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2C-Adrenergic but Not Serotonin 1B Receptors
Department of Pharmacology and Therapeutics (P.M.C.L.,
M.H.G.) McGill University Montreal, Quebec,
Canada H3G 1Y6
Departments of Medicine and Cellular
and Molecular Medicine (P.R.A.) Neuroscience Research
Institute University of Ottawa Ottawa, Ontario, Canada K1H
8M5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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2C-adrenergic and serotonin 1B
(5-HT1B) receptors in the opossum kidney (OK) cell line, we have
isolated a 3.073-kb OK-GRK2 clone encoding a 689-amino acid protein
that shares 94.2% amino acid identity with rat GRK2. Northern blot
analysis revealed the presence of GRK2 mRNA transcripts of 5.0 and 3.0
kb in OK cells. In intact OK cells, preincubation (45 min) with agonist
(5-HT or UK 14304, 1 µM) reduced the maximal
inhibition of forskolin-induced cAMP accumulation mediated by
endogenous 5-HT1B and 2C-adrenergic receptors by
12 ± 2% or 17 ± 4%, respectively. In transfected OK cells
overexpressing OK-GRK2, agonist-induced desensitization of the
2C-adrenergic receptor, but not the 5-HT1B
receptor, was enhanced by 2- to 4-fold. Conversely, in cells
overexpressing the kinase-inactive mutant OK-GRK2-K220R,
2C-adrenergic receptor desensitization was
selectively abolished, whereas desensitization of the 5-HT1B receptor
was slightly enhanced. Similarly, depletion of GRK-2 protein by stable
transfection of full-length antisense OK-GRK2 cDNA blocked the
desensitization of 2C-adrenergic receptors but not of
5-HT1B receptors. These results represent the first evidence of the
coexistence of GRK2-dependent (for 2C receptors) and
GRK2-independent (for 5-HT1B receptors) mechanisms of desensitization
in intact cells and demonstrate the selectivity of GRK2 for distinct
Gi-coupled receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Acute receptor desensitization has historically been viewed as two separate mechanisms: heterologous and homologous desensitization. Heterologous desensitization is a process whereby activation of one type of receptor leads to the desensitization of nonstimulated receptors, whereas homologous desensitization refers to the loss of responsiveness of the stimulated receptor only. These rapid forms of desensitization appear to involve primarily the phosphorylation of the receptor on serine and threonine residues located in the intracellular cytoplasmic loops (1, 2, 3). There are at least two distinct classes of kinases involved in this phosphorylation process: the second-messenger kinases [e.g. protein kinase C (PKC), protein kinase A (PKA), and second-messenger independent receptor kinases termed G protein-coupled receptor kinases (GRKs) (2)].
GRKs are a family of serine/threonine kinases that phosphorylate G
protein-coupled receptors in the ligand-activated state (5, 6). They
belong to a family of kinases comprising six members (GRK1-GRK6) whose
activities are differentially regulated by G protein ß
-subunits,
phospholipids, or posttranslational modifications. Each kinase sequence
has a centrally located catalytic domain flanked by an amino terminus
that has conserved features and a carboxyl terminus that is highly
variable. The C-terminal domain directs localization of the kinases to
the membrane or receptor, by isoprenylation (via a CAAX box in GRK1),
association with membrane-bound G-ß-dimers (GRK2, GRK3), or by
direct interactions with membrane phospholipids (GRK4, 6) (5, 6, 7, 8, 9).
Mutations that inactivate either membrane translocation or kinase
activity inhibit GRK-induced receptor phosphorylation and
uncoupling (10, 11). The hallmark of these kinases is their specificity
for activated (e.g. agonist-bound) receptors. Once
phosphorylated, target receptors have an increased affinity for
ß-arrestins, and the binding of ß-arrestin to the
GRK-phosphorylated receptor is thought to prevent receptor coupling to
G proteins (5, 6, 9). Thus, unlike receptor phosphorylation by PKC or
PKA that directly uncouples the receptor, GRK-induced uncoupling
requires both GRK and ß-arrestin molecules that recognize the
appropriate receptor (9).
