(Received for publication, October 4, 1996, and in revised form, January 23, 1997)
From the Department of Physiology and Biophysics,
State University of New York, Health Sciences Center, Stony Brook,
New York 11794-8661, the
Department of Biological Chemistry,
University of Michigan Medical School, Ann Arbor, Michigan 48109, the
** Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania
17822, and the
Department of Pharmacology,
Mt. Sinai School of Medicine, City University of New York,
New York, New York 10029-6574
Recombinant adenylyl cyclase isozyme Types I, II,
VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3-polyphosphates (Désaubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028-14034). Inhibitory potencies were dependent on isozyme type, the
mode of activation of the respective isozymes, and on P-site ligand.
For the nucleoside derivatives potency typically followed the order
2
,5
-dideoxyadenosine (2
,5
-ddAdo) >
-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade)
9-(tetrahydrofuryl)-adenine
(9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase,
which was essentially insensitive to inhibition by 9-CP-Ade. For the
adenine nucleoside 3
-polyphosphates inhibitory potency followed the
order Ado < 2
-dAdo < 2
,5
-ddAdo and 3
-mono- < 3
-di- < 3
-triphosphate. Differences in potency of these ligands were noted
between isozymes. The most potent ligand was 2
,5
-dd-3
-ATP with
IC50 values of 40-300 nM. The data demonstrate
isozyme selectivity for some ligands, suggesting the possibility of
isozyme-selective inhibitors to take advantage of differences in P-site
domains among adenylyl cyclase isozymes. Differential expression of
adenylyl cyclase isozymes may dictate the physiological sensitivity and
hence importance of this regulatory mechanism in different cells or
tissues.
Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a domain referred to as the P-site from its requirement for an intact urine moiety (1-4). Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5, 6), inhibition kinetics (7, 8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain. The observation that purified native and recombinant Type I adenylyl cyclases are inhibited by P-site ligands, although exhibiting decreased sensitivity to inhibition (4-6, 11), establishes the locus of the P-site on the enzyme per se and that inhibition is not via cell surface receptors or G-proteins.
P-site-mediated inhibition has been characterized pharmacologically (1,
2, 4, 12-16). Inhibition requires an intact adenine moiety, and
potency of inhibition is increased substantially for deoxyribose and
especially 3-phosphorylribose adenine nucleosides. Inhibitory potency
follows the order: 3
-mono- < 3
-di < 3
-triphosphate and
adenosine (Ado) < 2
-deoxy (d)1-Ado < 2
,5
-ddAdo, with 2
,5
-dd-3
-ATP being the most potent ligand and
exhibiting an IC50 ~40 nM (15). In addition,
tolerance for large substitutions at the 3
-position and for other
ribose modifications has been demonstrated (1, 2, 4).
We reported previously that levels of 2-d-3
-AMP and 3
-AMP varied
considerably in different tissues and were dependent on the metabolic
state of the animal (17). Moreover, sensitivity of adenylyl cyclases to
inhibition by these nucleotides varied among rat tissues. We suggested
that diversity in P-site-mediated inhibition depended on the adenylyl
cyclase isozyme and that this diversity may well dictate differences in
pharmacological and possibly physiological influence of this inhibition
in the respective tissues (17). This is also suggested from studies in
which P-site agonists, e.g. 2
,5
-ddAdo or 9-THF-Ade, have
been used to alter function in a variety of cell systems,
e.g. (14, 18-23). Although effects on cell function and on
cellular cAMP levels have been uniformly consistent with P-site
inhibition of adenylyl cyclase, potencies of a given compound differed
among systems, and unexpected potencies of some ligands were noted in
others. Since it was not possible in those earlier studies to establish
which adenylyl cyclase isozyme(s) was(were) present, it is possible
that variations in expected behavior may well have been due to
differences in levels of expression of the several isozymes.
