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 cataly
Trang 1PRIORITY PAPER
Dimerization of mammalian adenylate cyclases
Functional, biochemical and ¯uorescence resonance energy transfer (FRET) studies
Chen Gu1, James J Cali2and Dermot M F Cooper1,3
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
1Mammalian 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 two
transmembrane cassettes also associate to facilitate the
functional assembly and tracking of the enzyme The older
literature had suggested that adenylate cyclases might 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 adenylate 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 assemblies than a simple
mono-meric arrangement 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 adenylate
cyclases [2,3] Independent, hydrodynamic analyses of
detergent-solubilized adenylate cyclase preparations also
indicated molecular masses of about 220 kDa for the
catalytic units [4±7], which, given the minimal protein
molecular masses of 120 kDa, again suggested 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 form 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 inwardly rectifying K+(Kir) channel subunits to form ATP-sensitive K+channel complexes, which contain four subunits each of SURs and Kir[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 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 these 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 of
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: dermot.cooper@uchsc.edu
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)
Trang 2AC8 did not cotraf®c to the plasma membrane with the
second transmembrane domains of AC2 and AC5 This
latter conclusion was arrived at independently by functional
assays [21] In our experiments, we were 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 energy transfer
(FRET) between partial and full-length cyan ¯uorescent
protein (CFP)- and yellow ¯uorescent protein (YFP)-tagged
molecules in live cells to search for persistent and intimate
interactions These studies lead us to conclude that
mam-malian adenylate cyclases do form dimers (or higher order
assemblies) the regions responsible are the hydrophobic
domains and this aggregation may contribute to the
associ-ation of adenylate cyclases with cellular regulatory factors
M A T E R I A L S A N D M E T H O D S
cDNA plasmid constructs and cell culture
Portions of AC8 were subcloned into N-terminal or
C-terminalenhancedgreen¯uorescentprotein(GFP)vectors
(Clontech) using convenient restriction enzyme digestion
sites or PCR-based strategies, as described previously [20]
In AC8D582)594, a region from Y582 to L594 in the C1
domain of AC8 was deleted; in AC8D1126)1248[20], a region
from R1126 to P1248 in the C-terminus of AC8 (in the C2
domain) was deleted; in AC6D553)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 Nhe1 and
BglII, from pECFP and pEYFP vector (Clontech) 8Tm1/
CFP/Tm2 was obtained by subcloning 8NTm1 of 8NTm1/
CFP between the restriction enzyme sites Nhe1 and Age1,
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 [2-3H]adenine
(1.5 lCi per well) to label the ATP pool The cells were then
washed once and incubated with a nominally Ca2+-free
Krebs buffer (900 lL per well) containing 120 mMNaCl,
4.75 mM KCl, 1.44 mM MgCl2, 11 mM glucose, 25 mM
Hepes, and 0.1% bovine serum albumin (fraction V)
adjusted to pH 7.4 with 2MTris base The use of Ca2+
-free Krebs buffer in experiments denotes the addition of
0.1 mM EGTA to the nominally Ca2+-free Krebs buffer
All experiments were carried out at 30 °C in the presence of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (500 lM), and Ro 20±1724 (100 lM), which were preincu-bated with the cells for 10 min prior to a 1-min assay Cells were preincubated for 4 min with the Ca2+-ATPase inhib-itor, thapsigargin, at a ®nal concentration of 100 nM This treatment passively empties intracellular Ca2+ stores, establishing a low basal [Ca2+]i and primes the cells for CCE [24] Assays were terminated by addition of 5% (w/v,
®nal concentration) trichloroacetic acid and the percent conversion of [3H]ATP to [3H]cAMP was measured as previously described previously [22] Means SD of triplicate determination are indicated
GFP ¯uorescence imaging The procedure was described previously [20] Transfected 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/Pi (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4
and 1.8 mM KH2PO4, 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
bySLIDEBOOK3.0 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 mM
Tris/HCl (pH 7.4), 150 mMNaCl, 1% Triton X100 (or 1% Nonidet P-40) and protease inhibitor cocktails) for 1 h at
4 °C, and then centrifuged (100 000 g; OptimaTM 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 mM NaCl, once with 1 mL 50 mM Tris/HCl (pH 7.4) and 150 mM NaCl, and eluted with 50 lL 2 ´ 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 the 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
Trang 3basis 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 particular optical
system and was determined using cells that express either
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 (FRETC) image was
obtained by subtracting CFP and YFP 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
R E S U L T S
