kDa protein was detected (Fig. 1B). While the lysates from the later times after IPTG induction had higher forskolin-stimulated enzyme activity, they did not have increased amounts of 60-kDa full-length protein. This suggested that the majority of adenylyl cyclase activity from IC(1)IIC(2) was proteolyzed after 4-19 h of IPTG induction and that the proteolyzed product was catalytically active. Expression of IC(1)IIC(2) in protease-deficient strains, BL21DE3 (Lon), SG22094 (Lon, Clp), or SG21163 (Lon, htpR) did not enhance the accumulation of full-length 60-kDa protein (Fig. 1B, data not shown for SG21163)(23, 24) .

A 30-kDa proteolytic fragment of IC(1)IIC(2) was detected using antiserum C2-1077, suggesting that there is a prominent cleavage at the junction between IC(1) and IIC(2). To investigate whether the complex of IC(1) and IIC(2) was part of a catalytically active species of the proteolyzed IC(1)IIC(2), HA1 and hexo-histidine-tagged IC(1) and IIC(2) were expressed separately. Using the monoclonal antibody from 12CA5, the 30- and 31-kDa proteins were detected in high speed supernatants of lysates from E. coli that expressed IC(1) or IIC(2) (expected molecular mass as 27 and 31 kDa, respectively), indicating that the IC(1) and IIC(2) protein were stable, soluble proteins. Adenylyl cyclase activities of E. coli lysates that expressed either IC(1) or IIC(2) were not different from those of lysates of E. coli that carried the control vector (0.01 nmolbulletminbulletmg). However, mixing of the lysates, each expressing one of these constructs, resulted in high enzyme activity (2-9 nmolbulletminbulletmg, Fig. 1C). The enzyme activity correlated generally with expression (monitored by immunoblot) of IC(1) and IIC(2) from cells (Fig. 1C).

Purification of IC(1) and IIC(2)

IC(1) could be purified by Ni-NTA (Fig. 2A). The enriched IC(1) could be further purified using a FPLC Mono Q column and Superdex 200 gel filtration. The 30-kDa protein was the adenylyl cyclase (indicated by arrow and verified based on enzyme activity and immunoblot, Fig. 2, B and C). A 29-kDa protein was a major contaminant. The recovery of forskolin-stimulated adenylyl cyclase activity was only 5%, and the yield was 50 µg from each liter of E. coli culture.


Figure 2: Purification of IC(1) and IIC(2)-Delta3. A, purification on a Ni-NTA column. Lysates of E. coli that expressed IC(1), IIC(2), and IIC(2)-Delta3 (1.5 ml) were passed through a 0.3-ml Ni-NTA column, and proteins that flowed through the column were collected. The column was subsequently washed with 1.5 ml of wash 1 buffer, Tbeta(5)NP, and that of wash 2 buffer, Tbeta(5)NIP. The column was then eluted with 1.5 ml of Tbeta(5)NIP. Lane 1, load; lane 2, flow-through; lane 3, wash 1; lane 4, wash 2; and lane 5, eluate. Total adenylyl cyclase activities (nmolbulletmin) of IC(1), IIC(2), and IIC(2)-Delta3 lysates were 72, 38, and 75, respectively. Activities of flow-throughs, washes, and eluates are given as a percentage of adenylyl cyclase activity applied. Immunoblot was performed with antibody, 12CA5. Data are representative of two experiments. B, Coomassie Blue stain of purified IC(1) (0.2 µg) and IIC(2)-Delta3 (5 µg). C, immunoblot of purified IC(1) and IIC(2)-Delta3 (100 ng each).



The same procedure did not succeed in the purification of IIC(2). The majority of the adenylyl cyclase activity from IIC(2) did not bind to Ni-NTA, probably due to proteolysis or masking of the hexo-histidine tag. When lysates containing IC(1) were mixed with lysates containing IIC(2) and applied to Ni-NTA column, most of adenylyl cyclase did not bind to the column (data not shown). This indicated that the binding between IC(1) and IIC(2) was not strong enough for copurification of IC(1) and IIC(2).

To purify IIC(2), we used IIC(2)-Delta3, a construct that deleted 36 N-terminal amino acids of IIC(2), residues that are not conserved among mammalian adenylyl cyclases. HA1 and hexo-histidine-tagged IIC(2)-Delta3 protein was expressed as a soluble protein, based on immunoblot, and formed G- and forskolin-regulated adenylyl cyclase when mixed with lysate containing IC(1)in vitro (Fig. 1C). IIC(2)-Delta3 could be purified by Ni-NTA and, after subsequent chromatography on FPLC-Mono Q, 95% pure protein (29 kDa) was obtained (Fig. 2B). Its identity was confirmed by immunoblot (Fig. 2C). The recovery of adenylyl cyclase activity was about 35%, and the yields for IIC(2)-Delta3 proteins were 2 mg from each liter of E. coli culture.