The regulation of G protein-coupled receptors by GRKs has been
extensively documented in heterogeneous expression systems using
overexpression of kinase and receptor cDNAs (5, 6, 7, 8, 9). Phosphorylation of
receptors by GRKs has been shown to occur and regulate many G
protein-coupled receptor systems such as ß2- and
2A-adrenergic receptors, m2 muscarinic acetylcholine
receptors, rat olfactory receptor, and rhodopsin photoreceptor systems
(9, 12, 13, 14, 15). While some substrate specificity is observed in these
studies, the question remains whether the endogenous levels of
GRK/ß-arrestin are sufficient to mediate agonist-induced homologous
desensitization in nontransfected cells. Recently, GRK3 (ßARK2) was
shown to selectively regulate and desensitize thrombin-mediated
calcium mobilization in Xenopus oocytes (16) whereas GRK2
(ßARK1) and GRK3 were demonstrated to desensitize the inhibition of
voltage-dependent calcium channels mediated by the
2-adrenergic receptor in isolated intact chick sensory
neurons (17). In addition, Shih and Malbon (18) elegantly demonstrated
that, depending on the heterologous expression system used, either the
second messenger-dependent kinases (PKC or PKA) or the GRKs played
a predominant role in promoting agonist-induced uncoupling of the
ß2-adrenergic receptor. This raises the issue of
physiological relevance in the modulation of receptors by these
kinases. Hence, we decided to examine the influence of endogenous GRK
on the homologous desensitization of serotonin 1B (5-HT1B) and
2C-adrenergic receptors endogenously expressed in the
opossum kidney (OK) cell line (19, 20, 21, 22).
OK cells are an epithelial kidney cell line derived from the
North American opossum, Didelphis virginiana (23). These
cells endogenously express several G protein-coupled receptors
including 5-HT1B, 2C-adrenergic, dopamine-D1, and
PTH receptors (19, 21, 24, 25). These receptor subtypes each undergo
agonist-promoted desensitization with respect to either inhibitory or
stimulatory actions (20, 22, 26, 27). For example, both 5-HT1B and
2C-adrenergic receptors mediate inhibition of adenylyl
cyclase in membrane preparations and undergo acute homologous
desensitization of this response after sustained exposure to agonist
(20, 22). We have addressed whether the desensitization observed can be
detected in intact cell preparations by specifically measuring cAMP
generation in whole cells.
The objectives of the present study were to determine whether 1)
endogenous opossum GRKs were expressed in this cell line and 2) whether
they play a role in the homologous desensitization process of the
endogenously expressed G protein-coupled receptors. We have identified
in OK cells a GRK-2 homolog, OK-GRK2,2 and shown
that OK-GRK2 selectively mediated desensitization of the
2C-adrenergic receptor response, but did not interfere
with desensitization of 5-HT1B receptor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-ZAP II cDNA library constructed using OK cell RNA. A
3.073-kb cDNA clone (named OK-GRK2) that shared 71.5% nucleotide
identity with rat GRK2 cDNA sequence (Fig. 1
) was identified in 10 of 10 positive
clones. The OK-GRK2 cDNA contains a predicted open reading frame that
encodes a 689-amino acid protein with 94.2% and 80.8% amino acid
sequence identity with rat GRK2 and GRK3, respectively. The amino acid
sequences of OK-GRK2 and rat GRK2 share 99% identity in the catalytic
domain (Fig. 2, upper box),
90% in the amino terminus, 95% in the carboxyl terminus (Fig. 2, lower box); hence, the clone was designated OK-GRK2. In
these domains amino acid substitutions were conservative
(e.g. aliphatic-aliphatic); however, in less well conserved
regions, nonconservative substitutions were observed.
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, two distinct GRK-2-related mRNA
species with molecular sizes of approximately 5.0 kb and 3.0 kb were
detected. The intense band at 5.0 kb may correspond to the predominant
OK-GRK2 cDNA clone in Fig. 1
, whereas the smaller species may represent
an alternatively spliced variant or use of an alternate polyadenylation
site.
