Báo cáo khoa học: Dimerization of mammalian adenylate cyclases Functional, biochemical and ¯uorescence resonance energy transfer (FRET) studies pot

PRIORITY PAPER
Dimerization of mammalian adenylate cyclases
Functional, biochemical and ¯uorescence resonance energy transfer (FRET) studies
Chen Gu
1
, James J. Cali
2
and Dermot M. F. Cooper
1,3
1
Neuroscience Program University of Colorado Health Sciences Center, Denver, CO, USA;
2
Promega Corp., Madison, WI, USA;
3
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO, USA
1
Mammalian adenylate cyclases are predicted to possess
complex topologies, comprising two cassettes of six trans-
membrane-spanning motifs followed by a cytosolic, catalytic
ATP-binding domain. Recent studies have begun to provide
insights on the tertiary assembly of these proteins; crystal-
lographic analysis has revealed that the two cytosolic
domains dimerize to form a catalytic core, while more recent
biochemical and cell biological analysis shows that the t wo
transmembrane c assettes also associate to facilitate the
functional assembly and tracking of the enzyme. The older
literature had suggest ed that adenyla te c yclases m ight form
higher order aggregates, although the methods used did not
necessarily provide convincing evidence of biologically
relevant events. In the present study, we have pursued this
question by a variety of approaches, including rescue or

suppression of function by variously modi®ed molecules,
coimmunoprecipitation and ¯uorescence resonance energy
transfer (FRET) analysis between molecules in living cells.
The results strongly suggest that adenylate cyclases dimerize
(or oligomerize) via their hydrophobic domains. It is
speculated that this divalent property may allow adenylate
cyclases to participate in multimeric signaling assemblies.
Keywords: adenylate cyclase; dimerization; ¯uorescence
resonance energy transfer; green ¯uorescent protein;
immunoprecipitation.
The interjection of stimulatory and inhibitory G-protein
modules between receptors and effector increased the
complexity of the a denylate cyclase signaling system, while
at the same time greatly expanding the perceived, regulatory
responsiveness of these systems [1]. Coincident with the
discovery of an increased number of signaling components,
Rodbell and colleagues proposed that these elements
occurred in higher order a ssemblies t han a simple mono-
meric a rrangement of receptor, G protein and effector.
Using radiation inactivation analysis, Schlegel et al.pro-
posed that adenylate cyclase existed in dynamic, multimeric
protein arrays of receptors, G proteins and ade nylate
cyclases [2,3]. Independent, hydrodynamic analyses of
detergent-solubilized adenylate c yclase preparations also
indicated molecular m asses of a bout 2 20 kDa for the
catalytic units [4±7], which, given the minimal protein
molecular masses of  120 kDa, again sugge sted a higher
order assembly of adenylate cyclases. Mammalian adenylate
cyclases are in the family of ATP-binding cassette (ABC)
transporters and share their overall structure [8] which, by

analogy, further raises the possibility that they might
multimerize. Many members of this family, such as the
transporters for glutamate [9], glucose [10] and serotonin
[11] are oligomeric. These proteins can f orm more complex,
heterooligomeric structures with more elaborate functions.
For instance, the cystic ®brosis transmembrane conduct-
ance regulator (CFTR), forms a dynamic macromolecular
complex, in which a PDZ domain-containing protein
(CAP70) facilitates CFTR±CFTR interaction to potentiate
chloride channel activity [12]. Another member of this
superfamily, the sulfonylurea receptor (SUR) associates
with inward ly rectifying K
+
(K
ir
) channel subunits to form
ATP-sensitive K
+
channel complexes, which contain four
subunits each of SURs and K
ir
[13,14].
Adenylate cyclase is now known to be capable of
intramolecular dimerization. The molecule is a twice-
repeated motif of six-transmembrane segments followed
by a cytosolic ATP-binding domain . These two ATP-
binding domains are highly homologous, and they must
associate for catalytic activity and regulation by G-proteins
[15,16]. The crystal structure of t hese catalytic domains has
been solved [17,18]. Dimerization between catalytic domains

