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From the Departments of
Received for publication, December 10, 2001
Despite important roles in myocardial hypertrophy
and benign prostatic hyperplasia, little is known about acute effects
of agonist stimulation on Desensitization (dampening of receptor responsiveness to continued
agonist exposure) is thought to involve a number of inter-related yet
distinct mechanisms occurring at the receptor and more broadly in the
signal transduction pathway (7). Receptor desensitization may occur
within seconds to minutes of agonist stimulation, and is generally
considered to result from receptor uncoupling from downstream effectors
due to receptor phosphorylation. Phosphorylation of activated GPCRs by
G protein-coupled receptor kinases (GRKs) leads to increased binding of
In contrast to the To accurately characterize acute agonist-mediated regulation of human
Materials--
Dulbecco's modified Eagle's medium (DMEM) and
fetal bovine serum were obtained from Invitrogen (Grand Island, NY).
[125I]HEAT, [3H]inositol,
[125I]iodoazidoprazosin, and
[32P]orthophosphate were from PerkinElmer Life Sciences
(Boston, MA). Dowex AG1-X8 was from Bio-Rad (Hercules, CA). PMA and
phentolamine were from Sigma. Norepinephrine, prazosin, and
5-methylurapidil were from Research Biochemical International (Natick,
MA). Bisindolylmaleimide I was from Calbiochem (La Jolla, CA). The
cDNA for GRK2 cloned into pcDNA1 was provided by Robert Lefkowitz.
Construction of Probes for RNase Protection Assays of Human
RNA Isolation and RNase Protection Assays--
Total RNA was
extracted from human tissues (prostate, heart) and SK-N-MC cells using
RNA STAT-60 (Teltest Inc., Friendswood, TX). Each RNA sample was
quantitated spectrophotometrically at 260 and 280 nm and stored at
Construction of HA-tagged Cell Culture, Stable Transfection, and
Saturation binding and competition analysis of transfected cell
membranes was performed with the radiolabeled
Ligand binding on intact cells was performed as described by Lattion
(8) with a few modifications. Cell monolayers grown in 12-well plates
were washed once with DMEM and treated with drugs, washed once again
with DMEM and then incubated with 2 nM [3H]prazosin in 0.5 ml of DMEM at either 4 °C for
12-15 h to determine cell surface binding or at 37 °C for 30 min to
determine total cellular receptor content. Phentolamine
(10 Measurement of Total Inositol Phosphate Production--
Rat-1
cells stably expressing either HA-
Final GRK-mediated desensitization assays were performed in transiently
transfected cells, since GRK transfection in stable cell lines resulted
in unexpected accelerated cell death (a condition not seen in any other
assay). All final IP assays were normalized for Photoaffinity Labeling of 32P Labeling and Immunoprecipitation of
Statistical Analysis--
Results are expressed as mean ± S.E. Statistical significance was analyzed by two-factor ANOVA (where
appropriate) followed by a two-tailed unpaired t test, with
p < 0.05 considered to be significant.
Profile of Human To focus our studies on the full-length functional
Construction, Stable Expression, and Characterization of
HA- To facilitate phosphorylation studies of the wild type human
Ligand binding and IP signaling of HA-
Acute Agonist-mediated Desensitization of the Human
1a-Adrenergic Receptor Is Primarily Independent of
Carboxyl Terminus Regulation
IMPLICATIONS FOR REGULATION OF 
1aAR SPLICE
VARIANTS*
§,
,¶**
Pharmacology and Cancer
Biology, ¶ Anesthesiology, and ** Surgery, Duke
University Medical Center, Durham, North Carolina 27710
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1a-adrenergic receptor
(1aAR) signaling and function. Regulatory mechanisms are
likely complex since 12 distinct human 1aAR
carboxyl-terminal splice variants have been isolated. After determining
the predominance of the 1a-1AR isoform in
human heart and prostate, we stably expressed an epitope-tagged 1a-1AR cDNA in rat-1 fibroblasts and subsequently
examined regulation of signaling, phosphorylation, and internalization
of the receptor. Human 1aAR-mediated inositol phosphate
signaling is acutely desensitized in response to both agonist and
phorbol 12-myristate 13-acetate (PMA) exposure. Concurrent with
desensitization, 1aARs in
32Pi-labeled cells are rapidly phosphorylated
in response to both NE and PMA stimulation. Despite the ability of PKC
to desensitize 1aARs when directly activated with PMA,
inhibitors of PKC have no effect on agonist-mediated desensitization.
In contrast, involvement of GRK kinases is suggested by the ability of
GRK2 to desensitize 1aARs. Internalization of cell
surface 1aARs also occurs in response to agonist
stimulation (but not PKC activation), but is initiated more slowly than
receptor desensitization. Significantly, deletion of the
1aAR carboxyl terminus has no effect on receptor internalization or either agonist-induced or GRK-mediated receptor desensitization. Because mechanisms underlying acute agonist-mediated regulation of human 1aARs are primarily independent of
the carboxyl terminus, they may be common to all functional
1aAR isoforms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1a-Adrenergic receptors
(1aARs)1 are G
protein-coupled receptors (GPCR) that mediate sympathetic nervous
system responses such as smooth muscle contraction and myocardial
inotropy (1). NE stimulation of 1ARs predominantly
activates Gq and results in membrane polyphosphoinositide
hydrolysis by activation of phospholipase C
; the resultant second
messengers IP3 and DAG mobilize intracellular calcium and
activate protein kinase C (PKC), respectively (2). Three
1AR subtypes (1a, 1b, and
1d) have been cloned and pharmacologically characterized
in several expression systems (for review, see Ref. 2). Clinically,
activation of 1aARs has been implicated in the dynamic
component of benign prostatic hyperplasia leading to bladder outlet
obstruction and in the development of myocardial hypertrophy (3-5).