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, A and B). As observed
for in vitro-translated OK-GRK2 (data not shown), OK-GRK2
had the same mobility as purified bovine GRK2 (molecular mass, 80 kDa),
but was weakly detected in nontransfected OK cells, suggesting that the
endogenous level of OK-GRK2 is relatively low. Several clones for each
construct were isolated for further characterization; however, only
some of these maintained appropriate levels of expression during the
course of experiments, including the overexpressed sense construct
clones GRK-3 and GRK-18 and the kinase-inactive clone KI-5, which each
displayed increased level of GRK expression compared with wild-type OK
cells. The level of OK-GRK2 expression was greater in the GRK-18 clone
than in GRK-3 cells (Fig. 4A). The antisense clone AS-4 was the only
clone that maintained a depleted level of GRK2 expression as compared
with the wild-type (W.T.) OK cells (Fig. 4B).
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). After examination of the time course
of desensitization, cells were pretreated for 45 min using 1.0 to 1000
nM 5-HT, followed by a 12-min assay for receptor-mediated
inhibition of forskolin-stimulated cAMP accumulation using a maximal
concentration of 5-HT (1 µM). The agonist preincubation
time of 45 min was chosen as a point of maximal acute desensitization,
consistent with the half-time observed previously (20). The endogenous
2C receptors were also shown to undergo homologous
desensitization in the parental OK cell line when pretreated with
either 0.1 or 1 µM UK14304, a specific
2-adrenergic agonist (Table 1). In multiple assays, UK 14304 induced
significant desensitization at both 0.1 and 1.0 µM
concentrations, whereas 5-HT-induced desensitization was less potent
(Table 1). The 5-HT1B and 2C receptors displayed a
maximal level of desensitization of 12 ± 2 or 17 ± 4%,
respectively, similar to the extent of desensitization obtained by
others (20, 22). This assay was used to examine agonist-induced
desensitization in the GRK clones.
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). In these
experiments, no significant differences were observed among the clones
compared with wild-type OK cells in forskolin-induced stimulation of
cAMP level (-fold basal) or the extent of inhibition (%
forskolin-stimulated cAMP level) by 5-HT or UK 14304 (1
µM) in vehicle-pretreated cells, except for the KI-5
clone (Table 1 legend). This suggests that the clones retained receptor
signaling properties equivalent to the wild-type OK cell line. The
sense GRK-3 and GRK-18 clones that overexpress OK-GRK2 (Fig. 4A)
displayed enhanced desensitization of the 2C-adrenergic
response by 2- to 4-fold compared with wild-type OK cells
(P < 0. 02 for GRK-3 and P < 0. 002
for GRK-18 at both concentrations of UK 14304). By contrast,
desensitization of the 5-HT1B-induced response in the GRK clones was
similar to OK cells except for the GRK-18 clone that more strongly
expresses GRK2, in which a slight enhancement of desensitization
compared with OK cells was observed at 1.0 µM 5-HT
(P < 0. 02). Although the extent of desensitization at
0.1 µM 5-HT achieved significance (P <
0.05) in the GRK-18 clone, but was not significantly different from
that in wild-type OK cells. Thus, the 5-HT1B response was much less
sensitive to overexpression of OK-GRK2 than the
2C-induced response. In the kinase-inactive mutant clone
KI-5, desensitization of 2C receptors by 0.1 and 1.0
µM UK 14304 was completely abrogated and was
significantly reduced compared with OK cells (P < 0.
05). By contrast, the desensitization of the 5-HT1B response appeared
unchanged or slightly enhanced. The KI-5 clone displayed a significant
desensitization of the 5-HT1B receptor at 0.1 µM 5-HT
(P < 0. 05) that was not present in the wild-type OK
cells (Table 1), suggesting a slight enhancement in the potency of 5-HT
for desensitization. Another GRK2-K220R-positive clone, KI-19 (n =
3) had a similar profile as KI-5 at both concentrations of agonist. The
desensitization of 2C receptors was reduced by >50% as
compared with the parental cell line, but 5-HT1B receptor
desensitization was unaffected (data not shown); however, with
increased passage in culture this response and mutant GRK2 expression
in this clone were lost. In the AS-4 clone in which endogenous OK-GRK2
protein level was reduced (Fig. 4B), agonist-mediated desensitization
of the 2C-adrenergic receptor was abolished at 0.1
µM UK14304, although a small but significant
desensitization occurred at 1.0 µM (P <
0. 05); at this concentration, desensitization was clearly inhibited
compared with OK cells (P < 0. 01; Table 1). These
observations indicate that the 2C receptors are
regulated by OK-GRK2, whereas regulation of 5-HT1B receptor
desensitization involves primarily GRK-independent processes. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunits is highly conserved (34, 35). Thus, the
clone has the structural characteristics of a functional GRK. Northern blot analysis revealed two mRNA transcripts of approximately 5.0 and 3.0 kb. Similarly, the message size we observe was approximately the same as found in the bovine brain (32). The presence of the second smaller band at 3.0 kb might represent alternative splicing of a large intron in the untranslated regions of the mRNA or alternate usage of 3'-polyadenylation sites. The gene for the human GRK2 was recently cloned and was found to be composed of 21 exons interrupted by 20 introns (36). If this structural complexity is conserved in the opossum GRK2 gene, it may explain the smaller mRNA seen at 3.0 kb.
