(Received for publication, August 2, 1994; and in revised form, October 13, 1994)
From the
Guanylyl cyclase-A (GC-A), a receptor for A-type natriuretic peptide (ANP), contains an extracellular ligand-binding domain, a single transmembrane domain, and intracellular protein kinase-like and cyclase catalytic domains. Expression of the putative cyclase catalytic region (HCAT) resulted in the formation of an active enzyme that migrated as a homodimer on gel filtration columns; treatment with sodium trichloroacetate caused dissociation of the dimer and a loss of cyclase activity. Co-transfection of HCAT and full-length GC-A led to elevated basal intact cell cGMP concentrations and increased cell homogenate guanylyl cyclase activity. However, atrial natriuretic peptide-induced elevations of cGMP and cyclase activity were inhibited by the introduction of HCAT. Alanine scanning mutagenesis of highly conserved residues within HCAT identified one mutation (D893A) that destroyed enzyme activity but not the ability of the mutant subunit to form homodimers. The mutant subunit inhibited the cyclase activity of wild-type HCAT (approximately 70%) as well as that of full-length GC-A (approximately 85%) in co-expression studies where the amount of wild-type HCAT or full-length GC-A was not altered. Unlike co-transfection with wild-type HCAT, co-transfection of HCATD893A and GC-A did not result in elevated basal intact cell cGMP concentrations. For the first time we describe deletion and point mutations within the plasma membrane family of guanylyl cyclase receptors that result in the formation of effective dominant negative proteins.
Effective inhibitors of specific guanylyl cyclase signaling pathways have not been available, but would prove valuable in determining the functions of cGMP and of the individual cyclase receptors in the many cells throughout the body where the role of this cyclic nucleotide is not understood.
Characteristic of the adenylyl
and guanylyl cyclases is the existence of at least two cyclase
catalytic consensus domains within an active enzyme. The various forms
of vertebrate adenylyl cyclase contain two internal cyclase homology
domains(1, 2, 3, 4, 5, 6) ,
where the separate expression of either region results in a loss of
enzyme activity(7) . Cytoplasmic forms of guanylyl cyclase
apparently require the co-expression of two subunits ( and
)
for activity(8, 9) , each of which contains a cyclase
homology domain(10, 11, 12) , and plasma
membrane forms of guanylyl cyclase have been suggested recently to
exist as dimers or higher-ordered structures even in the absence of
ligand(13, 14, 15) .
Since two consensus
cyclase catalytic domains may be required for enzyme activity, the
construction of cyclase mutants that retained the ability to
oligomerize but not the ability to signal in response to ligand or the
ability to form cGMP would act as dominant negative mutations. Such
approaches have proved successful with various proteins, including the
family of receptor tyrosine
kinases(16, 17, 18, 19) . In these
cases, truncation mutants have been constructed by deletion of the
intracellular catalytic domain; the resulting extracellular domain
fragment is a potent dominant negative subunit which can combine with
the full-length receptor to form an inactive hybrid. The topological
resemblance of membrane guanylyl cyclase and receptor tyrosine kinases
led two groups to design a similar cytoplasmic truncation mutant of
GC-A, ()and although the full-length and mutant receptors
appeared to associate, the ANP-induced elevations of cyclic GMP were
not effectively blocked at high concentrations of ANP (13, 14) . Dominant negative mutations have been
generated of the soluble, heterodimeric form of guanylyl cyclase, but
given their inherent differences in structure, these constructs are of
no use in inhibiting membrane guanylyl cyclase.
Here, we considered the possibility that an extracellular truncation mutant would continue to dimerize with the full-length receptor and in so doing act as a dominant negative protein. This turned out to be the case, but since the cytoplasmic fragment contained cyclase activity, intracellular cGMP was elevated in the basal state. We then tested a series of point mutants and found one (D893A) that inactivated the cyclase when combined with wild-type receptor. Thus, the introduction of the point mutant not only interrupts ligand signaling, but also blocks cGMP production by the receptor.