Notwithstanding the importance of 1aARs in several
pathophysiological states, surprising little is known about mechanisms
underlying 1aAR expression and function. Transcriptional mechanisms unique to 1aARs have been shown to be
important in maintaining full 1AR responsiveness to
agonist in rat neonatal myocytes where long term (24-72 h) NE
stimulation leads to up-regulation 1aAR mRNA and
receptor protein expression, concurrent with down-regulation of
1b and 1dAR subtypes (5, 6). While
important, such long-term studies do not examine acute regulation of
1aAR signaling in response to agonist stimulation,
giving rise to the question whether 1aARs are subject to
acute processes such as agonist-induced desensitization and receptor phosphorylation.
-arrestin to the receptor complex, uncoupling receptors from
G proteins and, in some cases, leading to internalization of the
phosphorylated receptors. Phosphorylation, desensitization,
internalization, and down-regulation (decreased expression) of
the 1bAR subtype in response to either agonist or PMA
exposure have been extensively demonstrated in native and cell models
(8-12). Significantly, these processes are mediated through the
carboxyl terminus of the 1bAR. Truncation of its carboxyl terminus significantly decreases agonist- and PMA-mediated phosphorylation, desensitization, and internalization of the
1bAR, and recent studies have pinpointed critical
serine residues within that receptor region that are involved in each
of these regulatory processes (8, 11-13).
1bAR, current knowledge regarding
agonist-mediated regulation of the 1aAR subtype is
severely lacking. One study of bovine 1aARs recently
suggested that the 1aAR, similar to the
1b subtype, is subject to agonist-induced
desensitization and phosphorylation (14). Mechanisms underlying
regulation of 1aARs, however, are potentially more
complex than those of the 1b subtype. Unlike
1b and 1dARs which are each expressed as a single isoform, several distinct 1a isoforms have been
isolated in addition to the original "wild type"
1aAR, 12 in humans and 4 in rabbit (15-18). Although
several of these variants give rise to non-functional truncated
polypeptides with only six transmembrane domains, there are four fully
functional 1aAR isoforms that exhibit ligand binding and
signaling characteristics essentially identical to those of the wild
type receptor (15-17). It is of particular interest that all
functional 1aAR splice variants are identical except at
the distal ends of their carboxyl termini, where each differs in
sequence and length. The study of 1aAR regulation in
endogenous systems may prove difficult, however, since several 1aAR carboxyl-terminal splice variants are concurrently
expressed in every tissue studied thus far (15-17). Additionally,
expression of severely truncated 1aAR isoforms can
decrease signaling by full-length receptors, possibly through
interference with trafficking and membrane localization of full-length
receptors (17).
1aAR signaling, we directly profiled expression levels of functional full-length 1aAR splice variants in human
heart and prostate and identified 1a-1 as the
predominant 1aAR isoform in these tissues. We then
stably expressed the 1a-1AR as an epitope-tagged fusion
protein in rat-1 fibroblasts to examine effects of acute (
30 min) NE
and PMA stimulation on 1aAR signaling and to
subsequently explore the roles of PKC and several GRK family members,
receptor phosphorylation, and receptor internalization in acute
regulation of 1aARs. In parallel we constructed,
expressed, and tested a fully functional carboxyl-terminal truncated
1aAR (whose sequence is common to all functional
1aAR splice variants) to characterize any regulatory
features that may be independent of carboxyl-terminal variation. Our
findings suggest that acute agonist-mediated regulation of human
1aARs is primarily independent of the carboxyl terminus and could therefore be common to all functional 1aAR isoforms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1aAR Carboxyl-terminal Splice Variants--
To profile
1aAR carboxyl-terminal splice variant expression in
human heart and prostate, cDNA templates for isoform-specific RNase
protection assay probes were constructed. Polymerase chain reaction
(PCR) using Pfu polymerase (Roche Molecular Biochemicals, Indianapolis,
IN) was used to amplify the 3' cDNAs ends of each functional human
1aAR splice variant: 1a-1 (wild type),
1a-2, 1a-3, and 1a-4. A
common 5' amplification primer (containing the internal
1aAR EcoRV restriction site) located 322 bp
upstream of the 1aAR carboxyl terminus splice site was
used in conjunction with variant-specific downstream primers
corresponding to distal 3' end of coding sequence and containing
introduced XbaI restriction sites. PCR products were
digested with EcoRV and XbaI and subsequently ligated into a pcDNA3 (Invitrogen, Carlsbad, CA) construct
containing the wild type 1aAR to generate full-length
1aAR splice variant constructs. Digestion of each splice
variant construct with EcoRV and ApaI provided
isoform-specific fragments that were subsequently ligated into pGEM7Z+
(Promega, Madison, WI). Modified sequences of each PCR construct were
verified by dideoxy DNA sequencing using the fmol DNA Cycle
Sequencing System (Promega).
70 °C until use. Radiolabeled antisense isoform-specific
1aAR probes were transcribed from linearized cDNA
constructs using RNA polymerase T7, and [-32P]UTP as
previously described (19). In addition to probes for each
1aAR splice variant subtype, a control
1aAR probe (located upstream of carboxyl-terminal splice
site in 1aAR second exon) and a human cyclophilin
control probe (Ambion, Inc., Austin, TX) were used; RNase protection
assays were conducted as previously described (19).
1aAR and Carboxyl
Terminus Truncated Mutants--
To facilitate phosphorylation studies,
sequence of the hemagglutinin (HA) epitope was added to the amino
terminus of the human 1a-1AR cDNA using PCR
mutagenesis. The 5' (sense) 59-mer oligonucleotide (5'-AAAAGAATTCATGTACCCATACGACGTCCCAGACTACGCCGTGTTTCTCTCGGGAATG-3') contained a synthetic EcoRI restriction site to
facilitate cloning, sequence encoding the 9-residue HA epitope
(YPYDVPDYA; bold italics) immediately downstream of the
1aAR start codon, and 19 bases corresponding to bp 4 to
22 of the 1aAR (underlined). The 3' (antisense) primer
was 5'-GAGCAGCCTCACTGAGAAGTGCGT-3', corresponding to bases 796 to 763 of the 1aAR receptor (GenBankTM accession
number 4501960). The resulting PCR product was digested with
EcoRI and Eco47III and subcloned into the
mammalian 1aAR expression vector
pcDNA3:1aAR; the final construct is called pcDNA3:HA-1a. A carboxyl-terminal-truncated
1aAR mutant (pcDNA3:HA-T348) was generated by
introducing a STOP codon after Arg348 of
HA-1a in pcDNA3:HA1a using the
Transformer Site-directed Mutagenesis Kit
(CLONTECH, Palo Alto, CA). Modified sequences of
each construct were verified by dideoxy DNA sequencing.
