(Received for publication, October 20, 1995; and in revised form, December 29, 1995)
From the
A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E.(1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms.
The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA.
The four GRK4
proteins have been expressed, and all incorporate
[H]palmitate. GRK4 is capable of augmenting the
desensitization of the rat luteinizing hormone/chorionic gonadotropin
receptor upon coexpression in HEK293 cells and of phosphorylating the
agonist-occupied, purified
-adrenergic receptor,
indicating that GRK4 is a functional protein kinase.
G protein-coupled receptor kinases (GRKs) ()are a
family of serine/threonine protein kinases that phosphorylate G
protein-coupled receptor proteins (1, 2) . Receptors
are phosphorylated by GRKs in vitro on multiple serine and
threonine residues on intracellular loops and/or the carboxyl-terminal
tail. GRK-phosphorylated receptors are bound by arrestin proteins,
leading to the functional removal of the receptor from the signaling
pathway by preventing further receptor coupling to G
proteins(3) . Agonist-occupied, activated G protein-coupled
receptors are phosphorylated by GRKs, but inactive receptor proteins
are not recognized by these kinases as
substrates(1, 2) . The GRKs, along with arrestin
proteins that bind to phosphorylated receptors and prevent coupling to
G proteins, mediate homologous desensitization of hormonal
responses(1) .
Six GRKs have been identified by purification
and by cloning. Rhodopsin kinase (GRK1) in the visual system
phosphorylates light-bleached rhodopsin on its carboxyl-terminal
tail(1, 3) . Arrestin bound to phosphorylated
rhodopsin prevents the further activation of transducin, dampening
activation of retinal cGMP phosphodiesterase(3) . The
``-adrenergic receptor kinases,'' GRK2 and GRK3, were
identified by their functional ability to phosphorylate
-adrenergic receptors and are widely distributed
throughout the body(2) . GRK2 and GRK3 have been shown to
phosphorylate a variety of G protein-coupled hormone and
neurotransmitter receptors (2) . Together with the somatic
-arrestin proteins, GRK2 and GRK3 have been implicated in the
uncoupling of receptors from their respective G proteins, a mechanism
leading to homologous desensitization(1, 3) .
A
putative member of the family of G protein-coupled receptor kinases was
recently identified by a positional cloning strategy, due to the
proximity of its gene to the Huntington's disease
locus(4) . Originally named IT11, this sequence has been
renamed GRK4 in an effort to systematize the nomenclature of these
enzymes(1) . GRK4 is most similar in sequence to mammalian GRK5 (5) and GRK6 (6) and to Drosophila GPRK2(7) . Together, these sequences define a subfamily of
GRK enzymes that is distinct from the more extensively characterized
rhodopsin kinase and -adrenergic receptor kinase subfamilies. Both
GRK5 and GRK6 have been functionally expressed and shown to
phosphorylate several G protein-coupled
receptors(5, 6, 8, 9, 10) .
Functional activity of the GRK4 kinase has not yet been reported, and
GRK4 is the least well understood member of the GRK family. Sallese et al.(11) have recently described an alternatively
spliced exon encoding 32 amino acids in the amino-terminal region of
human GRK4. In this paper, we define the structure of the human GRK4 gene and show that the GRK4 gene transcript
undergoes extensive alternative splicing to generate four distinct
forms of GRK4 mRNA that encode four forms of the GRK4 protein. We
demonstrate the functional ability of GRK4 to augment desensitization
of the LH/CG receptor in transfected cells and to phosphorylate the
agonist-occupied, purified
-adrenergic receptor.
The amino-terminal coding region of the GRK4 cDNA was amplified from human testis first-strand cDNA using the primers 5`-ctcctcggtctcgcagaatcc (in the 5`-untranslated region) and 5`-aggtagttatgggcaactcta (antisense to RVAHNYL), while the carboxyl-terminal coding region was amplified using the primers 5`-aacatgggatccccccctttctgtcctgat (encoding PPFCPD) and 5`-gcaccggaattctcagcattgcttgggttc (antisense to EPKQC*). Product DNA bands were cut from agarose gels and TA subcloned into the pCRII vector (Invitrogen) or digested with BamHI and EcoRI (underlined sites) and subcloned into pBSII (Stratagene).