Characterization of Purified IC(1) and IIC(2)-Delta3

Purified IC(1) and IIC(2)-Delta3 proteins by themselves had little enzyme activity (Table 1). Mixing of IC(1) and IIC(2)-Delta3 proteins resulted in G- and forskolin-stimulated activity (Table 1, Fig. 3, A and B). As it did for IC(1)IIC(2), GTPS-G acted synergistically with forskolin in activating mixed IC(1) and IIC(2)-Delta3, while 2`-d-3`-AMP inhibited the activity (Fig. 3, C and D). NaCl inhibited the activity of mixed IC(1) and IIC(2)-Delta3 (IC = 300 mM, not shown). The highest turnover number for adenylyl cyclase activity of mixed IC(1) and IIC(2)-Delta3 (when activated by G and forskolin simultaneously) was 8.2 s, similar to rates of the purified native and recombinant type I adenylyl cyclase(19, 25, 26) . Thus, the purified soluble adenylyl cyclase has the proper catalytic and regulatory properties and could be used as a model system for the biochemical and structural analysis of mammalian adenylyl cyclase.




Figure 3: Enzyme activity of a mixture of IC(1) and IIC(2)-Delta3 (0.2 µg each). A, activation by forskolin; B, activation by G-GTPS; C, synergistic activation by G-GTPS and forskolin; D, inhibition by 2`-deoxy-3`-AMP. The concentration of GTPS-G is 200 nM in C. Sum (Fsk+G) is the sum of adenylyl cyclase activities observed in the presence of forskolin or GTPS-G alone; Fsk + G is adenylyl cyclase activity observed in the presence of both GTPS-G and forskolin. The means ± S.E. are representative of two experiments.



Increased concentrations of IIC(2)-Delta3 markedly increased adenylyl cyclase activity when added to a fixed amount of IC(1) (6 nM) (Fig. 4A). The half-saturable concentration (EC) of IIC(2)-Delta3 for forskolin- and G-GTPS-activated activity was 0.8 and 1.3 µM, respectively. When G and forskolin were used together, EC of IIC(2)-Delta3 fell about 10-fold to 0.08 µM. This suggested that the synergistic effects of G-GTPS and forskolin on enzyme activity reflected an increase in the affinity of IC(1) and IIC(2)-Delta3 for each other.


Figure 4: Complementation of adenylyl cyclase activity. A, complementation of IC(1) by IIC(2)-Delta3. B, complementation of IM(1)C(1)-(1-570) or IM(1)C(1)-(1-484) by IIC(2)-Delta3. C, complementation of IM(1)C(1) or IIM(2)C(2) by IC(1). Purified IC(1) (20 ng), or Sf9 cell membranes (20 µg) containing IM(1)C(1)-(1-570) or IM(1)C(1)-(1-484) were mixed with the indicated quantities of IIC(2)-Delta3 on ice for 10 min before the assay. Sf9 cell membranes containing IM(1)C(1) or IIM(2)C(2) (20 µg) were mixed with the indicated quantities of IC(1). Adenylyl cyclase assays were performed at 30 °C in the presence of 100 µM forskolin (A, Fsk, B, and C) or 100 µM forskolin and 200 nM GTPS-G (A, Fsk+G). The means ± S.E. are representative of two experiments.



Purified IC(1) or IIC(2)-Delta3 were subjected to gel filtration on Pharmacia Superdex 200 using TE(1)D(1)N as the buffer. A major peak of adenylyl cyclase activity consistent with a globular 30-kDa protein was observed, half of the size for the enzyme activity of lysates containing IC(1)IIC(2) (Fig. 5A). The molecular size did not shift for the mixture of IC(1) and IIC(2)-Delta3, presumably due to the low affinity between two molecules (Fig. 5A).

To investigate whether a complex of IC(1) and IIC(2)-Delta3 could be detected, gel filtration was performed in the presence of forskolin under the conditions for the enzyme assay (forskolin, 1 mM ATP, and 10 mM MgCl(2) and the minimal concentration (100 mM) of NaCl; Fig. 5B). A major peak of adenylyl cyclase activity consistent with a globular 30-kDa protein was observed when the purified IIC(2)-Delta3 was applied alone, half of the size for the enzyme activity of lysates containing IC(1)IIC(2). When IC(1) alone was applied, a major peak of adenylyl cyclase activity consistent with a globular 55 kDa was observed; the shift from 30- to 55-kDa protein was due to the lower concentration of NaCl. This suggested that IC(1) might exist as a dimer or as a nonglobular protein at lower salt concentrations (100 mM). When the mixture of IC(1) (1 µg) and IIC(2)-Delta3 (50 µg) was tested, a peak of adenylyl cyclase activity consistent with 45-kDa proteins was observed. The shift in elution profile suggested that IC(1) and IIC(2)-Delta3 did interact. The low apparent molecular mass (45 kDa instead of the expected 60 kDa) could be accounted for by dissociation of the complex of IC(1) and IIC(2)-Delta3 and/or an unusual shape of the complex.