1AR Ligand
Binding--
Cells were grown in monolayers and maintained in DMEM
supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) in 5% CO2 at 37 °C. For
stable expression of receptors, plasmids
pcDNA3:HA-1a and pcDNA3:HA-T348 were
individually transfected into rat-1 fibroblasts by electroporation
(Gene Pulsar II, Bio-Rad Inc.; optimal conditions were 250 mvolts and
950 µF capacity, resulting in a time constant of 12-15 ms). Clones
resistant to G418 (800 µg/ml) were isolated and tested for receptor
expression. Selection was maintained in clonal cell lines with 400 µg/ml G418.
1AR
antagonist [125I]HEAT (300 and 120 pM for
saturation and competition binding, respectively) as previously
described (20). The 1AR agonists norepinephrine and
oxymetazoline, and antagonists prazosin and 5-methylurapidil were
utilized for competition analysis. Resultant curves were fit using
noniterative regression analysis with PRISM software (Graphpad, San
Diego, CA).
4 M) was used to determine nonspecific
prazosin binding. Following binding, cells were washed three times with
ice-cold PBS containing 0.1% bovine serum albumin, and scraped in 1 ml
of water; 3H was counted in 7 ml of scintillation mixture
using a Wallac 1409 Liquid Scintillation Counter (Wallac, Gaithersburg, MD).
1a or HA-T348
receptors were labeled with [3H]inositol for 20-24 h
with 2.5 µCi/ml in DMEM supplemented with 3% fetal bovine serum,
penicillin (100 units/ml), and streptomycin (100 µg/ml). After
labeling, cells were washed with PBS and when necessary incubated in
PBS for various times at 37 °C; pretreated cells were exposed to
various drugs during this incubation as indicated. Cells were then
quickly washed with PBS, placed in PBS with 20 mM LiCl and
immediately stimulated by NE addition for the indicated times. NE was
added to the cells from ×100 stocks in 10 mM ascorbic acid
while only ascorbic acid was added to unstimulated cells. Inositol
phosphates were extracted as described by Martin (21) and separated
using Dowex AG1-X8 anion exchange (formate) columns. After washing the
columns twice with water, total inositol phosphates were eluted in 1 M ammonium formate, 0.1 M formic acid, and combined with 15 ml of scintillation mixture; 3H was
quantitated using a liquid scintillation counter.
1aAR
density and cell count at the time of assay. GRK overexpression was
confirmed by Western analysis. Rat-1 or COS-7 cells were plated at
40,000 cells per well in 12-well plates, grown overnight, and transfected with 0.25 µg of pcDNA3 containing
HA-1aARs or HA-T348 with or without 0.25 µg of
pcDNA1 containing GRK2 or GRK6; the total transfected DNA was kept
at 0.75 µg (per 40,000 cells) by adding pcDNA3. Following 18 h of transfection, cells were washed with Hank's balanced salt
solution, labeled with [3H]inositol in 10% fetal bovine
serum, and assayed for total inositol phosphate production in DMEM
essentially as described for stable cells.
1aARs--
Membranes
from cells expressing either HA-1a or HA-T348 receptors,
prepared for ligand binding as described above, were resuspended at a
concentration of 1 µg of total protein/µl. 175 µg of total protein were incubated with 6 nM
[125I]iodoazidoprazosin (200 µl total volume) in the
dark at room temperature for 60 min in the presence or absence of
prazosin (1 µM). Following incubation, samples were
UV-irradiated for 10 min, centrifuged at 14,000 × g
for 10 min, and resuspended in 6 × SDS-PAGE sample buffer.
Proteins were separated by electrophoresis on a 10% SDS-polyacrylamide
gel (Protogel, National Diagnostics). 125I-Labeled
receptors were detected by exposure to X-Omat AR film (Kodak) for
24-48 h.
1aARs in Cells--
Equal numbers of rat-1 fibroblasts
stably expressing either HA-1a or HA-T348 receptors were
grown to confluency in 6- or 12-well cluster plates. Cell monolayers
were washed three times with phosphate-free DMEM, then incubated in the
same medium containing 32Pi (0.2 mCi/ml) for
2 h at 37 °C. Different drugs were added during this incubation
as indicated under "Results." Following incubation, cells were
washed three times with ice-cold PBS, solubilized in ice-cold lysis
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mM
Na3VO4, 10 mM
Na4P2O7, 10 mM NaF, and
Complete Protease Inhibitor Mixture (Roche Molecular Biochemicals)),
and centrifuged at 14,000 × g for 10 min at 4 °C.
To reduce background, solubilized proteins were incubated with protein
G-agarose (Roche Molecular Biochemicals) for 1 h at 4 °C. After
removal of nonspecific precipitated material, the supernatant was
incubated with 3F10 rat monoclonal antibody (Roche Molecular
Biochemicals) against the HA epitope tag at 4 °C. After 1 h,
protein G-agarose beads were added to the sample mixture and incubation
was continued overnight. Isolated immune complexes were washed twice
with ice-cold lysis buffer, twice with high salt buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Nonidet P-40, 0.05% sodium deoxycholate), and once with low salt buffer (50 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40, 0.05% sodium
deoxycholate). After removal of buffer, immune complexes were
resuspended in 6 × SDS sample buffer and were separated by
electrophoresis on a 10% SDS-PAGE gel. After autoradiography,
32P was quantitated with a PhosphorImager and analyzed
using ImageQuant gel image analysis software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1aAR Isoform Expression in
Prostate and Heart
1aAR isoform that is predominantly expressed in human
prostate and heart, we directly examined expression of the four
full-length 1aAR splice variants in these tissues, as
well as in SK-N-MC cells (the only currently available human cell line
that endogenously expresses 1aARs, albeit at very low
levels). RNase protection assays (without prior PCR amplification) were
performed using 1aAR probes designed to contain both
isoform-specific carboxyl-terminal sequences (WT 1a-1,
1a-2, 1a-3, and 1a-4) and
sequences common to all functional 1aAR isoforms (Fig.
