The atrial natriuretic peptide receptor (NPR-A) coupled to
guanylyl cyclase activity (GC-A) (
)is a prototype for a
family of membrane-bound guanylyl cyclases currently numbering 6 in
mammals (A-F). The B and C cyclases appear to be receptors for
C-type natriuretic peptide and heat-stable enterotoxins/guanylin,
respectively, while D, E, and F remain orphan
receptors(1, 2, 3, 4) .
Members
of the family contain a single apparent transmembrane domain that
separates an amino-terminal extracellular, ligand binding domain from
an intracellular region that contains both a cyclase catalytic domain
and a protein kinase homology domain (KHD)(1) . The function of
the KHD is not fully understood, but since ATP is required for
transduction of the ANP binding signal to activation of cyclase
catalytic activity, and since various mutations within the KHD
interrupt ANP/ATP signaling(5, 6, 7) , it has
been suggested that the protein kinase homology domain binds
ATP(6) . ATP appears to act without conversion to ADP or other
metabolites since non-hydrolyzable analogues of ATP also substitute for
ATP(8, 9) . The KHD of the cyclases resembles the
kinase homology domain 2 of the JAKS; an invariant Asp found in the
catalytic domain of known protein kinases is a different amino acid in
either the cyclases or kinase homology domain 2 of the JAKS. Although
the requirement of ATP and peptide ligand for activation of GC-A is
somewhat analogous to the guanine nucleotide-dependent activation of
adenylyl cyclase by hormones, non-hydrolyzable analogues of GTP
activate adenylyl cyclase in the absence of hormone, and sub-micromolar
as opposed to millimolar concentrations of nucleotide are
effective(10) .
During previous attempts to purify GC-A, the
ANP/ATP activation signal has vanished, even though ANP has continued
to bind to the receptor (11, 12) . Thus, it was
suggested that additional factor(s), specifically ATP binding
proteins(8) , are required for transduction of a hormone
binding signal to activation of the cyclase catalytic domain. Now we
show, however, that after purification of detergent-solubilized GC-A to
apparent homogeneity, the cyclase retains ability to be activated by
ANP/ATP.
EXPERIMENTAL PROCEDURES
Materials
Sf9 cells (13) were
obtained from the American Type Culture Collection. BaculoGold virus
was from Pharmingen. cDNA encoding FLAG-GC-A was kindly provided by Dr.
Michael Chinkers (Vollum Institute, Oregon Health Sciences Center).
Lubrol 12A9 (from ICI) was a gift from Dr. Elliott Ross (University of
Texas Southwestern Medical Center at Dallas). Polyoxyethylene-10-lauryl
ether was from Sigma, M1 and M2 immunoaffinity columns were from IBI,
wheat germ agglutinin-agarose was from Vector, and Q-Sepharose FF was
from Pharmacia.
I-ANP,
[
-
P]GTP, and [
H]cGMP
were from DuPont NEN, and rat ANP (99-126) was from Peninsula.
Reagents used for radioimmunoassay of cGMP have been previously
described(14) .
Expression of FLAG-GC-A in Sf9 Cells
The
baculovirus expression vector PVL-FLAG-GC-A was cotransfected with
BaculoGold virus into Sf9 cells, and the recombinant virus was then
cloned according to Kitts et al.(15) . Sf9 cells
infected with the recombinant virus expressed
0.7-1 pmol of
I-ANP binding site/mg of particulate protein, while no
significant binding was detected in uninfected cells.
Solubilization of FLAG-GC-A
48 h
post-infection with the recombinant virus, Sf9 cells expressing
FLAG-GC-A were pelleted and frozen in liquid nitrogen. The frozen
pellets were thawed in the presence of a solution containing 50
mM HEPES, pH 7.5, 100 mM NaCl, 40% glycerol, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM
-glycerol
phosphate, and 5 mM sodium azide (buffer A). The thawed cells
were broken in a Dounce homogenizer, and crude particulate fractions
were obtained and washed by centrifugation (30,000
g)
in the same buffer. The particulate fraction was resuspended in buffer
A at a concentration of 20-30 mg of protein/ml. FLAG-GC-A was
then solubilized by rocking the solution for 60 min at 4 °C in
buffer A containing 1% Lubrol 12A9 or polyoxyethylene-10-lauryl ether.
After 60 min, the detergent extract was diluted with buffer A to 0.5%
of either non-ionic detergent and centrifuged at 100,000
g. The supernatant fluid was obtained and diluted to 0.1%
detergent with buffer A. Approximately 20-40% of the
I-ANP binding sites detected in the particulate fraction
were solubilized. Higher recoveries (up to 70-80% of
I-ANP binding sites could be obtained at higher
detergent:protein ratios; however, enzyme solubilized under these
conditions lost ANP and ATP sensitivity (not shown).