Receptor Specificity of OK-GRK2
Bylund and co-workers have shown that homologous
desensitization of endogenously expressed 5-HT1B and
2C-adrenergic receptors occurs in intact OK cells with
approximate half-times of 60 or 30 min, respectively (20, 22). Using a
45-min agonist preincubation period, we detected a similar extent of
desensitization of these receptors in intact OK cells. Although rapid
desensitization via receptor uncoupling occurs within minutes and is
rapidly reversible, GRK-mediated desensitization has a slower time
course and may involve both uncoupling and receptor internalization,
both processes that are potentially regulated by GRK (37). It is
unlikely that receptor down-regulation is involved, since this requires
a longer time course and higher agonist concentration (3, 20).
Using this desensitization paradigm our observations demonstrate a
Gi-coupled receptor specificity of OK-GRK2 in the OK cell line.
Overexpression of OK-GRK2 enhanced agonist-induced desensitization of
the 2C-adrenergic receptors at 1 µM
UK14304, whereas minimal enhancement of 5-HT1B receptor desensitization
was observed only in the GRK-18 clone that more strongly overexpressed
OK-GRK2. Thus, the cloned OK-GRK2 is functionally active and potent in
mediating 2C receptor desensitization, but only weakly
influences desensitization of the 5-HT1B receptor at the highest level
of expression. The inhibitory action of mutant or antisense GRK2
expression suggests the importance of the apparently low level of
endogenous OK-GRK2 in 2C-adrenergic receptor
desensitization. Kinase-deficient GRK2 mutants have been shown to
preclude agonist-induced desensitization of several receptor subtypes,
including endogenously expressed ß2-adrenergic receptors
(but not for PGE2 receptors) in bronchial cells (10), and the acute
desensitization of m2-muscarinic or angiotensin II receptors in
transfected HEK-293 cells (13, 38). Our results indicate that the
kinase-inactive OK-GRK2-K220R mutant prevents the desensitization of
the endogenously expressed 2C-adrenergic receptor, but
not the 5-HT1B receptor. GRK2 has been shown to bind to ß-subunits
of G proteins and to phosphatidyl inositol bisphosphate via its
pleckstrin homology domain (34, 35, 39). OK-GRK2-K220R might sequester
B-subunits to prevent GRK2 from translocation to the receptor,
thus blocking its function. Alternately, kinase-inactive GRK2 may
interact directly with phosphorylation sites on the receptor to
sterically hinder access of endogenous GRKs, including OK-GRK2. Thus,
overexpression of kinase-inactive GRK2 could block the action of
multiple GRKs. Depletion of OK-GRK2 using antisense RNA expression
resulted in a loss of detectable OK-GRK2 protein and a complete
attenuation of agonist-mediated desensitization of
2C-adrenergic receptors. These results imply that, under
physiological levels of expression, 2C-adrenergic
receptor is selectively regulated by OK-GRK2, consistent with the
presence of several conserved phosphorylation sites for GRK in the
third cytoplasmic loop (21). The opossum 2C-adrenergic
receptor is a homolog of the human 2C4 subtype (21),
which has never been shown to undergo homologous desensitization (12).
Taken together, these data provide compelling evidence for functional
activity of OK-GRK2 in the agonist-induced desensitization of the
endogenous 2C-adrenergic receptor in OK cells. In the
same cells, OK-GRK2 did not substantially modulate desensitization of
the 5-HT1B receptor.