To evaluate this diversity in response, we are reporting the first
investigation of P-site-mediated inhibition of several recombinant
types of adenylyl cyclase. This study compares potency of several
P-site ligands to inhibit Type I (24), Type II (25), Type VI (26), Type
VII (27), and three splice variants of Type VIII (28, 29) adenylyl
cyclases. These adenylyl cyclase isozymes were selected because they
represent forms of the enzyme exhibiting distinct regulatory
characteristics (30). For example, all are activated by
Gs but Types I and VIII are activated also by
Ca2+/calmodulin. When Type I enzyme is activated by
Gs
it is inhibited by G
(11, 31, 32), whereas when
Type II adenylyl cyclase is activated by Gs
its activity
is increased further by G
(31, 32). The Type VI enzyme responds
to Gs
(26) but is not activated by
Ca2+/calmodulin or G
.
Both detergent-solubilized and particulate preparations of adenylyl cyclase from rat and bovine brains were prepared and assayed as described previously (4, 5, 8, 9, 17). Enzyme assays were conducted in duplicate or triplicate. IC50 values were derived from inhibition curves comprising six to eight concentrations of inhibitor in two to eight experiments for each condition.
Preparation of Expressed Recombinant Adenylyl CyclasesMembranes and membrane extracts were prepared as described previously (25, 26, 32) from fall army worm ovarian (Sf9) cells infected with baculovirus encoding Type I, Type II, or Type VI adenylyl cyclase or vector alone (thyroid peroxidase controls). Cells were lysed by N2 cavitation, and the particulate fraction was collected by centrifugation. The membranes from Sf9 cells encoding Type I adenylyl cyclase (32) were resuspended in a buffer containing 20 mM HEPES, pH 8, 1 mM EDTA, 6% sucrose, 5 mM MgCl2, and 1 mM dithiothreitol. Membranes from Sf9 cells encoding Type II, Type VI, and thyroid peroxidase (25, 26) were resuspended in a buffer containing 20 mM HEPES, pH 8, 1 mM EDTA, 2 mM dithiothreitol, 200 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of leupeptin and aprotinin. Detailed procedures for the preparation of Type II and Type VI enzyme have been described (33). All membranes were diluted at least 10-fold with a buffer containing 20 mM HEPES, pH 8, and 5% glycerol before being diluted further into the adenylyl cyclase assay containing the same buffer.
Enzyme Type VII (27) and the splice variants of Type VIII (29) were
expressed in HEK 293 cells (ATCC CRL 1573) as described previously. HEK
cells expressing Type VII enzyme were washed in phosphate-buffered
saline (prepared from Sigma tablets P-4417), scraped from dished into a
small portion of the buffered saline, collected by centrifugation, and
then stored at 80 °C. Membranes were prepared following lysing of
the cells by N2 cavitation as described previously
(28).
9-Cyclopentyl-9H-adenine was synthesized by amination of the corresponding 6-chloropurine that was itself prepared by alkylation, as adapted from Montgomery and Temple (34). A mixture of 6-chloropurine (5.57 g, 36 mmol), cyclopentyl bromide (6.56 g, 44 mmol), and K2CO3 in dimethyl sulfoxide (80 ml) was stirred at ambient temperature for 3 days. The reaction medium was diluted with water (700 ml), extracted with EtOAc (3 × 150 ml), washed with brine, dried over MgSO4, and purified by flash chromatography on silica gel; elution was with EtOAc:hexane (9:1) yielding 4.10 g (51%) of 6-chloro-9-cyclopentyl-9H-purine. A solution of 6-chloro-9-cyclopentyl-9H-purine (0.67 g, 3 mmol) and ammonia (20 mmol) in ethanol was heated at 90 °C for 24 h. The medium was evaporated in vacuo, and the residue was dispersed in brine (20 ml), extracted with EtOAc (3 × 30 ml), washed with brine, dried over MgSO4, and purified by flash chromatography on silica gel, eluting with EtOAc:MeOH (95:5), yielding 0.36 g (59%) of 9-CP-Ade. The methanesulfonate salt was prepared by adding one equivalent of methanesulfonic acid to a solution of the free base in isopropyl alcohol. The crude salt was recrystallized from isopropyl alcohol.