Purification of FLAG-GC-A
All
purification procedures were performed at 4 °C. Detergent extracts
(200 ml) were rocked with 6 ml of M2 immunoaffinity resin for 2-3
h and then washed batchwise by centrifugation (10 times, 30 ml) with
buffer B (buffer A containing 0.05% detergent, 0.25 mM ATP,
150 mM NaCl, and 0.025% phosphatidylcholine). FLAG-GC-A was
eluted from the resin by rocking with 6 ml of buffer B containing 0.1
mg/ml of FLAG peptide for 30 min. After centrifugation at 3000
g, FLAG-GC-A was collected in the supernatant fluid. The above
elution procedure was repeated 10 times. The eluates were then pooled
and filtered through a 0.45-µm filter, and the filtrate was then
mixed with 2 ml of Q-Sepharose FF for 30 min and centrifuged. FLAG-GC-A
was collected in the supernatant fluid. The Q-Sepharose FF resin was
then washed three times with 10 ml of buffer B by centrifugation, and
additional FLAG-GC-A was also collected in the supernatant fluid. The
supernatant fluids were then pooled and rocked with 10 ml of wheat germ
agglutinin-agarose for 2 h and centrifuged. The pellet was washed
batchwise (10 times, 40 ml of buffer B) by centrifugation. FLAG-GC-A
was then eluted from the resin by rocking with 10 ml of buffer B
containing 0.3 MN-acetylglucosamine for 20 min.
After centrifugation, FLAG-GC-A was collected in the supernatant fluid.
The procedure was repeated five times. The supernatant fluids were then
pooled and filtered through a 0.45-µm filter. The filtrate was then
made 2.25 mM with respect to CaCl
and added to 2
ml of M1 immunoaffinity resin. The slurry was rocked for 2-3 h
and washed batchwise by centrifugation (10 times, 10 ml of buffer B
containing 2.25 mM CaCl
), and then purified
FLAG-GC-A was eluted from the resin by rocking 30 min with 1 ml of
buffer B containing 2 mM EDTA. FLAG-GC-A was collected in the
supernatant fluid after centrifugation. The elution procedure was
repeated four times. Purified FLAG-GC-A was subsequently stored at
-80 °C. The purification procedures were routinely performed
within 20-24 h to minimize losses in ANP/ATP sensitivity.
I-ANP Binding Assay
ANP binding
activity was determined as described previously(12) , with the
exception that 1% polyethyleneimine-treated Whatman GF/C filters were
used for filtration. Nonspecific binding was determined using 1
µM non-radioactive ANP (99-126), and data were
analyzed by nonlinear curve fitting using the LIGAND computer
program(16) .
Guanylyl Cyclase Assay
Guanylyl cyclase
activity was determined by incubating samples (0.5-1 fmol
I-ANP binding sites) at 37 °C for the indicated time
intervals in 0.1 ml of a reaction mixture consisting of 25 mM HEPES, pH 7.5, 50 mM NaCl, 0.1% bovine serum albumin,
0.25 mM 1-methyl-3-isobutylxanthine, 6 mM
MgCl
, 5 mM creatine phosphate, 4 units of creatine
phosphokinase, and 0.5 mM GTP. To determine the enzyme
activity using MnGTP as substrate, 0.1% Triton X-100 and 6 mM MnCl
also were added. The reaction was initiated by
the addition of enzyme samples and terminated by the addition of 0.45
ml of 0.11 M zinc acetate, followed by 0.45 ml of 0.11 M Na
CO
. After centrifugation at 3000
g, 5 or 45 µl of the supernatant fluid were
assayed by radioimmunoassay for cyclic GMP content(14) . For
the MgGTP dependence of the ANP/ATP experiment (Fig. 5),
[
-
P]GTP was used as substrate, and the
reaction was terminated by 0.1% SDS. The amount of
[
P]cGMP was determined as described previously
for adenylyl cyclase assays(17) , with the modification that
only 0.2 ml of H
O was added in the initial wash of the
Dowex.
Figure 5:
Kinetics of guanylyl cyclase activation by
MgGTP. Guanylyl cyclase activities were determined in crude detergent
extracts (40 fmol
I-ANP binding sites) (A) or
purified FLAG-GC-A preparations (107 fmol
I-ANP binding
sites/assay) (B) with (
) or without (
) 1 mM caged ATP and 1 µM ANP in the presence of the
indicated concentrations of MgGTP. The assay was performed for 5 min at
37 °C in the presence of a constant concentration of free
MgCl
(6 mM), using
[
-
P]GTP as substrate as described under
``Experimental Procedures.'' Results are mean ± S.E.
of duplicate assays.