is even preserved in the much simpler trypanosomal
adenylate cyclase, which possesses a single transmembrane
spanning segment [19]. Recently, we showed by a variety of
functional and imaging techniques that the two transmem-
brane clusters, quite independently of the cytosolic compo-
nents, interacted persistently, which dictated the traf®cking
and functional assembly of adenylate cyclase, AC8 [20]. The
interaction between the transmembrane domains was
isoform speci®c, as the ®rst transmembrane domain o f
Correspondence to D. M. F. Cooper, Department of Pharmacology,
Box C-236, University of Colorado Health Science Center, 4200 East
Ninth Ave, Denver, CO 80262, USA. Fax: + 303 315 7097,
Tel.: + 303 315 8964, E-mail:
Abbreviations: CFTR, cystic ®brosis transmembrane conductance
regulator; SUR, sulfonylurea receptor; FRET, ¯uorescence resonance
energy transfer; PVDF, poly(vinylidene di¯uoride); GFP, green
¯uorescent protein; YFP, yellow ¯uorescent protein; CFP, cyan
¯uorescent protein; ABC, ATP-binding cassette; CCE, capacitative
Ca
2+
-entry.
(Received 12 November 2001, accepted 28 November 2001)
Eur. J. Biochem. 269, 413±421 (2002) Ó FEBS 2002
AC8 did not cotraf®c to the plasma membrane with t he
second transmembrane domains of AC2 and AC5. This
latter conclusion was arrived at independently by functional
assays [21]. I n our exper iments, we wer e also intrigued to
®nd that the second set of transmembrane segments
homodimerized strongly, although they were retained in
the ER. These observations, along with the earlier bio-

chemical data, prompted us to consider the possibility that
adenylate cyclases might dimerize .
Here, we have used a variety of approaches ranging from
either suppression or rescue of function by inactive or active
partial molecules, respectively, intermolecular coimmuno-
precipitation and ¯uorescence resonance e nergy t ransfer
(FRET) between partial and full-length cyan ¯uorescent
protein (CFP)- and yellow ¯uorescent protein (YFP)-tagged
molecules i n live cells to search for persistent and intimate
interactions. These studies lead us to conclu de that mam-
malian adenylate cyclases do form dimers (or higher o rder
assemblies) the regions responsible are the hydrophobic
domains and this aggregation may contribute to the associ-
ation of adenylate cyclases with cellular regulatory factors.
MATERIALS AND METHODS
cDNA plasmid constructs and cell culture
Portions of AC8 were subcloned into N-terminal or
C-terminal enhan ced green ¯uorescent protein (GFP) vectors
(Clontech) using convenient restriction enzyme digestion
sites or PCR-based strategies, as described previously [20].
In AC8
D582)594
, a region from Y582 to L594 in the C1
domain of AC8 was deleted; in AC8
D11 26)1248
[20], a region
from R1126 to P1248 in the C-terminus of AC8 (in the C2
domain) was deleted; in AC6
D553)666
, a region from S553 to

F666 in the C1 domain of AC6 was deleted. These three
deletions were generated by a PCR-based strategy. GFP/
AC8, GFP/8Tm2C2, 8NTm1C1/GFP, GFP/8Tm2, CFP/
8Tm2, YFP/8Tm2, GFP/C2, 8NTm1/GFP, GFP/8C1 and
8NTm1 were as described in [20]. CFP/AC8 and YFP/AC8
were obtained by switching the GFP of GFP/AC8 into CFP
and YFP between the restriction enzyme sites Nhe1and
BglII, from pECFP and pEYFP vector (Clontech). 8Tm1/
CFP/Tm2 w as obtained b y subcloning 8NTm1 of 8NTm1/
CFP between the restriction enzyme sites Nhe1andAge1,
which are both located right before the CFP of CFP/8Tm2.
Similarly, 8Tm1/YFP/Tm2 was obtained by subcloning
8NTm1 of 8NTM1/YFP into YFP/8Tm2. HEK 293 cells
were maintained as described previously [22].
Measurement of cAMP accumulation
In intact cells, cAMP accumulation was measured accord-
ing to the method of Evans et al. [23], as described
previously [22] with some modi®cations. Cells on 24-well
plates were incubated (60 min at 37 °C) with [2-
3
H]adenine
(1.5 lCi per well) to label the ATP pool. The cells were then
washed once and incubated with a nominally Ca
2+
-free
Krebs buffer (900 lL per well) containing 120 m
M
NaCl,
4.75 m
M

KCl, 1.44 m
M
MgCl
2
,11m
M
glucose, 25 m
M
Hepes, and 0.1% bovine serum albumin (fraction V)
adjusted to pH 7.4 with 2
M
Tris base. The use of Ca
2+
-
free Krebs buffer in experiments denotes the addition of
0.1 m
M
EGTA to the nominally Ca
2+
-free Krebs buffer.
All experiments were carried out at 30 °C in the presence of
phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine
(500 l
M
), and Ro 2 0±1724 (100 l
M
), which were p reincu-
bated with the cells for 10 m in prior to a 1-min assay. Cells
were preincubated for 4 min with the Ca
2+

-ATPase inhib-
itor, thapsigargin, at a ®nal concentration of 100 n
M
.This
treatment passively empties intracellular Ca
2+
stores,
establishing a low basal [Ca
2+
]
iand
primes the cells for
CCE [24]. Assays were terminated by addition o f 5% (w/v,
®nal concentration) trichloroacetic acid a nd the percent
conversion of [
3
H]ATP to [
3
H]cAMP was m easured as
previously described previously [22]. Means  SD of
triplicate determination are indicated.
GFP ¯uorescence imaging
The procedure w as described p reviously [20]. T ransfected
HEK 293 cells were plated on glass coverslips coated with
E-C-L cell Attachment Matrix (Upstate, Lake Placid, NY,
USA; 1 : 100 dilution, 2 h). Forty-eight hours after trans-
fection, the coverslips were loaded onto an Atto¯uor cell
chamber (Molecular Probes, Eugene, OR) and 0.5 mL
NaCl/P
i