1A). With each probe, levels of individual isoform mRNA expression (isoform-specific fragment) were quantitated relative to all other functional 1a
isoforms (common sequence fragment). As shown in Fig. 1, B
and C, the 1a-1 (wild type 1a)
predominates in human prostate (81 ± 2%), heart (89 ± 4%), and SK-N-MC cells (85 ± 1%). 1a-4 mRNA
represents 6-11% of the remaining 1aAR mRNA pool,
whereas 1a-2 and 1a-3 are rare in all
tissues/cells studied (3-9% combined). Because of its overwhelmingly
predominant expression, we subsequently focused our studies on
regulation of the wild type 1a-1 receptor (hereafter
referred to as 1aAR).
View larger version (41K):
[in a new window]
Fig. 1.
Relative expression of
1aAR carboxyl-terminal splice variant
mRNA. A, schematic of probes used in RNase
protection assays containing sequences common to all variants as well
as isoform-specific sequences. B, representative RNase
protection assay results using RNA from human prostate, heart, and
SK-N-MC cells with size and identity of 1aAR
isoform-specific (a-1, a-2, a-3, and a-4) and common protected
fragments indicated. Exon2 (Ex2) denotes
1aAR second exon control probe that does not distinguish
between isoforms and therefore represents total 1aAR
mRNA pool (shown in unlabeled lane for each tissue). C,
quantitation of 1aAR isoform mRNA expression in
human prostate, heart, and SK-N-MC cells. Data are mean ± S.E.,
n = two to five independent experiments.
1aAR and Carboxyl-Truncated Mutant HA-T348
1aAR, we constructed two specialized mutant
1aARs: 1) an amino terminus HA epitope-tagged
1aAR (HA-1a), and 2) a mutated
HA-1a in which the carboxyl terminus was truncated after
amino acid 348, eliminating the last 118 amino acids of the
1a-1AR (HA-T348). HA-T348 retains expression of the
palmitoylated Cys345 (22), but is truncated well upstream
of the 1aAR carboxyl-terminal splice site present in all
functional 1aAR splice variants just after
Arg422. Thus, the truncated construct represents sequence
common to all functional 1aAR isoforms, regardless of
splice variant identity. HA-1a and HA-T348 constructs
were each stably transfected into rat-1 fibroblasts (cells lacking
endogenous 1ARs), and several stable clones were
isolated for each construct with receptor expression levels varying
from 0.1 to 1.5 (HA-1a) and 0.2 to 2.8 (HA-T348) pmol/mg
of total protein. Receptor expression levels were routinely measured to
ensure they did not change over time. Unless otherwise stated, clonal
lines expressing similar levels of HA-1a and HA-T348 (1.5 and 1.8 pmol/mg protein, respectively) were utilized in this study. However, assays performed on two additional clones of each cell
type with lower levels of receptor expression (HA-1a,
400 and 200 fmol/mg protein; HA-T348, 700 and 350 fmol/mg protein) demonstrated similar responses of 1aARs to agonist- and
PMA-induced desensitization (data not shown). This suggests that these
phenomena are, to a large degree, independent of receptor density in
these cells.
1a and HA-T348
were characterized and compared with those of the wild type
1aAR (untagged, full-length) to determine potential
effects of epitope tagging and carboxyl-terminal truncation on the
receptor. Binding affinities of both HA-1a and HA-T348
for agonists (NE and oxymetazoline) and 1AR antagonists
(prazosin and 5-methylurapidil) are indistinguishable from those of the
untagged wild type receptor (Table I). In
cells labeled with [3H]inositol and stimulated with
varying concentrations of NE, both HA-tagged receptors are capable of
stimulating production of total inositol phosphates in a
dose-dependent manner to the same extent as wild type
1aARs, with EC50 0.36 ± 0.03 and
0.24 ± 0.05 µM for HA-1a and
HA-T348, respectively, compared with 0.31 ± 0.09 µM
for the wild type 1aAR. The ~100-fold difference
between NE EC50 values and receptor affinities
(pKi) at the 1aAR is not unique to
this receptor. Similar differences between agonist potency over ligand
affinity has been demonstrated for other GPCRs (e.g.
2-3-fold increase for full agonists of the m3 muscarinic acetylcholine
receptor (23) and a nearly 10,000-fold increase for isoproterenol at
the 2AR (24)). The 1AR antagonist
prazosin (1 µM) prevents NE-induced increases in IP
production above basal values at wild type, HA-tagged, and truncated
1aARs (data not shown). Thus, addition of the HA epitope
tag to the amino terminus and/or truncation of the 1aAR
carboxyl terminus has little effect on ligand binding or second
messenger production of the human 1aAR.
Pharmacological characteristics of HA-1a and HA-T348
compared to wild type 1aAR
Acute Desensitization of 1aARs
We next examined the rate of total IP accumulation following NE
stimulation as a function of time in rat-1 fibroblasts expressing HA-1a or HA-T348. Total IP production rates of both
full-length and truncated receptors are identical (Fig.