Iodination of Purified Receptor
Purified
FLAG-GC-A was incubated with 100 µCi of
[
I]NaI for 20 min at room temperature in the
presence of 0.1 M Tris-Cl, pH 6.8, and 1 pellet of IODOBEAD
(Pierce). After removing the supernatant fluid from the IODOBEAD, free
[
I]NaI was removed by a G-25 spin column. The
sample was then electrophoresed using an 8% separating gel by the
method of Laemmli(18) . The gel was stained with
silver(19) , vacuum-dried, and subjected to autoradiography
with HYPER-FILM-MP (Amersham).
Protein Determination
Protein content of
the crude membrane fraction and the detergent extract was determined by
amido black staining (20) using bovine serum albumin (Pierce,
no. 23209) as a standard. The protein content of the purified FLAG-GC-A
was determined by densitometry according to Merril and
Pratt(21) , as modified as follows. Purified FLAG-GC-A and
bovine serum albumin standards (0.1-10 ng) were electrophoresed
on SDS-polyacrylamide gels and then silver stained(19) . After
drying between transparent cellulose sheets (Bio-Rad), the protein
content of the FLAG-GC-A sample was determined by comparing the
intensity of staining with that of the bovine serum albumin. The
intensity of staining was determined in a densitometric scanner
(Molecular Dynamics model 300A) under conditions where the intensity
was linearly proportional to the protein content.
RESULTS AND DISCUSSION
Expression of Recombinant FLAG-GC-A in Sf9
Cells
To facilitate rapid purification of the receptor, the
FLAG epitope (DYKDDDDK) was introduced just to the carboxyl side of the
signal peptide, thereby representing the amino terminus of the
processed cyclase. The insertion of the FLAG epitope did not appear to
alter biological activity of the enzyme since the number of
I-ANP binding sites/mg protein or the relative cyclase
activation in response to ANP/ATP in Sf9 membrane preparations was
equivalent whether wild-type or FLAG-GC-A cyclase was expressed (data
not shown).ANP/caged ATP maximally activated FLAG-GC-A at 48 h
following infection with recombinant baculovirus, at which time
0.6-1.0 pmol of
I-ANP binding sites/mg of protein
in the crude particulate fraction were obtained (Table 1). With
the described protocol to grow Sf9 cells (2% fetal bovine serum in
IPL-41 in suspension), 0.7-1.0 nmol of
I-ANP
binding sites/liter of culture were routinely obtained.
Solubilization of Epitope-tagged
GC-A
Initial experiments using many types of detergents
such as Triton X-100 and deoxycholate to solubilize GC-A resulted in
complete losses of ANP/ATP sensitivity (not shown). Of the various
detergents tested, Lubrol 12A9 or polyoxyethylene-10-lauryl ether
resulted in the highest ANP/ATP-responsive cyclase. However, high
detergent/protein ratios during the initial solubilization decreased
responsiveness to ANP/ATP. The ANP/ATP response of GC-A also was very
sensitive to the concentration of glycerol, and the response to ANP/ATP
was largely attenuated when solubilization buffers contained 20%
glycerol but was retained in 40% glycerol. For the highest recovery of
ANP/ATP-responsive cyclase, FLAG-GC-A was routinely solubilized with 1%
detergent at protein concentrations of greater than 20 mg/ml and 40%
glycerol. Under these conditions, 41% of the initial
I-ANP binding activity and 7% of the particulate protein
were solubilized (Table 1). The K
for
I-ANP binding for the solubilized and the membrane-bound
receptor was the same under these conditions (Table 1).
Purification of Ligand-sensitive
FLAG-GC-A
The main objective of this study was to determine
whether or not GC-A, serving as a prototype for the membrane guanylyl
cyclases, could signal in the absence of other protein factors. Batch
procedures using peptide competition (M2 immunoaffinity resins), N-acetylglucosamine competition (wheat germ
agglutinin-agarose), and EDTA chelation (M1 immunoaffinity resin) were
used to minimize the time required for purification. The capacity of
the M2 immunoaffinity resin in buffer B restricted the amount of
I-ANP binding sites applied to 40 pmol/ml resin. Typically, 40-50%, 60%, and 15-20% of FLAG-GC-A was
recovered at the M2 immunoaffinity, wheat germ agglutinin-agarose, and
M1 immunoaffinity steps, respectively. The final yield was typically
about 0.2%.