Affinity-purified GRK4 antipeptide antibody raised against a peptide
sequence from GRK4 was obtained from Santa Cruz Biochemicals. The
antigen peptide (IPWQNEDCLTMVPSEKEVEP) is interrupted in the GRK4
and GRK4
sequences by the addition of exon XV, and this antibody
was found to recognize only GRK4
and GRK4
(data not shown).
Immunoblotting was performed as described(5) , using the crude GRK4-CT antiserum at 1:2500 dilution or affinity-purified antibody at 1 µg/ml. Briefly, protein samples were separated by SDS-PAGE on 10% acrylamide gels, transferred to nitrocellulose membranes, and blocked by incubation with 3% bovine serum albumin in phosphate-buffered saline. Primary antiserum in 3% bovine serum albumin was allowed to bind for 1 h and washed, and the membrane was incubated for 1 h with goat anti-rabbit IgG-alkaline phosphatase conjugate (Pierce) for 1 h. Avidin-alkaline phosphatase conjugate was included to detect biotinylated molecular weight standards (Bio-Rad). After washing, the membrane was developed using Western blue reagent (Promega).
Recombinant GRK4 baculoviruses were
produced by cotransfection of Sf9 cells with pVL1393 vectors bearing
individual GRK4 inserts and BaculoGold baculovirus DNA (Pharmingen).
Expression of GRK4 forms in Sf9 cells was induced by infection of 1.5
10
cells/ml with the appropriate recombinant
baculovirus at a multiplicity of infection of 5. Cells were grown for
48 h after infection; harvested by centrifugation; and stored frozen in
20 mM HEPES, pH 7.2, 5 mM EDTA, and 20 mM NaCl supplemented with a mixture of protease inhibitors (2
µg/ml aprotinin, 10 µg/ml benzamidine, 4 µg/ml leupeptin, 1
µg/ml pepstatin, and 100 µM phenylmethylsulfonyl
fluoride) (buffer A).
Human -adrenergic receptors were purified from
baculovirus-infected Sf9 cells by alprenolol affinity chromatography
and reconstituted into phospholipid vesicles as described(23) .
Receptors were reconstituted in vesicles of 100% phosphatidylcholine or
in vesicles of 5% phosphatidylinositol 4,5-bisphosphate
(PIP
) and 95% phosphatidylcholine. Lipids were all 99% pure
grade from Sigma.
All product bands were subcloned and sequenced. Comparison of the DNA sequences of the short and long PCR-amplified clones indicated that the longer clones were analogous to the shorter clones and to the previously reported GRK4 sequence, with additional nucleotide sequences occurring precisely at the predicted gaps, for each of the amino- and carboxyl-terminal products. These insertions each maintained the open reading frame, and deduced amino acid sequences of these insertions corresponded well to sequences found in GRK5 and GRK6 (Fig. 1). The amino-terminal insertion is identical to that reported by Sallese et al.(11) .
Figure 1:
Human GRK4 alternative exons. The
deduced amino acid sequences of the alternatively spliced exons within
the amino- and carboxyl-terminal regions of GRK4 were aligned with the
cognate regions of bovine GRK5(5) , human GRK6(6) , and
bovine rhodopsin kinase (RhK)(37) . Residues identical
in all sequences are indicated (*), as are residues conserved among at
least three sequences (). One-letter amino acid code is used.
Conserved amino acids are defined as follows: A, G, P, S, T; C; D, E,
N, Q; I, L, M, V; F, W, Y; H, K, R.
Figure 2: Organization of the human GRK4 gene. Individual exons of the human GRK4 gene are shown as boxes, and tick marks indicate EcoRI sites. The alternative splicing of exons II and XV is indicated by the thin lines above and below the exon map. Exons were identified by sequencing from cosmid DNAs. Intron sizes were determined by PCR amplification of cosmid and genomic DNAs from primers in flanking introns. The size of intron A was estimated based on the overlapping cosmid restriction map, as exons I and II were not both present on any single cosmid and failed to amplify from genomic DNA. Exons were assigned to restriction fragments by Southern blotting of cosmid DNAs and compared with the known restriction map of this region of chromosome 4(15) .