2). Stimulation of cells with
105 M NE induces a rapid rise in IP levels
for 2 min, after which point the rate of accumulation decreases. This
not only suggests that desensitization of 1aAR occurs at
this early time point but also implies that the carboxyl-terminal tail
is not required for acute desensitization. In addition, signaling by
both receptors continues to rise at an identical, robust, and nearly
linear rate through at least 30 min of receptor stimulation. Continued
accumulation of total IPs throughout this time period indicates that
the agonist-sensitive pool of [3H]inositol-labeled
membrane lipids is not rapidly depleted by agonist treatment in these
experiments. It also suggests that non-acute mechanisms of
desensitization do not predominate over this period.
|
Time Course of Acute Agonist- and PMA-induced 1aAR
Desensitization
To address the issue of whether or not human 1aARs
undergo acute desensitization in response to agonist stimulation, we
examined the ability of HA-1a-expressing rat-1
fibroblasts to respond to a subsequent challenge with NE after initial
pretreatment with NE. Cells were treated with 105
M NE for 2 to 30 min in PBS without LiCl (to allow
recycling of IPs generated during pretreatment), washed, and then
restimulated with 105 M NE in the presence of
LiCl. NE-stimulated IP responses of pretreated cells were compared with
those of untreated cells to determine both the time course and extent
of HA-1a desensitization. Desensitization of
HA-1a to subsequent NE stimulation occurs within 2 min
of NE agonist pretreatment, reducing the IP response to 
60-70%
that of naive cells (Fig. 3A,
Table II). Longer periods of pretreatment with NE result in reduced, but continued receptor responsiveness; even
after 30 min of NE stimulation, signaling by HA-1a
persists at 50% naive levels. Significantly, parallel experiments
involving the carboxyl-truncated 1aAR, HA-T348, revealed
that both the rate and extent of agonist-induced desensitization are
essentially identical to the corresponding wild type responses (Fig.
3A, Table II). Thus, even very short NE pretreatment periods
can induce nearly maximal acute desensitization and this behavior is
observed even in the HA-T348 receptor lacking the carboxyl terminus.
The modest amount of additional desensitization that occurs between 5 and 30 min also appears similar in the full-length and truncated receptor. Although the intracellular carboxyl terminus has been implicated in regulation of several GPCRs including the
1bAR, these data suggest that the carboxyl terminus of
the human 1aAR does not play a similar role in
regulating agonist-induced desensitization.
|
|
).
Potential PKC Involvement in Acute Agonist-induced Desensitization
of 1aARs
Agonist stimulation of 1aARs leads to activation of
the second messenger-dependent kinase PKC, a kinase that
could potentially feed back to phosphorylate and desensitize the
receptors. To examine a possible role of PKC in 1aAR
desensitization, we utilized the active phorbol ester PMA to stimulate
PKC directly. PMA pretreatment of cells expressing HA-1a
or HA-T348 effectively reduces the IP response of both full-length and
truncated receptors to a subsequent stimulation by NE, although
HA-1a is desensitized to a greater extent than HA-T348
at each time point (Fig. 3B, Table II). PMA-induced desensitization is dose-dependent, with maximal effects
achieved with 107 M PMA (data not shown).
Similar to the time course of NE desensitization, PMA-induced
desensitization is rapid, with maximal desensitization occurring within
2 min of PMA treatment. Thus PKC could potentially play a role in
agonist-mediated desensitization of 1aARs. It is worth
noting, however, that while carboxyl-truncated 1aAR is
less sensitive to PMA-mediated desensitization than the full-length receptor, the truncated receptors sensitivity to agonist-mediated desensitization is not different from the full-length
1aAR. This suggests that second messenger activation of
PKC probably does not serve in an agonist-mediated desensitization
feedback loop.
To explore this topic further, we utilized the specific PKC inhibitor
bisindolylmaleimide I (GF 109203X) to block PKC activity. If PKC plays
a substantial role in agonist desensitization, pretreatment of
1aAR expressing cells with the PKC inhibitor should
reduce the desensitizing effects of NE pretreatment. Initial
dose-response experiments determined that 1 µM
bisindolylmaleimide I is sufficient to completely inhibit PMA-mediated
effects on 1aAR signaling (data not shown). When
[3H]inositol-labeled cells expressing either full-length
and truncated 1aARs were treated with
bisindolylmaleimide I complete inhibition of PMA-induced
desensitization of both receptors was seen (Fig. 4). In contrast, treatment with the PKC
inhibitor does not affect the degree to which either receptor is
desensitized in response to NE stimulation. These data indicate that
although direct PKC activation can lead to desensitization of
1aARs, it does not play a significant feedback role in
agonist-induced desensitization of 1aARs.
|
GRK-mediated Desensitization of 1aARs
After concluding that PKC does not function in a significant
feedback capacity in agonist-mediated 1aAR
desensitization, we examined the ability of members of the GRK family
to affect 1aAR signaling. In these experiments, IP
accumulation was assessed in rat-1 cells transiently expressing
HA-1a or HA-T348 with or without coexpressed GRK 2 and
GRK6 (Fig. 5). Coexpression with GRK2
desensitized both HA-1aAR and HA-T348 identically,
resulting in 63.5 ± 2.4% and 63.8 ± 2.6% of the activity
of receptor alone, respectively (Fig. 5, A and
B). In contrast, coexpression of GRK6 with
HA-1aAR resulted in no desensitization (Fig.
5C). The ability of GRK2 to desensitize
HA-1aAR was also confirmed in COS-7 cells where it was
found to be similar to that observed for HA-1bAR (data
not shown). Although perhaps coincidental, it is worth noting that
GRK-mediated desensitization (Fig. 5) and NE-induced (compare with
Table II and Fig. 3) desensitization were approximately the same. The
identical desensitization observed for full-length and truncated
1aAR reinforces the evidence presented above indicating that the carboxyl terminus plays little if any role in desensitization of this receptor in rat-1 cells.
|
) or with (
) GRK2; B,
HA-T348 without (
) or with (
) GRK2; and C,
HA-
) or with (
) GRK6. Cells loaded
with [3H]inositol were stimulated for 20 min with
various concentrations of NE. Total IPs were quantitated and plotted as
percent of response of cells expressing receptor alone. Data are
mean ± S.E. from three independent experiments, each performed in
triplicate.