The relative insensitivity of 5-HT1B receptor desensitization to
modulation of GRK2 activity indicates that at physiological and
moderately supraphysiological levels of GRK2, desensitization of the
5-HT1B receptor proceeds by a GRK2-independent mechanism. Concurrent
observation of GRK2-mediated desensitization of the 2C
receptor in the same OK clones indicates that GRK2 is active and
selective for the 2C receptor at the GRK2 protein level
found in intact cells. These results suggest that factors
(e.g. localization or accessibility, other regulatory
proteins) additional to suitability as a phosphorylation substrate may
determine sensitivity to GRK2. Alternately, it may be that the 5-HT1B
receptor is a weaker substrate in vitro for GRK2 than the
2C-receptor.
Protein kinases other than OK-GRK2 may mediate agonist-induced 5-HT1B receptor desensitization. Recently, it was shown that the dopamine D1A receptor was differentially regulated in terms of agonist-mediated desensitization by various GRKs when transfected in HEK-293 cells (40). The notion that another member of the GRK family participates in agonist-promoted attenuation of the 5-HT1B receptor is conceivable. A 380-bp GRK3-related cDNA fragment was identified in the OK cell line using PCR with degenerate oligonucleotides and could correspond to a second GRK subtype. This suggests that other GRKs might be expressed in the cell line that may be responsible for promoting homologous desensitization of the 5-HT1B receptor. This could be investigated using specific GRK monoclonal antibodies to determine GRK specificity as elegantly demonstrated by Oppermann and colleagues (41). However, the desensitization of the 5-HT1B receptor in the kinase-inactive clones suggests that other GRK subtypes that are susceptible to competition with the kinase-inactive mutant do not mediate this response. Another possibility is that the attenuation response for the 5-HT1B receptors may not necessarily be mediated by GRK-induced phosphorylation, but by a second messenger kinase such as PKC. Interestingly, the 5-HT1B receptor-mediated calcium response was shown to be uncoupled by acute preactivation of PKC in OK cells (42).
The role of GRK in vivo may supercede its role in receptor desensitization. Recently, transgenic mice overexpressing GRK2 specifically in cardiac tissue were generated. These mice not only exhibited reduced cardiac contractility in response to isoproterenol but the myocardial adenylyl cyclase activity was attenuated due to the reduced functional coupling of the ß2-adrenergic receptors (43). In contrast, mice overexpressing a GRK inhibitor displayed increased cardiac contractility. In addition, mice with a disruption of the GRK2 gene were shown to display severe cardiac malformations, suggesting a role in fetal development (44). These results suggest a pivotal role played by GRK2 physiologically.
To conclude, we have cloned a functional GRK2 from the opossum kidney
cell line and have shown that in intact cells OK-GRK2 displays
Gi-coupled receptor specificity in mediating the homologous
desensitization of endogenous 2C-adrenergic receptors,
but not of 5-HT1B receptors. Nevertheless, the 5-HT1B receptor is
phosphorylated in vitro by OK-GRK, suggesting the other
factors determine receptor susceptibility to GRK-mediated
desensitization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]ATP and [
-32P]ATP were
purchased from Amersham Corp. (Arlington Heights, IL). Serotonin,
3-isobutyl-1-methylxanthine, and forskolin were from Sigma
Chemical Co. (St. Louis, MO) and UK 14304 was purchased from RBI
Chemicals (Natick, MA). Tissue culture media and sera were from
GIBCO-BRL (Gaithersburg, MD), and OK cells were purchased from the ATCC
(Manassas, VA).
Cell Culture
OK cells were grown to 80–90% confluence with DMEM
(Hi-glucose) and supplemented with 8% FBS at 37 C in a humidified
atmosphere with 5% carbon dioxide. Media were changed 12–24 h before
experimentation.
PCR
Random hexamers were used as primers to reverse transcribe total
RNA (1 µg) isolated from the OK cell line using Superscript
reverse-transcriptase (GIBCO-BRL). The cDNAs were amplified by PCR [1
min at 95 C, 2 min at 54 C, 2 min at 72 C for 40 cycles] using
degenerate oligonucleotides, sense
(5'-GGCAAGATGTA(T/C)GC-(A/T/C/G)ATGAA-3') and antisense
(3'-AC(C/T)TCGGG(A/C/G/T)GCCATGTACCC-5') designed to highly conserved
regions of the catalytic domain of GRK2 [Drosophila, rat,
and bovine (29)]. The PCR reactions contained 1 µg of
reverse-transcribed OK cell mRNA, 1.5 mM MgCl2,
and 5x Hot Tub buffer (Amersham). Two distinct 380-bp cDNAs were
isolated (10–1 and A-1) and subcloned into the EcoRV site
of pBluescript (KS+, Stratagene, La Jolla, CA). DNA sequence analysis
using the Sanger method with a T7 polymerase-based DNA sequencing kit
(Pharmacia) revealed that they shared 81% and 66% identity,
respectively, at the nucleotide level to the rat GRK2.