Inactive mutant adenylate cyclases suppress
the activity of wild-type adenylate cyclasesin 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 Ca2+acting via calmodulin, binding to
the C-terminus [22] Issues of speci®city of intermolecular
interactions were addressed with AC5 or AC6, which are
inhibitable by Ca2+, apparently independently of
calmod-ulin [26] Adenylate cyclases can be divided into ®ve major
domains, the N-terminus, the ®rst 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 AC8D582)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, the entry of
Ca2+triggered by store depletion (capacitative Ca2+-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, AC8D582)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, AC8D582)594
dramat-ically suppressed activity under all stimulation conditions
(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, AC8D582)594, also suppressed the activity of AC5 and AC6 (Fig 1B) Conversely, AC8 activity was suppressed
by the corresponding, inactive mutant of AC6, AC6D553)666 (Fig 1B) 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-type AC8 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 were pretreated with thapsigargin (100 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 accumulation 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.
Trang 4when AC8 was coexpressed with AC8D582)594, we employed
a GFP-tagged form of AC8, GFP/AC8, which resembles
the wild-type both in terms of catalytic activity and
appropriate location in the plasma membrane [20]
Co-transfection with AC8D582)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
suppressed by coexpression with AC8D582)594(Fig 2C)
Rescue of inactive mutants by half molecules
of AC8in vivo
A corollary of the experiments described above involving
dominant negative suppression of adenylate cyclase activity
is the rescue of inactive, mutant molecules 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 the activity of AC8D582)594 expressed in HEK 293
cells Previously, we described a C-terminus deletion of
AC8, AC8D1126)1248 [22], which lacked part of the C2a
domain and the entire C2b region AC8D1126)1248 is completely inactive when expressed alone in HEK 293 cells,
as are AC8D582)594, GFP/8Tm2C2 (the eGFP tagged second half of AC8) 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 AC8D582)594and GFP/8Tm2C2 or AC8D1126)1248
and 8NTm1C1/GFP also only had background adenylate cyclase activity (Fig 3) In contrast, cAMP accumulation of cells cotransfected with either AC8D582)594and 8NTm1C1/ GFP or AC8D1126)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 by 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 coupling 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 with AC8D582)594and AC8D1126)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 transfected 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 transfected 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+ ).
Trang 5Homo- and heteromeric interactions revealed
by coimmunoprecipitation assays
To test whether AC8 molecules actually bind to each
other and to AC6 molecules, we performed
coimmuno-precipitation assays with various epitope-tagged adenylate
cyclase constructs We previously described the mutant
AC8D1)106, D1184)1248or Ô8M13Õ, which lacks the ®rst 106
residues at the N-terminus and the last 65 residues at the
C-terminus [22] It is constitutively active, independent of
Ca2+ and is targeted correctly [20,22] AC8D1)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 AC8D1)106, D1184)1248into 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 AC8D1)106,
D1184)1248, respectively, along with any associated proteins,
which were then run on SDS/PAGE and transferred to a
PVDF membrane Ab VIII-A 1229±1248 antibody was
used in the Western blots to determine whether AC8 was
present in association with either AC6 or AC8D1)106,
D1184)1248 Indeed, AC8 did associate with AC8D1)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 cyclase molecules
To narrow down the region of interaction between adenylate cyclase molecules, we cotransfected HEK 293 cells with AC8D1)106, D1184)1248and GFP-tagged parts of
Fig 3 Inactive mutants can be partially rescued by halves of AC8
in vivo Top: diagram of the constructs; the green box represents the
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 GFP/AC8 in the corresponding region Assays were
performed as in Fig 1 cAMP accumulation was measured by adding
20 l M forskolin and 4 m M Ca 2+ Experiments were performed three
times with similar results The asterisks indicate value that are
signi®cantly dierent from the background (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 with anti-(T7 tag)Ig Only AC8 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 membranes Cotransfection conditions are indicated on the top of each blot; molecular mass (kDa) is shown on the left of the blot (A) The asso-ciation between 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) The association between AC8 D1)106, D1184)1248 and dierent 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 nonspeci®c antibody band The arrow heads show the positions of GFP/8Tm2C2 (c 80 kDa) and GFP/8Tm2 (c 55 kDa) The upper bands are probably oligomeric forms The amount of AC8 expressed in all of the transfections was very similar (data not shown).