Potential Mechanisms Underlying NE- and PMA-induced
1aAR Desensitization
Phosphorylation of Full-length and Truncated 1aARs
in Cells--
Discovery of rapid desensitization of human
1aARs led us to explore possible mechanisms underlying
this phenomenon. Rapid phosphorylation of GPCRs by GRKs and/or second
messenger-activated kinases such as PKC is thought to lead to receptor
desensitization; therefore, we desired to examine phosphorylation of
full-length and truncated 1aARs in intact cells.
Experiments designed to identify 1aAR proteins showed
that photoaffinity labeling of membranes expressing
HA-1a with [125I]azidoprazosin yields a
single diffuse band centered ~60 kDa (Fig.
6, lane 1). This corresponds
to the size of the glycosylated wild type 1aAR reported
previously (25). Labeling of membranes derived from cells expressing
HA-T348 yields a diffuse band of ~48 kDa, corresponding to the
expected gel mobility of the truncated receptor (Fig. 6,
lane 2). Prazosin (1 µM) blocks labeling of both peptides, confirming specificity of the photoaffinity labeling reaction and identification of 1aARs (Fig. 6,
lanes 3 and 4).
|
Phosphorylation of 1aARs was examined in rat-1
fibroblasts stably expressing HA-tagged full-length or truncated
receptors that were equilibrated with inorganic 32P to
label intracellular pools of ATP. Following drug treatment, cells were
lysed and solubilized and the HA epitope-tagged receptors were
immunoprecipitated and subjected to SDS-polyacrylamide gel electrophoresis. Treatment of cells expressing HA-1a or
HA-T348 with either NE or PMA clearly resulted in increased
phosphorylation of the receptors relative to untreated cells (Fig. 6).
This phosphorylation response was both dose-dependent (data
not shown) and time-dependent (see below). Less
phosphorylation was observed for HA-T348 than for the
HA-1a (Fig. 6). This difference is quantitated in
carefully paired time courses presented below. Both
HA-1a and HA-T348 display some basal phosphorylation,
although basal phosphorylation of HA-T348 is reduced relative to
HA-1a and is not apparent at the exposure level shown in
Fig. 6. Pretreatment of cells with prazosin before NE stimulation
prevents agonist-mediated increases in phosphorylation of the receptor
(data not shown). The selective -AR antagonist propranolol
(104 M) does not block NE-stimulated
32P incorporation into the HA-1a, consistent
with the absence of -ARs in rat-1 cells; furthermore, treatment with
forskolin/isobutylmethylxanthine (104
M/103 M) does not change basal
phosphorylation of 1aARs, ruling out a possible role for
cAMP-dependent protein kinase A (PKA) in either basal or
agonist-mediated 1aAR phosphorylation (data not shown). These data demonstrate that 1aARs are phosphorylated in
response to both agonist stimulation and direct activation of PKC by
PMA. In addition, stimulation with either NE or PMA causes receptor phosphorylation at sites outside of the carboxyl tail of the
1aAR.
To characterize the temporal correlation between 1aAR
desensitization and receptor phosphorylation, we examined receptors from 32P-labeled rat-1 fibroblasts expressing either
full-length or truncated receptors that were treated with NE or PMA for
various periods of time (Fig. 7). In a manner that parallels time
courses of both NE- and PMA-induced desensitization of
1aARs, both drugs increase HA-1a
phosphorylation on a rapid time scale. Near-maximal
HA-1a phosphorylation in response to NE stimulation is
achieved within 2 min, with a slight increase at 5 min, and is
sustained throughout 30 min of treatment (Fig.
7A). PMA induces maximal
phosphorylation of HA-1a within the first 2 min of
treatment, with PKC-mediated phosphorylation remaining at maximal
levels after 30 min of treatment as well (Fig. 7A). Thus,
the time frame of phosphorylation of 1aAR is closely
correlated with the temporal occurrence of acute receptor
desensitization.
|
Interestingly, NE-induced phosphorylation of the truncated receptor
does display somewhat different behavior than full-length receptor. As
for HA-1a, phosphorylation of HA-T348 is maximal at
about 2 min of receptor stimulation during which time 32P
incorporation into the receptor rises 8.9-fold over basal levels (Fig.
7B). However, after 5 min receptor phosphorylation begins to
decrease until a steady level of 4.2-fold over basal is achieved after
20 min of receptor stimulation. It is important to remember that
HA-T348 remains maximally desensitized during this entire period (refer
to Fig. 3A). In addition, quantification of Fig. 7 data (in
arbitrary pixel units) suggests that NE-induced phosphorylation of the
truncated receptor is approximately half that observed for full-length
receptor. This observation could indicate that truncation has
eliminated potential phosphorylation sites. However, other explanations
are possible; for example, decreased solubilization or
immunoprecipitation of HA-T348 compared with full-length receptor. Even
if NE stimulation induces phosphorylation of sites in the carboxyl-tail
of 1aAR, the absence of any apparent role for the carboxyl-terminal tail in desensitization (Figs. 2, 3, and 5) suggests
the sites do not participate in controlling agonist-mediated desensitization. This does not preclude potential roles of other phosphorylation sites in other regulatory processes.
The time course of PMA effects on HA-T348 phosphorylation is similar to
that of the full-length receptor. Maximal 32P incorporation
was achieved between 2 and 5 min of treatment with PMA, continuing
through 30 min (Fig. 7B). Quantitation indicates that
PMA-induced phosphorylation of HA-T348 is about half that observed for
the full-length receptor. In contrast to agonist-mediated desensitization, the carboxyl-terminal tail of 1aAR does
influence PMA-induced desensitization (see Fig. 3B above). This fact is consistent with phosphorylation of the tail playing a role is PKC
mediated 1aAR desensitization. Nevertheless, PMA-induced phosphorylation and partial desensitization of HA-T348 suggests regions
other than the carboxyl-terminal tail also participate in these events.