Adenosine 33-ADP was synthesized by a modification of the methods
described by Mitchel et al. (35) and Sheridan et
al. (36) (Fig. 1). The principal modifications are
the use of sonication to facilitate the reaction, which otherwise would
have been allowed to continue for 150 h (35), and the use of anion
exchange chromatographic techniques to separate the several
polyphosphorylated derivatives of adenosine obtained. This modified
procedure represents a one-step preparation of nucleoside 3
-di- and
3
-triphosphates from the corresponding monophosphate and is
characterized by a short reaction time and convenient reaction
conditions.
A suspension of 3-AMP (1 g, 2.8 mmol) and phosphoramidic acid (1.63 g,
16.8 mmol) in 50 ml of formamide was sonicated under an argon
atmosphere overnight. The temperature was maintained at 20-25 °C by
putting the ultrasonication bath in a cold box at 4 °C. The medium
was cooled to 5 °C, diluted with 2 liters of cold water (5 °C),
and neutralized with triethylamine (2.8 ml, 20 mmol). Reaction products
were purified on QAE-Sephadex (HCO3
form) with a linear
gradient of triethylammonium bicarbonate (0.01-0.3 M)
followed by an isocratic elution with 0.3 M
triethylammonium bicarbonate and then another linear gradient with
triethylammonium bicarbonate (0.3-1 M) (Fig.
2). The appropriate fractions were lyophilized and then
coevaporated several times with methanol, yielding 380 µmol (14%) of
3
-ADP and 14.6 µmol 3
-ATP. Both nucleotides were isolated as their
respective sodium salts by precipitation in 1 M sodium
iodide in acetone from the methanol solution of the triethylammonium
nucleotide. The precipitate was centrifuged and washed three times with
cold acetone and dried in vacuo giving the sodium salts of
3
-ADP and 3
-ATP. No impurities were noted by high performance liquid
chromatography on a Spherogel TSK DEAE-5PW column eluted with a
gradient of triethylammonium bicarbonate. The following spectra are for
3
-ADP. 1H NMR (D2O)
3.81 (d, 2H,
J = 2.8 Hz, H-5
and H-5"), 4.62 (m, 1H, H-4
), 4.90 (m, 1H, H-3
), 5.87 (d, 1H, J = 6.5 Hz, H-1
), 8.13 (s,
1H, H-2), 8.29 (s, 1H, H-8). 31P NMR (D2O)
5.94 (dd, JP-H = 7.6 Hz,
JP-P = 20.6 Hz, P-1),
1.34 (d,
J = 20.5 Hz, P-2). NMR spectra were recorded with a Bruker AC250 at 250 MHZ for proton spectra and at 101 MHZ for 31P spectra, with a 85% solution of
H3PO4 as external standard.
Materials
[-32P]ATP was purchased from ICN
Pharmaceuticals. Lubrol-PX was filtered through alumina (Neutral, AG7,
from Bio-Rad) to remove peroxides. Dimethyl sulfoxide was redistilled
under partial vacuum over CaH2. 2
,5
-ddAdo and the
3
-polyphosphates of 2
,5
-ddAdo and of 2
-dAdo were synthesized by
methods reported previously from this laboratory (9, 15, 16). 9-THF-Ade
(SQ 22,536) was a gift from Salvatore Lucania, Bristol-Myers-Squibb,
Pharmacology Research Institute, P.O. Box 4000, Princeton, NJ
08543.
Previously reported data suggested
tissue-dependent differences in inhibitory potency of
P-site ligands and also showed that activation of the enzyme by various
agents resulted in different sensitivity of the enzyme to inhibition
(17). To ascertain whether these differences would be reflected in
specific isozymes, the potency of 2-d-3
-AMP was evaluated on
recombinant bovine Type I, Type II, and Type VI adenylyl cyclases (Fig.