Trang 6AC8 [20] The T7 tag antibody was used to pull down
AC8D1)106, D1184)1248and any coimmunoprecipitating
pro-teins, and anti-GFP living color peptide antibody was used
in the subsequent Western blotting to identify the associated
proteins AC8D1)106, D1184)1248strongly interacted with the
second half of AC8 (GFP/8Tm2C2, approximately 80 kDa)
and the second transmembrane cluster (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 AC8D1)106,
D1184)1248and GFP/8Tm2 and AC8D1)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 rigorous detergent treatment
and while positive results are informative, negative results
do not necessarily prove that weaker interactions do not
occur One approach to overcoming this problem is FRET
microscopy in living cells [25] FRET relies on sustained,
intimate associations between proteins at distances on the
order of 5 nm or less, although the chemical nature 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 earlier 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 YFP/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-CFP-tagged, was present in the tightly associated 8NTm1/8Tm2 complex at the plasma membrane This result suggests that the transmembrane 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 detected between the 8NTm1/ CFP and 8NTm1/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 could 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 transmembrane cluster and full-length AC8 CFP and YFP tagged constructs were 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.
Trang 7To 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 two transmembrane
clusters and both the CFP and YFP molecules were
correctly maintained, as the intact molecules could traf®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 cyclase can mediate
oligomerization When cells were cotransfected with
8Tm1/CFP/Tm2 and YFP/AC8, although they were
colocalized in the plasma membrane, no FRET was
detected (Fig 6C) This again suggests that even though
these molecules could dimerize, inadequate access between
the N-terminus of AC8 and the C1 region of different
molecules precluded the detection of FRET
D I S C U S S I O N
The present group of studies have convinced us that
adenylate cyclases dimerize and that functional
conse-quences can accompany this dimerization 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 mutant 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 limitations, the coimmunoprecipita-tion experiments reported here, along with the funccoimmunoprecipita-tional interactions that we encountered, did indicate that independent adenylate cyclase molecules 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 traf®cking to the 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 also traf®cked to the plasma membrane by itself, and formed multimers in the plasma membrane, as seen by FRET analysis Therefore, the hydrophilic and hydrophobic portion of mammalian adenylate cyclases may be considered to have two
Fig 6 Tracking 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 cotransfected in (D).
Trang 8roles 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 functional 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 fact that only half molecules can rescue inactive
adenylate cyclase mutants, and the rescued activity is
considerably less than that of the wild-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 the 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 cyclase can be proposed that
minimally comprises two adenylate 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 intramolecular
dimerization, makes it reasonable to speculate that the two
adenylate cyclase molecules are arranged in a head-to-tail
fashion when they dimerize This arrangement is also
consistent with previous data showing that the N-terminus
and C-terminus of AC8 appeared to interact to permit
regulation by Ca2+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 the cell
Nevertheless, one speculation that might be worth 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 Ca2+-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 Ca2+-sensitive adenylate cyclases and
CCE channels are intimately colocalized, with the result that
only Ca2+entering via CCE channels can regulate these
cyclases (including AC8) while the release of Ca2+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] What 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
association with four Kirsubunits [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 intriguing properties of Ca2+-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
A C K N O W L E D G E M E N T S
The authors thank M Rodbell
2 for the original stimulus for this study and Kent Fagan for useful comments on the manuscript This work was supported by NIH grants GM 32483 and NS 28389 (to
D M F C.).
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