Several differences were noted between the sequences obtained here
and the originally reported sequence(4) , and several
polymorphisms were noted within the samples analyzed here. All
numbering refers to the longest (GRK4) form reported here.
Gly
was observed in all cDNA clones from two patient cDNA
samples, in amplified genomic fragments from an independent patient,
and in cosmid BJ56, rather than Asp as described previously. The
original IT11A clone (4) was resequenced in this region and
found to encode Asp
as reported. In one patient cDNA
sample, all clones contained Leu
, Val
instead of Arg
, Ala
, which was found
in all other cDNA and genomic DNA samples. Cosmid L142C5 encodes
Ile
instead of Val. Cosmid BJ56 carries a silent C
T polymorphism within Ser
. Both cDNA samples, one genomic
DNA sample, and cosmid BJ56 all encode Ala
, while cosmid
BJ56w4-3 encodes Val
, the one previously reported
polymorphism in the human GRK4 sequence(4) .
Figure 3: Northern analysis of GRK4 mRNA. A, the tissue distribution of GRK4 mRNA was determined by Northern blotting using mRNA obtained from the indicated human tissues. The probe was the IT11A clone, which lacks both amino- and carboxyl-terminal alternative exons. Size standards indicated on the left are 9.5, 7.5, 4.4, 2.3, and 1.35 kb. B, the presence of alternative exon II (Alt-N) and alternative exon XV (Alt-C) in human testis mRNA was determined by hybridization with oligonucleotide probes antisense to sequences within exons II and XV, respectively. Size markers are as described for A.
The IT11A clone used as probe above contains neither alternative exon. To ascertain whether these alternative exons are also present in the mRNA in significant levels, Northern blots were probed using antisense oligonucleotide probes specific for exons II and XV. As shown in Fig. 3B, exon II and XV probes each recognized a 2.5-kb mRNA from human testis. The ready detectability of both amino- and carboxyl-terminal alternative exons in testis mRNA indicates that they are highly abundant within the GRK4 mRNA pool, implying that the longest mRNA form may be the most abundant. The two oligonucleotide probes recognized no other RNA bands in 15 other human tissues tested (data not shown). The similarity in size among the GRK4 splice variant mRNAs is not surprising as only small internal sequences are alternatively spliced.
Figure 4: Sf9 cell expression of GRK4 proteins. Sf9 cells infected with individual GRK4 baculoviruses were separated into soluble and membrane fractions. Equivalent volumes were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-GRK4-CT antiserum. Size standards indicated on the left are 200, 116, 97, 66, and 45 kDa.
Figure 5:
Palmitoylation of GRK4 proteins. Upper
panel, COS-7 cells transfected with individual GRK4 cDNAs were
labeled with a [S]methionine/cysteine mixture.
Immunoprecipitated GRK4 proteins were separated by SDS-PAGE, and dried
gels were exposed to x-ray film for 1 day. Lower panel, COS-7
cells transfected with individual GRK4 cDNAs were labeled with
[
H]palmitate. Immunoprecipitated GRK4 proteins
were separated by SDS-PAGE, and the gel was treated with fluor. The
dried gel was exposed to x-ray film for 2 months. A representative
experiment (of three) is shown.
We therefore chose to examine
the ability of GRK4 proteins to modulate signaling by the rat LH/CG
receptor transiently expressed in HEK293 cells. Individual GRKs were
cotransfected with LH/CG receptor cDNA, and cAMP accumulation was
assessed after stimulation of [H]adenine-labeled
cells with human CG (Fig. 6). In the absence of added GRK cDNAs,
human CG produces a dose-dependent cAMP accumulation (data not shown).