(137 m
M
NaCl, 2.7 m
M
KCl, 10 m
M
Na
2
HPO
4
and 1.8 m
M
KH
2
PO
4
, pH 7.4) was added. Images were
captured at room temperature for GFP ¯uorescence
(excitation, 480/20 nm; emission, 510/20 nm). The ¯uores-
cence imaging workstation consisted of a Nikon Eclipse TE
300 microscope equipped with a 100 ´ 1.4 N.A. oil immer-
sion objective lens, thermoelectrically cooled charged-
coupled device Micromax 5 MHz camera (Princeton
Instruments), z-step motor and dual ®lter wheels controlled
by
SLIDEBOOK
3.0 software (Intelligent Imaging Innovation,
Denver, CO, USA). Binning 1 ´ 1 mode and 500 ms
integration times were used. The criteria for imaging
analysis was that only cells with medium and low expression

levels were captured and counted.
Co-immunoprecipitation and Western blotting
HEK 293 cells transfected with various constructs were
solubilized in 1 mL immunoprecipitation buffer ( 50 m
M
Tris/HCl (pH 7.4), 150 m
M
NaCl, 1% Triton X100 (or 1%
Nonidet P-40) and protease inhibitor cocktails) for 1 h at
4 °C,andthencentrifuged(100000g;Optima
TM
TL
ultracentrifuge, Beckman). The supernatant was incubated
(2±4 h, 4 °C) with 5 lg anti-(T7 tag) Ig (Novagen) and
100 lL protein A±agarose beads (Pierce). The beads were
washed three times with 1 mL immunoprecipitation buffer
plus 350 m
M
NaCl, once with 1 mL 50 m
M
Tris/HCl
(pH 7.4) and 150 m
M
NaCl, and eluted with 50 lL2´
sample buffer. The immunoprecipitates were resolved by
SDS/PAGE, transferred to a poly(vinylidene di¯uoride)
(PVDF) membrane, and subjected to Western blotting
using either Ab ACVIII-A 1229±1248 antibody (as des-
cribed previously [22]), or Living color peptide antibody
(Clontech, 1 : 100 dilution; as described previously [20]).

FRET measurements
The manipulation of cells expressing YFP- and CFP-tagged
proteins and imaging procedures were all t he same as those
for GFP imaging. FRET between CFP and YFP was mea-
sured and calculated for the entire image on a pixel-by-pixel
414 C. Gu et al. (Eur. J. Biochem. 269) Ó FEBS 2002
basis using a three-®lter ÔmicroFRETÕ method as described
previously [20,25]. Brie¯y, to measure FRET, three images
were acquired through YFP, CFP and FRET ®lter
channels. The raw FRET images consist of both FRET
and non-FRET components (the donor and acceptor
¯uorescence bleeding through the FRET ®lter). The extent
of cross-bleeding is characteristic of the particu lar optical
system and was determined using cells that express e ither
CFP/8Tm2 or YFP/8Tm2. In several experiments we
found that 55.3  0.8% of CFP and 1.28  0.06% of
YFP ¯uorescence can bleed through the FRET channel.
Therefore, to calculate the cross-over image, CFP and
YFP images were multiplied by, respectively, 0.565 and
0.014. Finally, the corrected FRET (FRET
C
) image was
obtained by subtracting CFP and Y FP cross-over images
from raw FRET images and is presented as a quantitative
pseudocolor image. All manipulations with images were
performed after subtraction of the background images.
RESULTS
Inactive mutant adenylate cyclases suppress
the activity of wild-type adenylate cyclases
in vivo

In a multimeric assembly requiring the integrity of the
whole complex for full function, it might be expected that
one inactive subunit would exert a dominant-negative
effect on activity. We evaluated this possibility with
adenylate cyclases, focusing largely on AC8, which can
be stimulated by Ca
2+
acting via calmodulin, b inding to
the C -terminus [22]. Issues of speci®city of intermolecular
interactions were add ressed w ith AC5 o r AC6, which are
inhibitable by Ca
2+
, apparently independently of calmod-
ulin [26]. Adenylate cyclases can be divided into ®ve major
domains, the N-terminus, the ®rs t transmembrane cluster
(Tm1), ®rst cytoplasmic loop (C1), second transmembrane
cluster (Tm2) and second cytoplasmic loop (C2) (see later).
The C1 and C2 regions are further subdivided into the
highly conserved catalytic C1a and C2a regions and the less
conserved C1b and C2b domains. In previous studies, by
deleting part of the C1 region we generated an inactive
mutant of AC8, termed AC8
D58 2)594
[22]. We wondered
whether this mutant might suppress the activity of
cotransfected wild-type AC8. Transfection of HEK 293
cells with wild-type AC8 resulted in a dramatic increase in
cAMP accumulation in response to forskolin, t he en try of
Ca
2+