Complementation of IC(1) and IIC(2)-Delta3 by the Halves of Adenylyl Cyclases

Although IC(1) and IIC(2) did form a complex with adenylyl cyclase activity, we failed to detect enzyme activity when two cytoplasmic domains from type I enzyme (IC(1) and IC(2)) were used (linked or a mixture of IC(1) and IC(2)) (not shown). To investigate the relative affinity of IC(1) for either IC(2) or IIC(2), we tested the ability of IC(1) to complement the carboxyl-terminal half of type I or type II enzyme (IM(2)C(2) and IIM(2)C(2)) (Fig. 4C). As reported previously, the truncation mutants of type I and type II adenylyl cyclases that consisted of either the amino-terminal half (INM(1)C(1)-(1-570) and INM(1)C(1)-(1-484)) or the carboxyl-terminal half (IM(2)C(2) and IIM(2)C(2)) of the protein had no detectable adenylyl cyclase activity when expressed alone; however, coexpression of the amino- and the carboxyl-terminal halves resulted in G- and forskolin-regulated adenylyl cyclase activity (Fig. 1A)(21) . IC(1) and IIC(2)-Delta3 each had no adenylyl cyclase activity alone, and Sf9 cell membranes containing the amino- or carboxyl-terminal halves of adenylyl cyclase had enzymatic activities that were similar to the control cell membranes (that containing beta-galactosidase) (Fig. 4, B and C). When IIC(2)-Delta3 was added to Sf9 cell membranes containing either INM(1)C(1)-(1-570) or INM(1)C(1)-(1-484), there was up to a 40-fold increase in forskolin-stimulated adenylyl cyclase activity (Fig. 4B). In contrast, there was no increase in enzyme activity when IIC(2)-Delta3 was mixed with Sf9 cell membranes containing IM(2)C(2). When IC(1) was reconstituted with Sf9 cell membranes containing IIM(2)C(2), there was up to a 20-fold increase in forskolin-stimulated adenylyl cyclase activity (Fig. 4C). However, only a 3-4-fold increase in enzyme activity was observed when IC(1) was reconstituted with Sf9 cell membranes containing IM(2)C(2). The EC of IC(1) to reconstitute adenylyl cyclase activity of IIM(2)C(2) was at least 10-fold lower than that of IM(2)C(2).

There has been considerable speculation about the roles of the transmembrane domains of adenylyl cyclases(12) . The transmembrane domains target adenylyl cyclase to the plasma membrane for interaction with, and thereby regulation by, G proteins. Our studies indicate that the two cytoplasmic domains of mammalian adenylyl cyclases do not appear to have high affinity for each other. EC for IIC(2) to complex with IC(1) is 0.8 and 1.3 µM in forskolin- and G-stimulated activity, respectively.

Affinity between two natural linked cytoplasmic domains (IC(1) and IC(2)) is at least 10-fold less than that between IC(1) and IIC(2). Thus, the transmembrane domain (M(2)) could link and facilitate the interaction of the two cytoplasmic domains by creating a high local concentration. It remains to be determined whether the transmembrane domains have additional functions, such as altering the interaction between two cytoplasmic domains for regulations or serving as pore structures.


FOOTNOTES

*
This work was supported by the Cancer Research Foundation and Brain Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacological and Physiological Sciences, University of Chicago, 947 E. 58th St., Chicago, IL 60637. Tel.: 312-702-4331; Fax: 312-702-3774.

(^1)
The abbreviations used are: G, the alpha subunit of the G protein that stimulates adenylyl cyclase; Fsk, forskolin; GTPS, guanosine 5`-3-O-(thio)triphosphate; 2`-d-3`-AMP, 2`-deoxyadenosine 3`-monophosphate; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s); FPLC, fast protein liquid chromatography; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; HA1, hemagglutinin of influenza virus. Buffer compositions: T = 20 mM Tris-HCl, pH 8.0 at 4 °C; beta(5) = 5 mM beta-mercaptoethanol; D(1) = 1 mM DTT; E(1) = 1 mM EDTA; N = 100-500 mM NaCl; P = 0.1 mM phenylmethylsulfonyl fluoride; I = 20-150 mM imidazole, pH 7.0.


ACKNOWLEDGEMENTS

We thank Susan Gottesman for providing SG22094 and SG21163, Richard Lerner for hybridoma, 12CA5, and Chester Drum, Wolfgang Epstein, and Mitchel Villereal for their helpful suggestions.


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