As for full-length 1aARs, the overall time frame of both
NE and PMA-induced phosphorylation of HA-T348 receptors is closely
correlated with the temporal occurrence of acute receptor desensitization.
Agonist-induced Internalization of
1aARs--
Sequestration of receptors from the
extracellular surface into various intracellular compartments may occur
as a component of both agonist-dependent and
agonist-independent desensitization and/or resensitization processes.
To determine whether receptor sequestration plays a role in acute
desensitization of 1aARs, we performed radioligand
binding on intact rat-1 fibroblasts expressing HA-1a or
HA-T348 with the lipophilic 1AR antagonist
[3H]prazosin at 4 °C, as previously described. Ligand
binding at this temperature has been shown to inhibit partitioning of
lipophilic prazosin across cell membranes, thus preventing recognition
of receptors located intracellularly and allowing quantitation of cell
surface receptors; ligand binding at 37 °C, on the other hand,
allows detection and quantitation of total cellular receptors (26).
Effects of NE treatment were similar in cells expressing full-length
1aARs and those expressing the truncated receptor. During at least 10 min of agonist stimulation, there is no measurable decrease in the number of cell surface 1aARs (Fig.
8A). Between 10 and 30 min of
agonist exposure, however, surface receptor numbers decrease by 25%
and continue to slowly decline over the next 60 min. Total numbers of
either full-length or truncated 1aARs do not decline in
response to NE or PMA stimulation over this same time period,
indicating that loss of surface receptors following NE exposure is due
to internalization and not degradation of receptors (data not shown).
Thus, internalization of 1aARs occurs at a much slower
rate than does either phosphorylation or desensitization of the
receptors and, therefore, cannot be responsible for rapid 1aAR desensitization that is seen within the first
several min of agonist exposure.
|
Interestingly, PMA treatment has no effect on the number of full-length
or truncated 1aARs at the cell surface throughout 90 min
of exposure (Fig. 8B). The inability of PMA-mediated
stimulation of PKC to induce internalization of 1aARs
stands in stark contrast to its ability to induce phosphorylation and
dampen signaling of these receptors, indicating that these are sharply
distinct processes for the 1aAR. This also suggests that
increased receptor phosphorylation alone does not induce
1aAR internalization, but that other factors are
necessary to initiate this process. These 1aAR
regulatory processes are clearly not induced by PKC activity. Furthermore, when cells stably expressing 1aARs are
treated with the PKC inhibitor bisindolylmaleimide I prior to agonist
stimulation, there is no effect on the rate or extent of
agonist-mediated internalization of HA-1aARs (data not
shown). These data provide further evidence that, similar to
agonist-mediated 1aAR desensitization, agonist-induced internalization of 1aARs occurs through a
PKC-independent pathway.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
One unique feature of 1aAR expression is the
presence of at least 12 receptor isoforms in various species, as
opposed to single isoform expression of 1b and
1dAR family members. The four full-length functional
human isoforms (WT 1a-1, 1a-2,
1a-3, and 1a-4) differ in amino acid
sequence at their distal carboxyl termini. Although the physiological
significance of the 1aAR isoforms remains unclear
particularly since their pharmacologic binding properties and second
messenger coupling are identical, differences between their
carboxyl-terminal amino acid sequences suggest that each
1aAR isoform may be differentially regulated through
this receptor region. For example, 4 potential PKC sites are found in
the carboxyl terminus of the 1a-1, 3 each in isoforms 1a-2, 1a-3, and 1a-4 (15,
16). Studies of 1aAR desensitization in endogenous
systems face several potential difficulties, however, since all four
functional 1aARs and numerous nonfunctional truncated receptor isoforms are found in all tissues studied thus far. To focus
on the most abundant 1aAR isoform in human prostate and heart, we profiled expression of functional 1aAR isoform
mRNAs in these tissues. 1a-1 is clearly the
predominant splice variant in human prostate and heart, as well as in
SK-N-MC cells. Although Chang et al. (16) originally
suggested that the 1a-4 mRNA predominates in human
prostate (based on reverse transcriptase-PCR experiments), our direct
RNase protection assay results clearly demonstrate much higher
expression of the 1a-1 isoform in both prostate and heart. Therefore, we stably transfected epitope-tagged human
1a-1 AR cDNA in rat-1 fibroblasts to create a system
in which acute regulation of the 1a-1AR isoform can be
studied without confounding coexpression of other splice variants.
Our studies provide convincing evidence that human 1aARs
are subject to acute agonist-induced desensitization of IP signaling in
response to NE stimulation. Desensitization of 1aARs is
rapid (occurring within 2 min) and is characterized by a rightward
shift and lowering of the NE dose-response curve (see Table
II). We demonstrate that 1aAR phosphorylation
is significantly increased within 2 min of agonist stimulation and
closely correlates with acute agonist-mediated desensitization of
1aARs. This agonist-induced process is most likely
mediated through actions of at least one GRK as overexpression of GRK2
is able to diminish 1aAR-mediated IP signaling. These
data also suggest that receptor phosphorylation plays an important role
in acute agonist regulation of 1aAR signaling, as has
been proposed for GPCRs in general. It is possible that agonist-induced
desensitization and phosphorylation of 1aARs may be
concurrent yet independent events; indeed, the causal effect of protein
phosphorylation on receptor desensitization has not been definitively
demonstrated for any GPCR. However, mutagenic elimination of protein
phosphorylation sites in several GPCRs (including 1bARs)
does provide compelling evidence that the elimination of receptor
phosphorylation sites results in significantly reduced receptor
desensitization (8, 12).
Internalization of GPCRs from the cell surface may also serve to
desensitize receptor populations by removal of receptors from agonist
exposure and prevention of further signaling. Agonist stimulation does
induce internalization of human 1aARs (as demonstrated by loss of cell surface receptor binding). However, since this does not
occur until after at least 10 min of agonist exposure, it cannot be
responsible for rapid agonist-mediated 1aAR
desensitization that is seen after only 2 min. This finding corresponds
with observations that receptor internalization does not play a role in
rapid desensitization of several other GPCRs, including the
2AR (27), m3-muscarinic receptor (28), and
1bAR (8).