triggered by store depletion (capacitative Ca
2+
-entry;
CCE) or especially the combination of forskolin and CCE
(Fig. 1A). Replacing half of the AC8 cDNA with empty
vector caused no drop in cAMP accumulation.
As expected, cAMP accumulation of HEK 293 cells
transfected with the inactive AC8 mutant, A C8
D58 2)594
,
was no different from that of cells transfected with empty
vector, regardless of the stimuli (Fig. 1A). However, when
cotransfected with wild-type AC8, AC8
D582)594
dramat-
ically suppressed activity under a ll stimulation c onditions
(Fig. 1A). These results are consistent with the formation
of homomultimeric complexes of AC8 molecules. We
wondered whether a similar approach might reveal that
heteromultimeric complexes could form between different
isoforms of adenylate cyclase. Consequently, cells were
transfected with combinations of inactive or active AC8
and active or inactive AC5 and AC6 cDNAs. The inactive
AC8, AC8
D582)594
, also suppressed the activity of AC5 and
AC6 (Fig. 1B). Conversely, AC8 activity was suppressed
by the corresponding, inactive mutant of AC6,
AC6
D55 3)666

(Fig. 1 B). These results are consistent with
the formation of heterodimers.
Mutants do not misdirect wildtype adenylate cyclase
The dominant negative effects of cotransfected adenylate
cyclase mutants on adenylate cyclase activity could also
arise from either a decreased expression or a misdirection of
the wild-type adenylate cyclase. To test whether the location
and/or the amount of wild-typ e A C8 expressed was altered
Fig. 1. Suppression of adenylate cyclase activity by inactive mutants.
(A) AC8 activity can be suppressed by the coexpression of
AC8
D582)594
. HEK 293 cells were transfected with the same total
amount of the indicated cDNAs. The cDNA ratio in the cotransfec-
tions was 1±1. Transfected HEK 293 cells w ere pretreated with
thapsigargin (1 00 n
M
for 4 min) to activate CCE. cAMP accumulation
in the intact cells was measured for 1 min after adding, vehicle (Basal);
20 l
M
forskolin (Forsk); 20 l
M
forskolin and 4 m
M
Ca
2+
(Forsk/
Ca
2+

); or 20 l
M
forskolin, 10 l
M
prostaglandin E
1
and 4 m
M
Ca
2+
(Forsk/PGE
1
/Ca
2+
). (B) Suppression by an inactive mutant can occur
with other adenylate cyclases. The cDNAs are shown under each bar.
Assays were performed as in (A). cAMP a ccumulation in the trans-
fected HEK 293 cells was measured for 1 min after adding, 20 l
M
forskolin and 10 l
M
prostaglandin E
1
for transfections involving AC5
and AC6; 20 l
M
forskolin, 10 l
M
prostaglandin E
1

and 4 m
M
Ca
2+
for transfections involving AC8.
Ó FEBS 2002 Dimerization of adenylate cyclases (Eur. J. Biochem. 269) 415
when AC8 was coexpressed with AC8
D58 2)594
,weemployed
a G FP-tagged form of AC 8, GFP/AC8, which resembles
the wild-type both in terms of catalytic activity and
appropriate location in the plasma membrane [20].
Co-transfection with AC8
D58 2)594
, did not alter the plasma
membrane localization of GFP/AC8 (Fig. 2A,B) and the
expression level was also apparently quite similar. However,
just as with the wild-type, the activity of GFP/AC8 was
suppress ed by coe xpressi on with AC 8
D582)594
(Fig. 2C).
Rescue of inactive mutants by half molecules
of AC8
in vivo
A corollary of the e xperiments described above involving
dominant negative suppression of adenylate cyclase activity
is the rescue of inactive, mutant mole cules by complement-
ary, partial molecules. Tang and colleagues had shown that
there was complementation of enzymatic activities between
truncated AC1 and inactive point mutations [27]. Those