Construction of OK cDNA Library
Poly-A+ RNA was isolated from the OK cell line using the
QuickPrep Micro mRNA Purification kit (Pharmacia). For cDNA synthesis,
5 µg of Poly-A+ RNA was reverse-transcribed using Superscript
reverse-transcriptase, and the complete synthesis of double-stranded
DNA was performed using the oligo-dT Riboclone cDNA Synthesis kit
(Promega, Madison, WI). The blunt-ended cDNAs were fractionated and
EcoRI adapters were ligated followed by ligation into
EcoRI-predigested Lambda ZAP II arms (Stratagene). Gigapack
II Gold (Stratagene) extracts were used to package recombinant
-phage extracts.
cDNA Cloning
The oligo-dT-primed OK cell cDNA library was amplified and 106
plaques were plated, transferred to Hybond N+ membranes, and screened
with the 350-bp A-1 and 10–1 PCR fragments labeled using a randomly
primed labeling kit (Boehringer-Mannheim) with the following
hybridization conditions: 50% formamide, 50% dextran-sulfate, 1%
SDS, 5x NaCl-sodium citrate (SSC), 5x Denhardt’s, 10
mM Tris (solution A), and 100 µg/ml sonicated
salmon sperm DNA at 42 C overnight. The filters were washed with 2x
SSC/1% SDS at room temperature followed by a high-stringency wash with
0.1% SDS/0. 2x SSC at 65 C. The 10 clones identified by this method
were isolated by repeated plating and screening with the labeled PCR
fragments. The isolated clones were rescued with helper phage to yield
the cDNAs as inserts in pBluescript SK vector. All 10 clones were
restriction mapped and were found to be identical. One clone was
subjected to manual and automated sequencing in sense and antisense
orientations using T7 polymerase and oligonucleotides directed to
regions of the sequence, or reverse and universal primers. This
3.073-kb clone was highly homologous to rat GRK2 and was therefore
named OK-GRK2.
Northern Blot Analysis
Poly-A+ RNA was isolated from the OK cell line using the
QuickPrep Micro mRNA Purification kit (Pharmacia). Seven micrograms of
poly-A+ OK RNA and 3 µg of 0.24–9.5 kb RNA ladder (GIBCO-BRL) were
fractionated on a 1. 3% agarose-formaldehyde gel and transferred to
Hybond-N membrane (Amersham). The 2.1 kb KpnI fragment of
OK-GRK2 was labeled and hybridized to the membrane in solution A
overnight with 100 µg/ml sheared, sonicated salmon sperm DNA at 42 C.
The filter was washed with 0.1% SDS/0.2x SSC at 65 C and exposed for
4 days at -70 C to Kodak XAR film (Eastman Kodak, Rochester, NY) with
an intensifier.
Construction of K220R Mutant and Stable Transfections of Sense,
Antisense, and K220R Mutant in OK Cells
The EcoRI fragment of the OK-GRK2 gene was subcloned
into pSelect to use as a template for site-directed mutagenesis
(Altered-sites mutagenesis, Promega). The oligonucleotide
5'-ATCAAGACACCTCATGGCATA-3' was used to
incorporate the point mutation. The mutation was confirmed by DNA
sequencing using T7 polymerase (Pharmacia). The K220R mutant,
antisense, and wild-type OK-GRK2 cDNAs were subcloned into the
eukaryotic expression vector pcDNA-3 (Invitrogen, San Diego, CA) and
stably transfected into OK cells using the calcium phosphate
coprecipitation method. Geneticin-resistant clones were selected and
grown in DMEM supplemented with 10% FCS and 1.5 mg/ml Geneticin.
Positive clones were identified by Northern and Southern blot
analyses.