Purity of FLAG-GC-A
The silver-stained
FLAG-GC-A recovered after the above purification steps was the only
major band detected (Fig. 1), and densitometry of the
silver-stained gel suggests that it is greater than 95% pure. The
purity also was examined by the use of nonspecific radioiodination.
FLAG-GC-A was the only major iodinated species, although longer
exposures revealed a minor contaminant at about 70 kDa, a protein not
detected by silver staining. The specific activity of the receptor
preparation of 5.8 nmol of
I-ANP bound/mg protein is
comparable with previously purified ANP receptor (6-7 nmol/mg
protein), suggestive of a highly purified cyclase preparation (12, 22) .
Figure 1:
SDS-polyacrylamide gel electrophoresis
of FLAG-GC-A. Left panel, the membrane fraction (469 ng of
protein), Lubrol 12A9 extract (713 ng of protein), or purified
FLAG-GC-A (2.5 ng of protein) were electrophoresed and stained as
described under ``Experimental Procedures.'' Right
panel, [
I]NaI was incubated with buffer or
with purified FLAG-GC-A (0.6 ng) in the presence of IODOBEAD. The
labeled samples were electrophoresed and subjected to autoradiographic
analysis as described under ``Experimental Procedures.''
Molecular weight markers are myosin (200,000),
-galactosidase
(116,250), phosphorylase B (97,400), bovine serum albumin (66,200), and
ovalbumin (45,000).
Ligand Binding Characteristics of
FLAG-GC-A
With either the crude detergent extract or the
purified preparation of cyclase, a single class of high affinity
I-ANP binding sites (K
= 65
pM) was obtained (Fig. 2).
Figure 2:
Equilibrium binding of
I-ANP
to GC-A. The crude Lubrol 12A9 extract (A) (558 ng of protein)
or purified FLAG-GC-A (B) (0.375 ng of protein) were incubated
with increasing concentrations of
I-ANP at 25 °C for
60 min as described under ``Experimental Procedures.''
Nonspecific binding was determined in parallel incubation mixtures
containing 1 µM nonradioactive ANP. Binding data were
plotted after correction for the nonspecific binding (5-20% of
total binding). Inset, Scatchard plot. The respective
B
and K
values were 3.9
pmol/mg protein and 64 pM for the crude extract and 5.8
nmol/mg protein and 66 pM for purified
FLAG-GC-A.
Ligand/ATP Responsiveness of FLAG-GC-A
In
crude particulate fractions, ANP or caged ATP activated the cyclase by
1.3- or 1.7-fold, respectively, when added alone, but when added
together, the cyclase was activated approximately 16-fold. The
requirement for both ANP and the nucleotide, ATP, for maximum
stimulation of GC-A has been previously
reported(5, 8, 9) . After detergent
extraction, ANP and ATP continued to stimulate minimally when added
alone, but together stimulated the cyclase about 9-fold. However, the
detergent-solubilized GC-A is highly labile. Considerable ANP/ATP
stimulation (50%) was lost upon storage at 4 °C for 24 h, and only
about 20% remained after incubation at 25 °C for 60 min. (
)Following an approximate 1300-fold purification of
GC-A, the receptor remained responsive to ATP/ANP (Fig. 3).
About 10% or 30% activations of FLAG-GC-A were obtained when ANP or
caged ATP were added alone, respectively. When an equivalent number of
I-ANP binding sites (0.5-1 fmol ANP binding sites)
were added per assay, the amount of cGMP produced upon incubation with
ANP/ATP was approximately the same for the detergent extract or the
purified enzyme preparation (see also Fig. 4, A and B). Therefore, the maximal responsiveness to ANP/ATP did not
change significantly during the purification procedure. The basal
activities were 2-3-fold higher in the purified preparation, and
consequently the -fold stimulation by ANP/ATP decreased from
approximately 8-fold in the crude detergent extract to 3-4-fold
in the purified GC-A preparation. The guanylyl cyclase reaction was
linear for at least 5 min, and as shown in Fig. 5, ANP/ATP
appears to alter V
as a function of MgGTP. ATP
at millimolar concentration is required for maximal stimulation by ANP (Fig. 5). Although millimolar concentrations of ATP are quite
high when compared to the binding of GTP to G proteins, which regulate
the adenylyl cyclase system (EC
0.1 µM),
millimolar concentrations of ATP are near the cellular concentration of
the adenine nucleotide. Therefore, variations in intracellular ATP
concentrations could affect ANP-dependent activation of guanylyl
cyclase.
Figure 3:
ANP and caged ATP activate purified GC-A.