experiments were performed with membranes prepared
from Sf9 cells expressing various baculovirus-encoded
constructs [27]. We wondered whether halves of AC8 could
rescue t he activ ity of A C8
D582)594
expressed in HEK 293
cells. P reviously, we described a C -terminus deletion of
AC8, AC8
D1126)1248
[22], which lacked part of the C2a
domain and the entire C2b region. AC8
D11 26)1248
is
completely inactive when expressed alone in HEK 293 cells,
as are AC8
D582)594
, GFP/8Tm2C2 (the eGFP tagged second
half of A C8) and 8NTm1C1/GFP (the eGFP tagged ®rst
half of AC8; Fig. 3 [ 20]). The cAMP accumulation of
HEK 293 cells transfected with these constructs alone was
around 0.1% when the cells were stimulated by forskolin
and CCE (Fig. 3). Cells cotransfected with the combination
of either AC8
D582)594
and G FP/8Tm2C2 o r A C8
D11 26)1248
and 8NTm1C1/GFP also only had background adenylate
cyclase activity (Fig. 3). In contrast, cAMP accumulation of
cells cotransfected with either AC8
D582)594

and 8NTm1C1/
GFP or AC8
D1126)1248
and GFP/8Tm2C2, approached
0.5%, under the same assay conditions (Fig. 3). This result
shows that complementation of activity can occur between
separate molecules, presumably b y generating a complete
catalytic core, which, of course, suggests intimate access
between these constructs. However, rescued activity is only
about one-tenth of the activity of the full length wild-type
AC8. This inef®cient c oupling between molecules might
suggest that the physical association between two catalytic
domains from the same molecule is preferred. Such
inef®ciency might also underlie the lack of detectable
adenylate cyclase activities from cells cotransfected w ith
AC8
D58 2)594
and AC8
D1126)1248
(Fig. 3).
Fig. 2. The activity but not the expression of
eGFP-tagged AC8 changed in the presence of
AC8
D582)594
. HEK 293 cells were transfecte d
with the same total amount of the indicated
cDNAs. The cDNA ratio was 1±1 in the
cotransfection. 24 h after transfection, half of
the cells were plated on coated glass coverslips
for eGFP-imaging; another half were plated in

24-well plates for in vivo assays. (A) Cells
transfected with GFP/AC8 + vector. (B) Cells
transfected with GFP/AC8 + AC8
D582)594
.
(C) Assays were performed as in Fig. 1. The
cAMP accumulation in these t ransfected cells
was measured for 1 min after adding vehicle
(Basal), 4 m
M
Ca
2+
(Ca
2+
), 20 l
M
forskolin
(Forsk), or 20 lm forskolin and 4 m
M
Ca
2+
(Forsk/Ca
2+
).
416 C. Gu et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Homo- and heteromeric interactions revealed
by coimmunoprecipitation assays
To test wh ether AC8 molecules actually bind to each
other and to AC6 molecules, we performed coimmuno-
precipitation assays with various epitope-tagged aden ylate

cyclase constructs. We previously described the mutant
AC8
D1)106, D1184)1248
or Ô8M13Õ, w hich lacks t he ®rst 106
residues at t he N-terminus and the last 65 residues at th e
C-terminus [22]. It is constitutively active, i ndependent of
Ca
2+
and is targeted correctly [20,22]. AC8
D1)106,
D1184)1248
has a T7 epitope tag at its N-terminus, but it
does not possess the epitope for the AC8 speci®c
antibody, Ab VIII-A, which is directed against amino
acids 1229±1248 [28]. We also constructed an AC6 with a
T7 tag at its N-terminus. Wild-type AC8 was cotransfect-
ed with either AC6 or AC8
D1)106, D1184)1248
into HEK 293
cells and 48 h later, coimmunoprecipitation assays were
performed (see Materials and methods). The T7 tag
antibody was used to pull down the AC6 or AC8
D1)106,
D1184)1248
, r espectively, along with any associated proteins,
which were then r un on SDS/PAGE and transferred t o a
PVDF membrane. Ab VIII-A 1229±1248 antibody was
used in the W estern blots to determine whether AC8 was
present in association with either AC6 or AC8
D1)106,

D1184)1248
. Indeed, AC8 did associate with AC8
D1)106,
D1184)1248
, and to a lesser extent with AC6 (Fig. 4A). AC8
immunoreactivity was not detected in coimmunoprecipi-
tations from any single transfection (Fig. 4A). Thus, these
data are also consistent with the occurrence of both
heteromeric and homomeric interactions between adeny-
late cyc lase molecules.
To narrow down the region of interaction between
adenylate cyclase molecules, we cotransfected HEK 293
cells with AC8
D1)106, D1184)1248
and GFP-tagged parts of
Fig. 3. Inactive mutants can be partially rescued by halves of AC8
in vivo. Top: diagram o f the constructs; the green box represen ts t he
GFP molecule, the 12 small black bars represent the 12 putative
transmembrane segments of AC8. The names of the domains of AC8
are above the G FP/AC8 in the corresponding region. A ssays were
performed as in Fig. 1. cAMP accumulation was measured by adding
20 l
M
forskolin and 4 m
M
Ca
2+
. E xperiments were performed three
times with similar results. The asterisks indicate value that are
signi®cantly dierent from th e backgroun d (P < 0.05). The cDNAs