Figure 1: Schematic diagram of various GC-A constructs. Wild-type GC-A contains an extracellular ligand-binding domain, a transmembrane domain, and intracellularly, protein kinase homology (PKH), hinge, and catalytic homology domains. HCAT contains a small portion of the PKH domain and the entire hinge and catalytic domains. CAT encodes only the catalytic homology domain.
In order to obtain a concentrated lysate, typically 20-40 100-mm plates of transfected cells were pooled and lysed into 2 ml of harvest buffer to yield a supernatant fraction of approximately 10 mg protein/ml. This solution (100 µl) was loaded onto a Bio-Rad Bio-Sil SEC-125 column. The elution buffer was identical to the harvest buffer. Fractions were collected at 0.2-min intervals (200 µl/fraction). Protein standards from Pharmacia Biotech Inc. and Bio-Rad were chromatographed to obtain a calibration curve.
For electrophoresis of GC-A co-transfectants, 7.5% SDS-PAGE was used. For native gels, SDS was omitted from all buffers.
For whole-cell stimulations, transfected cells were split to 6-well (35-mm) plates, washed with serum-free medium, incubated at 37 °C in medium + 0.1 M IBMX for 10 min, and then incubated for 10 min in 1 ml of fresh medium, 0.1 mM IBMX plus various concentrations ANP. Stimulations were stopped with 1 ml 1 N perchloric acid, and cGMP was measured by radioimmunoassay, following cyclic nucleotide purification, as described previously(26) .
Figure 2: Migration of HCAT on gel permeation columns. A, migration of HCAT in the presence of 100 mM NaCl, 25 mM HEPES, pH 7.4, and 10 mM dithiothreitol. The soluble fraction (100 µl) of pSVLCAT-transfected COS cells was loaded onto a Bio-Sil SEC-125 column, and fractions (200 µl) were collected at 0.2-min intervals. Main panel, enzyme activity of the various fractions collected. Aliquots (50 µl) were assayed as described under ``Experimental Procedures.'' Elution positions of molecular mass standards are indicated. Inset, immunoreactivity of the same fractions. Aliquots (50 µl) of each fraction were run on 10% SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal antibody R1215, which recognizes the carboxyl terminus of HCAT. B, migration of HCAT in the presence of the sodium trichloroacetate. Gel filtration was performed as in part A, except the column was equilibrated with elution buffer containing 0.3 M sodium trichloroacetate. Samples were run on 10% SDS-PAGE and transferred to nitrocellulose for Western blot analysis. Molecular mass standards, also run in the presence of sodium trichloroacetate, are indicated.
CAT, the construct containing 58 fewer amino acids at the amino terminus was devoid of catalytic activity, and all immunoreactive material migrated at the void volume of the gel filtration column, suggesting that it was denatured (data not shown). Thus, amino acids contained within the hinge domain (or possibly at the very carboxyl terminus of the defined protein kinase homology domain) are integral to forming a properly folded active catalytic site, at least in COS cells. The hinge domain is highly conserved among membrane forms of guanylyl cyclase and resembles the coiled coil sequences which mediate association in some oligomeric proteins(27) ; thus it is possible that this region mediates dimerization of two catalytic domains to form an active site.
Figure 3:
The effects of co-transfection of HCAT and
of GC-A on cGMP concentrations and guanylyl cyclase activity in COS
cells. Cells were transfected with pCMV-GC-A and either pCMV5 or
pCMV5-HCAT. A, intact cells were treated with various
concentrations of ANP for 10 min, and cGMP concentrations were measured
by radioimmunoassay. B, cells were homogenized and assayed for
cyclase activity in the presence of 100 µM GTP, 5 mM MgCl, 1 mM ATP, and various concentrations of
ANP, and cGMP levels were measured by radioimmunoassay. For both
experiments, data for experimental co-transfectants (GC-A + HCAT) are presented as percent of control co-transfectants (GC-A + vector), and points represent duplicate or
triplicate determinations within a single representative
experiment.