Guanylyl cyclase activities were determined under conditions where
equivalent amounts of
I-ANP binding sites (0.5 fmol) were
added to each reaction mixture: crude membrane fraction (341 ng of
protein), Lubrol 12A9 detergent extract (175 ng of protein) and
purified FLAG-GC-A (0.118 ng of protein). The assays were performed at
37 °C for 15 min in the presence of 0.5 mM GTP and 6
mM MgCl
as described under ``Experimental
Procedures,'' with or without caged ATP (c-ATP, 1
mM) or ANP (1 µM) as indicated. Results are mean
± S.E. of triplicate assays. The values presented are from one
experiment, which is representative of two
experiments.
Figure 4:
Concentration dependence of guanylyl
cyclase activation by caged ATP or ANP. A, the equivalent of
0.683 fmol of
I-ANP binding sites of either crude
detergent extract (
) (175 ng of protein) or purified FLAG-GC-A
(
) (0.118 ng of protein) were incubated with the indicated
concentrations of caged ATP in the presence of 1 µM ANP.
Data are mean ± S.E. of duplicate assays. B. The
equivalent of 0.683 fmol of
I-ANP binding sites of either
crude detergent extract (
) (175 ng of protein) or purified
FLAG-GC-A (
) (0.118 ng of protein) were incubated with the
indicated concentrations of ANP in the presence of 1 mM caged
ATP. Data are mean ± S.E. of duplicate
assays.
Kinetics of Activation by MnGTP
Membrane
forms of guanylyl cyclases normally display positive cooperative
kinetics as a function of MnGTP. FLAG-GC-A also yielded positive
kinetics with respect to MnGTP in either the crude detergent extract or
after purification (Fig. 6). This contrasts to the sea urchin
guanylyl cyclase, which lost cooperative kinetics as a function of
MnGTP after purification in initial work (23) but is consistent
with later studies where the phosphorylated form of the sea urchin
cyclase was purified and retained positive cooperativity(24) .
Figure 6:
Kinetics of guanylyl cyclase activation by
MnGTP. Guanylyl cyclase activities were determined using equivalent
amounts of
I-ANP binding sites (0.1 fmol/assay) of crude
detergent extract (
) (35 ng of protein) or purified FLAG-GC-A
(
) (0.024 ng of protein) with the indicated concentrations of
MnGTP. The experiment was performed for 10 min at 37 °C in the
presence of a constant concentration (1.2 mM) free
Mn
. Results are mean ± S.E. of duplicate
assays. Inset, double reciprocal plot. Velocities are
expressed as the pmol of cyclic GMP formed per 10
min.
Other Activities of GC-A
Could the KHD
act as a protein kinase or as an ATPase? Using an ATPase assay (lower
detection limit, 3 nmol/min/mg protein)(25) , we have not
observed any detectable ATPase activities from 150 ng of our purified
preparation of FLAG-GC-A (not shown). Using nonspecific kinase
substrates such as histone IIIs and VIIs, we have not observed
detectable protein kinase activities in the purified preparations of
FLAG-GC-A (not shown). This is consistent with the loss of protein
kinase activity observed in the c-Kit receptor tyrosine kinase from the W
allele of the white spotting locus where a conserved Asp in the catalytic domain is mutated to
an Asn(26) . An Asn rather than an Asp is also found in the
same relative position in GC-A. Also, the absence of a critical lysine
residue known to be important for kinase activity (27) suggests
that protein kinase activity may be absent in the cyclases. That GC-A
contains the glycine loop implicated in nucleotide binding suggests
that it can bind ATP. The failure to detect protein kinase activity
with GC-A, however, is consistent with its missing several critical
residues found within the catalytic pocket of protein kinases (28) .GC-A represents a prototype of the particulate
guanylyl cyclase family where there are at least six subtypes
identified so far(1, 2, 3, 4) . The
results from this study initially show that the ligand/ATP are able to
activate this class of particulate receptor guanylyl cyclases
independent of other protein cofactors. In this unique signaling
system, which utilizes ATP as the signal tranduction molecule, the
domains for ligand binding, ATP regulation, and the effector guanylyl
cyclase are located in one molecule. This is in contrast to the G
protein-coupled receptor system, where the components for ligand
binding, signal transducing, and effector are located in separate
protein molecules. The mechanism of activation of GC-A is not yet
understood, but the ANP/ATP activation is known to be regulated by
phosphorylation (29, 30, 31) and
oligomerization(32, 33) . Although additional protein
factors therefore are not required for signal transduction, it also
seems likely that modulating factors will be discovered based on
precedence with other cell surface receptors.