transfected are indicated by the plus signs.
Fig. 4. Homo- and heteromeric interactions between adenylate cyclases
revealed by coimmunoprecipitation assays. Immunoprecipitations were
performed wit h a nti-(T7 t ag)Ig. O nly A C8
D1)106, D1184 )1248
and AC6
have a T7 tag at their N-terminal. The immunoprecipitated proteins
were run on SDS/PAGE and transferred onto PVDF m embranes.
Cotransfectio n conditions are indicated on the top of each blot;
molecular mass (kDa) is shown on the left of the blot. (A) The asso-
ciation b etween AC8 and either AC8
D1)106, D1184)1248
or AC6 was
tested. Ab ACVIII-A 1229±1248 antibody was used in the Western
blotting. (B) T he assoc iatio n betwe en AC 8
D1)106, D1184)1248
and
dierent parts of AC8 was tested. Living color peptide antibody
recognizing the eGFP molecule was used in the Western blotting. The
asterisk indicates the position of a no nspeci®c antibody band. The
arrow heads sho w the positions of GF P/8Tm2C2 (c. 80 kDa) and
GFP/8Tm2 (c. 55 kDa). The uppe r bands are p robably oligomeric
forms. The amount of AC8 expressed in a ll of the transfections was
very similar (data not shown).
Ó FEBS 2002 Dimerization of adenylate cyclases (Eur. J. Biochem. 269) 417
AC8 [20]. The T7 tag antibody was used to pull down
AC8
D1)106, D1184)1248
and any coimmunoprecipitating pro-
teins, and anti-GFP living color peptide antibody was used

in the subsequent Western blotting to identify the associated
proteins. AC8
D1)106, D1184)1248
strongly interacted with the
second half of AC8 (GFP/8Tm2C2, approximately 80 kDa)
and the second transmembrane c luster (GFP/8Tm2;
approximately 55 kDa), but not at all or only weakly with
either the ®rst transmembrane cluster (8NTm1/GFP), the
®rst cytoplasmic domain (GFP/8C1) or the second cyto-
plasmic domain ( GFP/8C2) (Fig. 4B). [The higher molec-
ular mass bands in the GFP/8Tm2C2 and AC8
D1)106,
D1184)1248
and G FP/8Tm2 and AC8
D1)106, D1184)1248
com-
binations were likely multimeric forms (Fig. 4B)]. These
coimmunoprecipitation experiments indicated that the
second transmembrane domain was the major region
responsible for bringing molecules together. However,
coimmunoprecipitation requires the retention of inter-
actions that will survive rather rigo rous detergent treatment
and while positive r esults are informative, negative r esults
do not necessarily prove that weaker interactions do not
occur. One approach to overcoming this problem is FRET
microscopy in living cells [25]. FRET r elies on sustained,
intimate associations between proteins at distances on the
order of 5 nm or less, although the c hemical n ature of the
interaction is not a major consideration. Consequently we
evaluated FRET analysis to probe the formation of

adenylate cyclase oligomers in live cells.
Higher order structures revealed by FRET microscopy
Using FRET microscopy, we had previously shown that
when tagged with CFP and YFP, the ®rst and second
transmembrane clusters of AC8 interacted with each other,
which resulted in the functional assembly of adenylate
cyclase and traf®cking to the plasma membrane [20]. We
also noted that the second transmembrane cluster of AC8
could form homooligomers, which were retained in the ER
[20]. This latter homomeric interaction of the second
transmembrane cluster reminded us of earlie r literature
which suggested that adenylate cyclase could dimerize or
oligomerize. To evaluate the possibility of dimerization
using FRET analysis, CFP/8Tm2 and YFP/8Tm2 were
cotransfected into HEK 293 cells with or without the
untagged ®rst transmembrane cluster, 8NTm1. As expected
from our previous studies, CFP/8Tm2 and YFP/8Tm2
associated with each other, yielding a strong FRET signal
from the ER (Fig. 5A). Upon the inclusion of 8NTm1, both
CFP/8Tm2 and Y FP/8Tm2 appeared at the plasma mem-
brane yielding a strong FRET signal (Fig. 5B). This result
indicated that more than one 8Tm2 molecule, one CFP-
tagged and one YFP-tagged, was present in the tightly
associated 8NTm1/8Tm2 complex at the plasma membrane.
This result suggests that the trans membrane domains can
mediate higher order assembly of adenylate cyclases. As a
corollary, we cotransfected 8NTm1/CFP, 8NTm1/YFP and
8Tm2 in HEK 293 cells. In this case, although the presence
of 8Tm2 ensured that appropriate intramolecular dimeriza-
tion occurred resulting in traf®cking to the plasma mem-