Western Blot Analysis
Western blot analyses were performed using cytosolic fractions
from the RNA-positive transfected clones. To prepare cytosolic
extracts, OK cells and clones were trypsinized and resuspended in 150
µl of lysis extraction buffer [150 mM NaCl, 50
mM Tris-HCl (pH 7. 8), 0. 01% sodium azide, 10
mM NaF, 30 mM sodium pyrophosphate] followed
by addition of 150 µl of lysis extraction buffer containing 4%
NP-40, 5 mM Na3VO4, 10 µg/ml
leupeptin, 10 mM benzamidine, 2 µg/ml soybean trypsin
inhibitor, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, 10 mM N-tosyl-1-phenylalanine
chloromethyl ketone, and 20 mM iodoacetamide. This
was incubated on ice for 30 min, and the supernatant was recovered
after centrifugation at 4 C, 15 min, at 12,000 x g. Using
BSA as a standard (Bio-Rad, Richmond, CA) for protein concentration, 50
µg of total protein were loaded in SDS-PAGE sample buffer, resolved
by a 10% SDS-PAGE, and electroblotted onto ECL nitrocellulose
membranes (Western blotting ECL kit, Amersham) for 2 h at 250 mA
constant current. Blots were preblocked in TBS-Blotto (150
mM NaCl, 20 mM Tris-HCl, pH 8.0, 5% nonfat
dried milk) at room temperature. The recombinant bovine GRK2 polyclonal
antibody (1:1000) was hybridized to the membranes in TBS-Blotto for
1 h at room temperature. Blots were washed in TBS-Blotto-0.5%
Tween 20 (4 x 15 min, 3 x 5 min for high stringency) or
TBS-Blotto-0.05% Tween 20 (4 x 15 min, or 3 x 5 min for
low stringency) and incubated with secondary antibody (horseradish
peroxidase-conjugated antirabbit IgG) 1:1000 for 1 h at room
temperature. Blots were washed in TBS-Blotto-Tween 20 as described
above and incubated with the Amersham ECL detection reagents and
developed for up to 10 min.
cAMP Assay
Measurement of cAMP was performed as described previously
with some modifications (31). Briefly, OK cells were plated in 24-well
dishes and propagated to near confluence for assay. The cells were
washed twice (5 min each) with 1 ml of DMEM/HEPES (serum-free DMEM + 20
mM HEPES, pH 7.2) and incubated with 0. 5 ml/well of
DMEM/HEPES for 45 min containing either no addition, 100
nM/1 µM 5-HT, or 100 nM/1
µM UK 14304. Typically, half of each plate was treated
with 5-HT and half with UK 14304 to measure actions of both compounds
under identical conditions. After incubation, medium was quickly
decanted and replaced with warm assay medium (DMEM/HEPES + 100
µM 3-isobutyl-1-methylxanthine) with agents (10
µM forskolin, 1 µM 5-HT, or 1
µM UK14304) and incubated for 15 min at 37 C. The 15-min
assay period was the minimal time to process in parallel multiple
plates of OK cells and clones. In control experiments, the cells were
washed twice with DMEM/HEPES before addition of assay medium: it was
determined that the 5-HT pretreatment did not alter subsequent basal or
forskolin-stimulated cAMP levels. The reactions were stopped by
removing the medium. The supernatants were collected and assayed for
cAMP by a specific RIA (ICN) as described (31). Standard curves
displayed an average IC50 value of 0.5 ± 0.2 pmol
using cAMP as standard. Data for cAMP assays are presented as mean
± SEM for triplicate wells. Percent desensitization was
calculated as follows {1 - [(F - XFA)/(F -
FA)]} x 100, where cAMP level was determined under the
following conditions: F = forskolin, FA = forskolin +
agonist; XFA = forskolin + agonist after the indicated agonist
pretreatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Medical Research Council (MRC), Canada. P.R.A. is recipient of the Novartis/MRC Michael Smith Chair in Neuroscience.
1 Present address: Astra Research Centre, 7171 Frederick Banting,
Ville St. Laurent, Canada H4S-1Z9. 
2 The GenBank accession number for OK-GRK2 cDNA is
AF087455.
Received for publication July 31, 1998. Revision received August 28, 1998. Accepted for publication September 28, 1998.
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REFERENCES |
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