Such inhibition by HCAT is most likely due to the formation of
GC-A/HCAT heterodimers which are reduced or deficient in the ability to
respond to ANP. However, to rule out the possibility that inhibition of
HCAT was due to a reduction in expression of GC-A, immunoblots of cell
homogenates were examined. Cells co-transfected with GC-A and either
vector or HCAT each expressed the same levels of GC-A (Fig. 4).
Routing of GC-A to the cell surface apparently also was not altered,
since immunoreactive GC-A in purified plasma membranes remained
constant as well (not shown). Binding of I-ANP to whole
cells was equivalent between GC-A/HCAT co-transfectants and cells
expressing GC-A alone. For GC-A/HCAT versus GC-A cells, K
values were 4.2
10
and 3.1
10
M, respectively,
and B
values were 2.4
10
and 2.0
10
M, respectively.
Therefore, since the amount of GC-A (estimated from immunoblots) is not
reduced by co-expression of HCAT, the equivalent binding values suggest
that ANP binds equally as well to monomeric as to oligomeric GC-A.
HCAT, therefore, did not inhibit cyclic GMP elevations or cyclase
activation by ANP as a result of an inhibition of ligand binding.
Figure 4: Western blot of co-transfectants to monitor protein expression. COS cells were transfected with pCMVGC-A and either vector (A), pCMV5HCAT (B), or PCMV5HCATD893A (C). Cells were harvested, as described under ``Experimental Procedures,'' and equal amounts of protein were run on 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal antibody R1215, which recognizes the carboxyl termini of both the fragments and full-length GC-A. Molecular weight markers are indicated, and GC-A and the fragments are designated by bullets.
Several
invariant or highly conserved residues throughout the catalytic domain
were targeted for alanine scanning mutagenesis; this included
Gly, an amino acid in the same relative position as the
glycine of rutabaga, the Drosophila adenylyl cyclase
mutant without activity, where glycine is mutated to arginine (28) . Gly
, along with charged residues
Asp
, Lys
, Asp
,
His
, Arg
, His
, and
Glu
were converted to alanine in HCAT. The HCAT point
mutants were then transfected transiently into COS cells and examined
by immunodetection to confirm protein size (data not shown) and assayed
for guanylyl cyclase activity (Fig. 5). One mutant protein,
E974A, displayed substantially higher activity (approximately
30-40-fold higher activities at 100 µm of MnGTP and
equivalent amounts of enzyme protein based on immunoblots) as compared
to wild-type HCAT. The remaining mutations, including G900A, resulted
in no detectable cyclase activity.
Figure 5:
Specific activity of various point mutants
of HCAT. Point mutants were introduced into HCAT as described under
``Experimental Procedures,'' transiently transfected in COS-7
cells, and assayed for guanylyl cyclase activity in the presence of
MnCl. Bars represent the mean of triplicate
determinations in a single representative experiment. The specific
activity of vector-transfected cells was less than 1 pmol
min
/mg protein.
Each of the mutant proteins was analyzed by gel filtration to determine apparent size. E974A, the superactive mutant, and one other mutant, D893A, eluted as homodimers (data summarized in Fig. 5). The remaining point mutants D877A, K887A, H909A, R940A, and H944A, as well as the mutant corresponding to rutabaga G900A, eluted in the void volume as high molecular weight aggregates, presumably due to protein denaturation. These conserved residues, as well as the rutabaga glycine, therefore, could play critical roles in protein folding.
Figure 6:
Co-transfection of HCAT and the point
mutant HCATD893A. COS-7 cells were transiently transfected with 1
µg of HCAT and either no additional DNA, increasing amounts of
HCATD893A, or increasing amounts of vector. Cells were harvested and
assayed for cyclase activity in the presence of
Mn/Triton X-100 as described under
``Experimental Procedures.'' Bars represent the
means ± standard error of triplicate determinations of a single
representative experiment.