In the presence of GRK2 cDNA, a GRK that is known to be active on many
G protein-coupled receptors, human CG-induced cAMP accumulation is
reduced by 38% at 200 ng/ml human CG. This loss of stimulation reflects
GRK-mediated processes occurring in the continued presence of the
hormone, which occur naturally but are augmented in the presence of
exogenous GRK2(9, 25) . Transfected GRK4
induces
a degree of desensitization (36% decrease at 200 ng/ml human CG) that
is comparable to that achieved by transfected GRK2. Similar results
were observed at 50 ng/ml human CG (data not shown).
Figure 6:
A, HEK293 cells transfected with LH/CG
receptor cDNA and individual GRK cDNAs (GRK2 (2) and GRK4 (4
)) or empty vector (V) were labeled with
[
H]adenine, and cAMP accumulation stimulated 200
ng/ml human CG was measured. Data were normalized to the activity of
cells lacking added GRKs, which varied from 3 to 8% conversion of ATP
to cAMP in individual assays. Data are shown as mean ± S.E. for
three individual experiments performed in triplicate. Values
significantly different (p < 0.05) from vector control, as
assessed in t tests, are indicated (*). B, HEK293
cells transfected with LH/CG receptor cDNA and individual GRK4 cDNAs
(GRK4, GRK4
, GRK4
, and GRK4
) or empty vector (V) were labeled with [
H]adenine, and
cAMP accumulation stimulated by 200 ng/ml human CG was measured. Data
are shown as mean ± S.E. for three individual experiments
performed in triplicate. Activity of cells lacking added GRKs was
defined as 100% (3-6% conversion of ATP to cAMP). Values
significantly different (p < 0.05) from vector control, as
assessed in t tests, are indicated
(*).
Individual GRK4
variant cDNAs were each cotransfected with LH/CG receptors to assess
their functional ability. Human CG-stimulated cAMP accumulation was
reduced in the presence of GRK4, GRK4
, and GRK4
to a
similar extent, with 34-43% reduction at 200 ng/ml human CG
(equivalent to that seen with GRK2). In contrast, GRK4
produced a
more modest 20% loss of LH/CG receptor signaling at 200 ng/ml human CG.
Similar results were observed at 50 ng/ml human CG for GRK4
,
GRK4
, and GRK4
, although GRK4
did not appear
significantly different from the control at this concentration (data
not shown). Thus, all four GRK4 variants appear capable of decreasing
signaling through the G protein-coupled LH/CG receptor.
Figure 7:
In
vitro phosphorylation of the -adrenergic receptor
by GRK4
. One pmol of
-adrenergic receptor (
2AR) reconstituted into phospholipid vesicles (either
100% phosphatidylcholine (PC) or 5% PIP
and 95%
phosphatidylcholine) was incubated with partially purified GRK4
in
the presence of 100 µM isoproterenol (ISO) or 100
µM propranolol (PRO). Reactions were separated by
SDS-PAGE, and dried gels were exposed to x-ray film. A representative
experiment (of three) is shown.
Characterization of the genomic organization of the human GRK4 gene verified the existence of two alternatively spliced
exons identified by polymerase chain reaction amplification of the GRK4
cDNA. The GRK4 gene is composed of 16 exons separated by 15
introns and spans over 75 kb of chromosomal DNA. Except for the first
intron, which is 16-20 kb long, the GRK4 introns average 4 kb in
length. In the human GRK2 gene, 21 exons are found over only
23 kb since all introns except the first average only 500 base
pairs(29) . Partial characterization of the mouse GRK3 gene reveals an organization identical to that of the GRK2 gene, but containing significantly longer average intron
sequences. ()Comparison of the structure of the GRK4 gene with those of GRK2 and GRK3 reveals that
all intron locations in the GRK4 gene are distinct from those
in the GRK2 and GRK3 genes. Even the exons composing
the conserved catalytic domain are completely distinct, which was
unexpected for such closely related protein kinases. However, cloning
of GRK cDNAs highly similar to GRK2 (dGPRK1) and GRK4 (dGPRK2) from Drosophila melanogaster(7) clearly demonstrates that
the precursors of the GRK2 and GRK4 genes diverged
before insects diverged from the lineage leading to chordates. GRK2 and
GRK4 may be seen as models for their two subfamilies of GRKs, and it
seems probable that the organization of the genes for the GRK4-related
GRK5 and GRK6 enzymes will be more similar to that of the GRK4 gene. GRK1 (rhodopsin kinase) is intermediate in similarity
between GRK2 and GRK4, so it will be interesting to compare the
structure of the GRK1 gene with those of GRK2 and GRK4.