brane, only weak FRET was de tected between the 8NTm1/
CFP and 8N Tm1/YFP elements (data not shown). These
data indicate that weaker associations occur between the
®rst transmembrane segments than between the second
transmembrane segments. Quite curiously, when the anal-
ogous experiment was performed with the full length CFP/
AC8 and YFP/AC8, even though they both located in the
plasma membrane, no clear FRET signal was detected
(Fig. 5C). This somewhat surprising result c ould be
explained by the fact that the two AC8 molecules associate
so that their N-termini are distant (> 5 nm) from each other
or that the N-terminus of AC8 is too long and ¯exible to
maintain a minimally effective distance for FRET to occur.
Fig. 5. Homomeric interactions between the
second transmem bran e cluster and full-length
AC8. CFPandYFPtaggedconstructswere
cotransfected into HEK 293 cells. Pictures in
each row were captured from the same cell.
The ®rst (CFP) and the second (YFP) columns
show the CFP ¯uorescence and YFP ¯uores-
cence, respectively. The third column (CFP/
YFP overlay) are the overlay of the CFP and
YFP images of the cell, which shows colocal-
ization. The FRET images are presented in the
fourth column (FRET
C
). FRET
C
is displayed
as a quantitative pseudocolor image. ALUFI,

arbitrary linear units of ¯uorescence intensity.
(A) Cotransfection of CFP/8Tm2 and
YFP/8Tm2. (B) Cotransfection of 8NTm1,
CFP/8Tm2 and YFP/8Tm2. (C) Cotransfec-
tion of CFP/AC8 and YFP/AC8.
418 C. Gu et al. (Eur. J. Biochem. 269) Ó FEBS 2002
To address the possibility that steric effects were
precluding the detection of FRET between two full length
adenylate cyclase molecules, we constructed a truncated
AC8 in which the two transmembrane clusters were linked
with either CFP or YFP (Fig. 6A). Remarkably, in this
molecule, the conformations of the t wo transmembrane
clusters and both the CFP and YFP molecules were
correctly maintained, as the intact molecules could t raf®c
separately to the plasma membrane (Fig. 6A,B). This
observation extends our previous ®nding that the two
transmembrane clusters, when coexpressed are necessary
and suf®cient for the plasma membrane targeting of AC8
[20]. Strikingly, coexpression of 8Tm1/CFP/Tm2 and
8Tm1/YFP/Tm2 in HEK 293 cells yielded not only the
expected colocalization, but also strong FRET signals in the
plasma membrane, which establishes dimer formation
(Fig. 6C). This is quite compelling evidence that the
transmembrane domains of adenylate c yclase can mediate
oligomerization. When cells w ere cotransfected with
8Tm1/CFP/Tm2 and YFP/AC8, a lthough they were
colocalized in the plasma membrane, no FRET was
detected (Fig. 6C). This again suggests that even though
these m olecules could dimerize, inadequate access between
the N-terminus of AC8 and the C1 region of different

molecules precluded the detection of FRET.
DISCUSSION
The present group of studies have convinced us that
adenylate cyclases dimerize and that functional conse-
quences can accompany this d imerization. The dominant
negative effects of inactive AC8 mutants on wild-type
activity, coupled with the rescue of inactive mutants by
complementary, but inactive, molecules led us to seek
structural correlates to this apparent multimolecular inter-
action, in which a catalytic center might be formed by the
C1a and C2a domains from different molecules. These
rescue experiments were reminiscent of earlier in vitro
experiments using truncation mutants of AC1, which
suggested that adenylate cyclase might dimerize [27]. In
that study, when a nonepitope-expressing, C-terminally-
truncated, active, AC1 was expressed along with a mutant
AC1 that possessed no enzymatic activity but that did
contain the C-terminal epitope, a signi®cant amount of the
enzymatic activity could be immunoprecipitated [27]. This
suggested that the functional C-terminal truncation m utant
and the inactive (epitope-containing) mutant associated, or
at least coimmunoprecipitated. Coimmunoprecipitation
experiments, by themselves, can suggest interactions
between molecules, although they do require persistent
interactions that can withstand detergent. Thus, a balance
must be established between the rigor that is required
to avoid nonspeci®c interactions and the lowering of
stringency that permits weak interactions to persist.
Notwithstanding these limitation s, the coimmunoprecipita-
tion experiments reported here, along with the functional