Figure 7:
Co-transfection of GC-A and HCATD893A.
COS-7 cells were transiently transfected with 0.1 µg of GC-A, and
0.1 or 0.5 µg of either HCATD893A or vector. Cells were harvested
and assayed for cyclase activity in the presence of
MnCl/Triton X-100 as described under ``Experimental
Procedures.'' Bars represent the means ± standard
error of triplicate determinations of a single representative
experiment.
Figure 8:
Effect of GC-A/HCATD893A co-transfection
on cGMP concentrations of intact cells and on guanylyl cyclase
activity. Cells were transfected with pCMV-GC-A and either vector or
pCMVHCATD893A. A, intact cells were treated with various
concentrations of ANP for 10 min, and cGMP concentrations were measured
by radioimmunoassay. B, cells were homogenized and assayed for
cyclase activity in the presence of 100 µM GTP, 5 mM MgCl, 1 mM ATP, and various concentrations of
ANP, and cGMP levels were measured by radioimmunoassay. For both
experiments, data for experimental co-transfectants (GC-A + HCATD893A) are presented as percent of control co-transfectants (GC-A + vector), and points represent duplicate
or triplicate determinations in a single representative
experiment.
Thus,
like HCAT, HCATD893A is a dominant negative inhibitor of signal
transduction by GC-A. In addition, and unlike HCAT, HCATD893A is also a
dominant negative inhibitor of cyclase catalysis. Presumably, aspartic
acid 893 is critical to enzyme activity, perhaps by contributing to the
structure of the active site, or participating, by benefit of its
charge, to catalysis itself. The importance of this residue is borne
out not only by its high conservation among all cyclases, but more
recently by the finding that alanine substitution made at the analogous
aspartic acid in the subunit of soluble guanylyl cyclase serves
as a potent dominant negative inhibitor when recombined with wild-type
subunit(29) . Elsewhere, it has been demonstrated that
catalysis by guanylyl and adenylyl cyclases proceed through the same
stereochemical course(30) , thus it will be interesting to see
if mutation of the analogous residue in adenylyl cyclase confers the
same inhibition.
Mutation by truncation is commonly used to engineer dominant negative mutants and has been attempted previously for membrane guanylyl cyclase. Chinkers and Wilson (13) and Lowe (14) each expressed the extracellular domain of GC-A, and when co-transfected with full-length GC-A it caused a shift to the right of the ANP dose-response curve. However, maximal cGMP elevations were not suppressed by the mutation. Given that GC-A may form tetramers, it remains possible that dimers of full-length/intracellular domain forms are capable of forming fully active tetramers.
Here, an intrinsically active intracellular fragment functions as a dominant negative inhibitor of signal transduction by full-length GC-A, even at high levels of ANP. Further mutation, alanine substitution at a highly conserved aspartic acid, also serves as a dominant negative inhibitor, in this case, of catalysis. Given the widespread expression of GC-A and its involvement in many endocrine and possibly paracrine functions, it would be desirable to examine this receptor in intact animals. These mutants may serve as useful reagents for examining GC-A function in vivo, much like dominant negatives have been used in analogous studies to examine the role of fibroblast growth factor acting through the fibroblast growth factor receptor (18) and RAF-1 kinase (31) to induce mesoderm development. In the regulation of blood pressure and volume via natriuresis and diuresis in the kidney, smooth muscle relaxation in the cardiovasculature, and aldosterone secretion by the adrenals, the importance of membrane guanylyl cyclase has been acknowledged, although the pathways have not been precisely detailed. The selection of tissue-specific promoters could lead to a dominant negative effect in limited areas in transgenic animals, allowing one to dissect the role of guanylyl cyclase receptors in multiple selected tissues. Finally, construction of distinct dominant negative mutants specific for either membrane or soluble forms of guanylyl cyclase should permit the two pathways to be studied independently, even if they are present in the same cell.