The GRK4 gene transcript undergoes alternative splicing to yield four distinct forms of GRK4 mRNA. This occurrence of alternative splicing is unique among the known GRK enzymes. The four GRK4 mRNAs arise from the presence or absence of exon II and/or exon XV. These alternative exons maintain the open reading frame and encode four GRK4 proteins that differ in the presence or absence of 32 amino acids in the amino-terminal region (11) or 46 amino acids in the carboxyl-terminal region. The locations of these alternative exons within the GRK4 protein suggest that they may play important functional roles in regulation of the enzyme, while the catalytic domains of all GRK4 forms are identical.
In this work, we
have demonstrated that GRK4 is an active protein kinase with the
activated receptor substrate recognition expected of a GRK enzyme.
GRK4 phosphorylated purified
-adrenergic receptor
in the presence of isoproterenol, but only using vesicles that
contained PIP
. GRK4 appeared essentially inactive at
phosphorylating
-adrenergic receptors reconstituted
into pure phosphatidylcholine vesicles. GRK5 autophosphorylation (26) and activity,
GRK6 activity,
and
GRK2 activity (23, 27) all exhibit similar lipid
cofactor requirements. PIP
-dependent activity appears to be
an under-appreciated common feature of GRKs, although its physiological
significance remains unexplored. The previous inability to measure the
activity of GRK4
(11) may be due to use of a soluble cell
fraction rather than detergent-extracted proteins, use of rhodopsin as
a substrate, or lipid composition of the rod outer segment membranes.
The carboxyl-terminal domain of GRKs is involved in their
subcellular localization. For GRK1 (rhodopsin kinase), the primary
amino acid sequence encodes a CAAX box that directs
post-translational farnesylation, proteolysis, and
carboxymethylation(30) . GRK1 is a cytosolic enzyme in the rod
outer segment, but translocates to the membrane upon light activation
of rhodopsin. A GRK1 mutant that lacks a CAAX motif fails to
be prenylated and exhibits a deficit in translocation(31) .
GRK2 and GRK3 also appear cytosolic in unstimulated cells, but
translocate to the cell membrane upon agonist activation of G
protein-coupled receptors(31) . This translocation is due to
association of GRK2 with free G protein -subunits and
PIP
through a carboxyl-terminal region that includes a
pleckstrin homology domain(23, 32, 33) .
Removal of this carboxyl-terminal region renders GRK2 unable to
translocate to activated receptors(33) . GRK4 contains neither
a CAAX motif for protein prenylation nor a G protein
-subunit-binding domain.
GRK5 and GRK6 also lack
prenylation or -subunit binding, but exhibit a significant
degree of association with cellular membranes. GRK5 has a highly basic
carboxyl-terminal sequence, which has been proposed to serve to anchor
GRK5 to phospholipids in the membrane(5) . GRK4 also has a
basic carboxyl-terminal sequence, which is present only in the two
variants containing exon XV (GRK4
and GRK4
). The absence of
this basic domain in GRK4
and GRK4
did not lead to striking
losses in the apparent membrane association of these proteins. The
carboxyl-terminal region of GRK6 has no basic region, but contains
cysteine residues that have been identified as sites for the
post-translational palmitoylation(19) . Palmitoylated GRK6 is
found only associated with cellular membranes(19) . All four
GRK4 proteins incorporate palmitate, presumably via their
carboxyl-terminal cysteine residue or on an internal cysteine residue
conserved with GRK6. While the functional role of GRK4 and GRK6
palmitoylation remains to be explored, this lipid clearly contributes
to membrane localization of the proteins. In addition, palmitoylation
and depalmitoylation are dynamic processes within the cell, so it is
possible that palmitoylation of GRK4 and GRK6 may be regulated in a
signal-dependent manner. Regulated palmitoylation and depalmitoylation
have been observed for other signal transduction
components(34, 35) .