interactions that we encountered, d id indicate that
independent adenylate cyclase molecu les interacted and
did so with speci®city. In this regard, the second transmem-
brane cluster seemed to play a dominant role in the
intermolecular interaction. The more discerning technique
of FRET analysis in live cells showed that in addition to
interacting with Tm1 and t raf®cking to t he plasma mem-
brane [20], homomeric interactions could occur between two
Tm2 domains in the plasma membrane, which meant that
the transmembrane domains of adenylate cyclase could
form higher order structures in the plasma membrane. This
concept was proven with our construct that retained only
the transmembrane domains with a CFP or YFP in the
middle of the two clusters. This construct a lso traf®cked to
the plasma membrane by itself, and formed multimers in the
plasma membrane, as s een by FRET analysis. Therefore,
the hydrophilic and hydrophobic portion of mammalian
adenylate cyclases may be considered to have two
Fig. 6. Tracking of the hydrophobic portion
of AC8. (A) The left panel shows the structural
diagram of 8Tm1/CFP/Tm2. The red and
blue cylinders represent the transmembrane
segments of the ®rst and second cluster,
respectively. The blue ball in the middle rep-
resents CFP. The right panel is the image of
8Tm1/CFP/Tm2 transfected cells. (B) The left
panel shows the structural diagram of
8Tm1/YFP/Tm2, which is the same as
8Tm1/CFP/Tm2 except that CFP is replaced
by a yellow ball, YFP. The right panel is the

image of 8Tm1/YFP/Tm2 transfected cells.
(C) and (D) are the FRET analyses arranged
as in Fig. 5. 8Tm1/CFP/Tm2 and 8Tm1/YFP/
Tm2 were cotransfected in (C). 8Tm1/CFP/
Tm2 and YFP/AC8 were cotran sfected in (D).
Ó FEBS 2002 Dimerization of adenylate cyclases (Eur. J. Biochem. 269) 419
roles. The hydrophilic portion is responsible for the
adenylate cyclase activity and its regulation, while the
hydrophobic portion governs the molecule's targeting and
oligomerization.
Whereas it seems reasonable to suggest that Tm2
domains bring molecules together, the f unctional rescue
studies seem to suggest that interactions between the two
catalytic domains are preferred within the same molecule
rather than between two molecules. This suggestion comes
from the f act that only half molecules can r escue inactive
adenylate cyclase muta nts, and the re scued activit y is
considerably less than that of the w ild-type, which suggests
an inef®cient interaction. Moreover, two full-length inactive
adenylate cyclase mutants, one mutated in the C1 loop and
the other mutated in t he C2 loop, cannot complement each
other's activity, which suggests that intermolecular interac-
tions between C1 and C2 loops do not occur in the natural
assembly of two adenylate cyclase molecules.
Based on these various ®ndings, a model for the higher
order assembly of adenylate c yclase can be proposed that
minimally comprises two aden ylate cyclase molecules. The
lack of FRET between two N-terminally-tagged molecules,
coupled with an inef®cient rescue by partial molecules,
along with the expectation that the N-terminus and C1 loop

would be close to each other, based on intramolecu lar
dimerization, makes it reasonable to speculate that the two
adenylate cyclase molecules are arranged in a head-to-tail
fashion when they dimerize. This arran gement is also
consistent with previous data showing that the N-terminus
and C-terminus of AC8 appeared to interact to permit
regulation by Ca
2+
acting via calmodulin [22].
Although the reported studies establish that adenylate
cyclase molecules dimerize, or form even higher order
structures, it is premature to speculate on the precise
advantages that this dimerization provides to t he cell.
Nevertheless, one speculation that might be w orth raising is
that adenylate cyclases could associate with other mem-
brane proteins. It is well known that many heteromultimer-
forming membrane proteins can homomultimerize in the
absence of their normal partners, as is the case with voltage-
gated Ca
2+
-channels, which can form functional assemblies
of varying properties [29]. A similar situation may occur
with adenylate cyclase. A substantial body of evidence
already shows that Ca
2+
-sensitive adenylate cyclases and
CCE channels are intimately colocalized, with the result that
only Ca
2+
entering via CCE channels can regulate these

cyclases (including AC8) while the release of Ca
2+
from
internal stores or ionophore-mediated intracellular calcium
ion concentration increases are quite ineffectual [30±32]. The
mechanism for this association is quite unclear [33]. W hat if
the multivalency of adenylate cyclase molecules provided
the basis for the association of adenylate cyclases with either
CCE channel proteins or scaffolding proteins, so that a
complete adenylate cyclase complex was an association
between adenylate cyclase molecules and CCE channel
proteins? Premises for this type of behavior by other
members of the ABC family of proteins include the ATP-
activated K
+
-channel discussed earlier, which is comprised
of a heterooctamer of four SUR protein subunits in
associationwithfourK
ir
subunits [13,14]. Thus the data
gathered presently, although initially appearing to introduce
a layer of cumbersome complexity to the structure of
adenylate cyclase, may actually be a step in resolving one of
the more intrigu ing properties of Ca
2+
-sensitive adenylate
cyclases, namely their essential colocalization with CCE
channels. At the same time, these ®ndings render more
prescient and add substance to a proposal ®rst raised over
20 years ago.

ACKNOWLEDGEMENTS
The authors thank M. Rodbell
2
for the original stimulus for t his study
and Kent Fagan for useful comments on the manuscript. This work
was supported by NIH grants GM 32483 a nd NS 28389 (to
D. M. F. C.).
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