3 and Table I). IC50 values
for inhibition of the rat brain2 enzyme by
2
-d-3
-AMP are shown for comparison. As expected, various stimulatory
agents resulted in substantially different activities, with the
greatest activity being uniformly obtained when assays were conducted
with 5 mM Mn2+ and 100 µM
forskolin. Reaction velocities with Mg2+ and forskolin were
roughly similar to those noted with Mn2+ alone, and
membranes assayed with Mg2+ exhibited the lowest
activities. Furthermore, the Type I enzyme was most potently inhibited,
whether activation was with Mn2+ (IC50 ~4
µM) or Mn2+ and forskolin (IC50
~6 µM). This greater sensitivity of the Type I enzyme
is consistent with previously published data with enzyme derived from a
number of tissues (1-4, 13, 14, 17). The small increase in
IC50 upon addition of forskolin is also consistent with
earlier experiments with the bovine brain enzyme (5) but is in contrast
with behavior of hepatic adenylyl cyclase (7) and the Type VI enzyme.
Although forskolin stimulated each adenylyl cyclase, its effect to
enhance potency of P-site ligands varied among isozymes. Hence, potency
is uncoupled from enzyme activity, suggesting that the processes of
catalysis and inhibition might not be causally linked.
|
One goal of these studies has
been to identify P-site ligands that could be used pharmacologically on
intact cells or tissues and exhibit isozyme-selectivity and possibly
tissue selectivity for inhibition of adenylyl cyclase. That such tissue
selectivity may occur is suggested from studies with a number of
tissues and with the several recombinant isozymes presented above.
Structure-activity relationships for P-site ligands have indicated a
tolerance for ribose substitutions and replacements. Several of the
membrane-permeable compounds that have been used by many investigators
in studies with intact cells and tissues, and these include:
9-(-d-arabinofuranosyl)adenine (Ara-Ade; IC50 ~100
µM) (1), 9-THF-Ade (IC50 ~10
µM; SQ 22-536), and 9-CP-Ade (IC50 ~20
µM) (13). These compounds were tested with several
recombinant adenylyl cyclases, and significant differences in
sensitivity were noted (Table II). The general order of
sensitivity to P-site-mediated inhibition was Type I > Type
VI
Type VIIIA > Type II. For the classical P-site
ligands, 2
-d-3
-AMP and 2
,5
-ddAdo there were smaller differences in
ligand potency among enzyme Types I, VI, and VIIIA than were noted with
the ribose-substituted ligands. For these ligands, each was
significantly less potent with the Type II enzyme than with the other
isozymes. An interesting compound was 9-CP-Ade, which was essentially
inactive with the Type II enzyme (Fig. 4). There was
greater than 1 order of magnitude difference in potency of 9-CP-Ade for
the Type II adenylyl cyclase (IC50 >1 mM)
compared with Type I (IC50 ~109 µM) and
Type VIIIA (IC50 ~117 µM) (Table II and
Fig. 4). This selectivity ratio of >10 is likely an underestimate
since in some experiments the IC50 for 9-CP-Ade was >4
mM, giving a selectivity ratio >40.
|
3
Although the above data indicate some
isozyme selectivity for the ribose-modified set of P-site ligands, less
pronounced differences in sensitivity were noted with the adenine
nucleoside 3-polyphosphates (Table III). Typically,
potency followed the order Ado < 2
-dAdo < 2
,5
-ddAdo and
for the 3
-phosphates: 3
-mono- < 3
-di- < 3
-triphosphate (cf. Refs. 15 and 16). A comparison is shown in Fig.
5 for inhibition of the Type VI adenylyl cyclase.
Although some differences were evident among the isozymes scanned in
this study, e.g. Type II enzyme was least sensitive to
P-site ligands, in general the more potent the compound the less
selectivity was noted. For some isozymes the impact of the removal of
the hydroxyl moieties was greater that with others. For example, the
IC50 values for the 2
-deoxy- and 2
,5
-dideoxynucleotides
for enzyme Types VII and VIIIA are little different, whereas for the
Type II enzyme the difference is 1 order of magnitude (cf.