Although the amino-terminal domain of the GRKs is thought to be involved in recognition by the kinase of the activated G protein-coupled receptor, much less is known about the role of this region of the kinases(1, 2) . An antipeptide antibody raised to the GRK1 amino terminus has been shown to block recognition of activated rhodopsin(36) . It remains possible that the amino-terminal splice variants of GRK4 differ in their receptor substrate preferences or activation-dependent recognition requirements. Testing these possibilities will require comparing the activity of GRK4 variants to recognize and phosphorylate several distinct receptor substrates.
Comparison of the ability of
the four GRK splice variant proteins to augment desensitization of the
LH/CG receptor in transfected cells revealed that all GRK4 forms are
functional. The GRK4, GRK4
, and GRK4
variants appeared
as active as GRK2, while the GRK4
variant appeared weaker. This
assay may be insensitive to subtle differences among the kinases, as
the expression level of the various enzymes was not directly
quantified. However, all of the GRK4 forms appear to have some
activity, so the alternative splicing does not render any forms
completely inactive. More quantitative comparisons among the four
variants will require purification of all four enzymes to equivalent
extents for assay against various substrate receptors.
The relative
abundance of the four native GRK4 proteins is unknown. In the
amplification of the GRK4 amino- and carboxyl-terminal regions, the
longer variant cDNA bands were more abundant, suggesting a predominance
of exon II- and exon XV-containing mRNAs. This is in contrast to the
report of Sallese et al.(11) , who observed that the
short amino-terminal form of GRK4 was most common in brain cDNA. Direct
immunochemical detection of GRK4 proteins in testis has proven
unsuccessful, although specific affinity-purified antibodies should
allow this question to be addressed. Affinity-purified antibodies
raised against a GRK4 carboxyl-terminal peptide obtained
commercially also failed to recognize native GRK4
and GRK4
proteins in testis membranes or cytosol (data not shown), supporting
the hypothesis that the longest (GRK4
) form may be the most
abundant form in testis.
It is noteworthy that GRK4 mRNA is
essentially limited to testis. Very low levels of GRK4 mRNA are present
in several other tissues, including brain(4, 11) . Of
the known GRKs, only the retinal GRK1 has such a limited tissue
distribution (37) and thus a defined native substrate receptor,
rhodopsin. The widespread and generally overlapping distributions of
GRK2, GRK3, GRK5, and GRK6 hamper efforts to understand their
individual functions. Little is yet known about the receptor substrate
preferences of GRKs, although certain receptors can be phosphorylated
equivalently by many GRKs(8, 9, 38) . The
limited distribution of GRK4 may facilitate definition of the G
protein-coupled receptors it regulates. Although it is not yet known
which of the three main testis cell types express GRK4, several
receptors are known to be present in each cell type(39) .
Leydig cells express LH/CG receptor and gonadotropin-releasing hormone
receptor. Sertoli cells contain follicle-stimulating hormone receptor
and glucagon receptor. The various germ cell stages express bombesin
BB receptors and a variety of olfactory-like receptors. In
addition, several G protein-coupled receptor mRNAs are expressed at
high levels in testis, although their cellular localizations have not
been defined. These include the adenosine A
receptor,
cannabinoid CB
receptor, and vasopressin V
receptor, as well as several ``orphan'' receptors. All
of these receptors are potential substrates for GRK4. Further studies
characterizing the function of GRK4 will focus on regulation of those
receptors expressed in the testis cell type(s) determined to contain
GRK4. Differential regulatory properties of the individual GRK4
variants may be more observable on those receptors to which the enzymes
normally have access.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33054[GenBank], U33055[GenBank], U33056 [GenBank](X75897[GenBank]), L03718[GenBank], and U33153[GenBank]-U33168[GenBank].