Table III). Analogously, the effect on inhibitory potency of the
sequential addition of phosphates to the 3
-position differed among
these isozymes. For the 2
-deoxy series the largest difference in
IC50 values for the Type II enzyme was between the 3
-di-
and 3
-triphosphate derivatives, whereas for the Type VI enzyme it was
between the 3
-mono- and 3
-diphosphate derivatives (Table III and Fig.
5). For enzyme Types VI, VII, and all three splice variants of Type VIII, IC50 values were little affected by the addition of
the third 3
-phosphate group. The data suggest that inhibition by this
class of ligand is a universal feature of adenylyl cyclase isozymes but
that the structure of the respective isozymes influences potency of
ligands, whether with ribosyl 2
- and 5
-hydroxyl or 3
-mono-, 3
-di-,
or 3
-triphosphate groups.
|
To begin establishing a basis for the known tissue dependence for
inhibition of adenylyl cyclases by P-site ligands, the efficacy of
several ribose-modified and 3-polyphosphorylated adenosines has been
tested on several recombinant forms of this enzyme family. The
rationale for the study is that the physiological importance of this
regulatory mechanism in a given tissue may be reflected in the
sensitivity of the adenylyl cyclase expressed in that tissue. The
degree of inhibition would depend on levels of P-site ligand, and these
may change as a result of changes in cell function. Our additional
consideration has been that ligands may be identified exhibiting
selectivity toward individual isozymes.
The influence of P-site inhibition in a given tissue may also be
influenced by factors affecting activity of adenylyl cyclases. Although
the data did not conform to the notion that sensitivity to inhibition
by P-site ligands increased as enzyme activity increased (7), they
indicated that different means of activation of adenylyl cyclase led to
differences in the IC50 values for inhibition
(cf. Table I). For example, enzyme activity with
Mn2+ + forskolin was uniformly greater than that with
Mn2+ alone, whereas the presence of forskolin led to
increased sensitivity to inhibition by 2-d-3
-AMP (Table I) or
2
,5
-ddAdo (not shown) with the Type VI enzyme but decreased
sensitivity with Types I and II enzyme. Arguably the enzyme
conformation resulting from activation by Mn2+ must differ
from those resulting from Mn2+ + forskolin,
Mg2+, GTP·
s, GTP·
i,
, and appropriate combinations of these. Different conformations
will exhibit greater or lesser sensitivity to inhibition by these
agents, and this influence will be isozyme-dependent and
may affect the physiological role of this inhibition.
Potency of P-site ligands obviously depends also on ligand structure.
This was particularly evident with the ribose-modified adenosine
derivatives as the comparison was extended to several isozymes (Table
II). For example, Type II adenylyl cyclase, unique in its regulation by
G-protein subunits and susceptibility to phosphorylation by protein
kinase C (31-33), was insensitive to inhibition by 9-CP-Ade and was
poorly sensitive to 9-THF-Ade. The differences in P-site potency among
the isozymes were more acutely noted with ribose-modified adenine
nucleosides than with the 3-nucleotides. The addition of the
3
-phosphates contributes substantial binding energy (10 kcal/phosphate; Ref. 37) and may minimize the effects of differences in
isozyme structure on ligand binding; that is, the differences among
isozymes, noted by IC50 values for 9-CP-Ade (Fig. 4), may
be masked by the increased binding affinities of the nucleoside
3
-polyphosphates (Fig. 5). An avenue for the development of
isozyme-selective ligands may be to exploit the differences established
here with the ribose-modified adenine nucleosides and the
dramatic inhibitory potency of the adenine nucleoside
3
-polyphosphates, thereby also aiding investigation of enzyme
structure and mechanisms of catalysis and inhibition.
We thank Dr. J. Marecek for helpful discussions.