Agonist-mediated desensitization of 1aARs occurs
independently of PKC effects, as demonstrated by the failure of PKC
inhibition to affect this process. A similar lack of phospholipase C
involvement in agonist-mediated desensitization of several other
phospholipase C-coupled receptors including substance P (29),
m3-muscarinic (30), thromboxane (31), and 1b-adrenergic
receptors (8) has also been demonstrated. In our hands, human
1aARs are nevertheless subject to PKC-mediated
desensitization as phorbol ester (PMA) pretreatment substantially
decreased IP production resulting from subsequent NE stimulation of the
1aAR. PMA treatment also leads to a rapid increase in
1aAR phosphorylation that closely correlates with the
onset of PMA-induced desensitization. In this context, it is noteworthy
that PMA treatment does not induce internalization of
1aARs from the cell surface. Conversely, PKC inhibition
does not affect agonist-mediated internalization of
1aARs from the cell surface. These data support the
hypothesis that exposure to agonist and PMA induces distinct and
possibly separate 1aAR regulatory responses. Although
our data do not support a specific role of PKC "feedback" in
agonist-induced desensitization of 1aARs, this second
messenger-dependent kinase may play a role in modulating the ability of 1aARs to signal following stimulation of
other PLC-coupled receptors within the same cell. This hypothesis is supported by recent demonstrations that stimulation of endothelin ETA receptors or bradykinin B2 receptors in
rat-1 fibroblasts leads to a marked increase in phosphorylation of
stably expressed 1bARs (32, 33).
Perhaps our most significant finding is that mechanisms underlying
acute agonist-dependent regulation of human
1aAR signaling appear to function independently of the
receptor carboxyl terminus. This is demonstrated by equal sensitivity
of the truncated and full-length 1aAR to NE-induced
desensitization. In addition, GRK2 desensitizes signaling by the
truncated and full-length receptor equally, suggesting that the
agonist-mediated pathway of desensitization remains intact in the
absence of the 1aAR carboxyl terminus. On the other
hand, sensitivity of the truncated receptor to PMA-induced desensitization is less than that of the full-length
1aAR. These data provide further evidence for the
hypothesis that agonist- and PMA-induced desensitization proceed
through separate, distinguishable mechanisms, and indicate that
although the 1aAR carboxyl terminus plays a substantial
role in PMA-mediated desensitization, it is not required in the
agonist-mediated pathway. This highlights a significant regulatory
difference between human 1aARs and the 1bAR subtype as the carboxyl-terminal of the
1bAR plays an indispensable role in mediating both
agonist- and PMA-induced phosphorylation and desensitization (8, 11,
12). In addition, our results place the human 1aAR as
the only Gq-coupled member of a very small group of GPCRs
whose mechanisms of desensitization are primarily independent of
carboxyl terminus regulation (e.g. follitropin receptor
(34), 2ARs (35), and the angiotensin II receptor (36)).
As acute desensitization of truncated 1aARs predicts, NE
and PMA treatment each lead to significantly increased phosphorylation of the truncated 1aAR. However, HA-T348 is modestly less
phosphorylated than the full-length receptor under all conditions. This
is not an unexpected result, given that potential phosphorylation sites were eliminated with truncation of the 1aAR after
Arg348. For example, the human 1a-1AR
contains 4 putative PKC phosphorylation sites within the carboxyl
terminus as well as several potential sites in other intracellular
regions of the receptor, including 1 in the second intracellular loop
and 4 in the third intracellular loop. Thus truncation eliminates half
of the potential PKC phosphorylation sites, perhaps explaining why the
truncated 1aAR cannot be desensitized as completely as
the full-length receptor. Of course the truncated receptor still
displayed substantial PMA-mediated desensitization concurrent with
rapid phosphorylation strongly suggesting involvement of sites not in
the tail.
Possible sites of GRK-mediated 1aAR phosphorylation are
more difficult to predict, since consensus sequences for receptor recognition and phosphorylation by individual GRKs are not as clearly
defined. Early studies suggested that GRK2 and GRK3 preferentially phosphorylate serines or threonines in proximity to acidic amino acids
(37) and Fredericks et al. (38) has observed that several receptors that are phosphorylated by GRKs contain a pair of acidic residues on the amino-terminal side of the phosphorylated residue. The
human 1aAR does not contain acidic pairs of residues,
however, several potential GRK phosphorylation sites (represented by
serines and threonines in the vicinity of acidic residues) are found
within the carboxyl terminus and, more interestingly, within
intracellular loops of the human 1aAR. Experiments
presented here indicate that elimination of some potential GRK
phosphorylation sites by truncation does not affect the ability of the
1aAR to undergo acute agonist-mediated desensitization
even if overall receptor phosphorylation is decreased. Furthermore,
agonist-induced phosphorylation of the truncated 1aAR
occurs concomitant with acute desensitization. These data are
consistent with the hypothesis that phosphorylation of only a discrete
subset of amino acids (retained within the truncated human
1aAR) regulates agonist-mediated desensitization of the
1aAR, although other sites may be phosphorylated that do
not affect receptor desensitization. It is worth noting that the
agonist-induced phosphorylation increase (over basal) was higher for
HA-T348 (8.9-fold) than for full-length HA-1aAR
(4.5-fold). It could certainly be the case that fold increases in
phosphorylation can influence desensitization as much as absolute
phosphorylation levels. If so, the fact that decreasing HA-T348
phosphorylation remains at 4.2-fold over basal even at the longest NE
exposure times (30 min), may explain continued maximal desensitization. On the other hand, at times far removed from acute desensitization it
is probable that mechanisms directed at downstream elements of the IP
cascade are functioning.
While our manuscript was in preparation, a study of phosphorylation of
the bovine 1aAR stabily expressed in rat-1 fibroblasts was published by Vazquez-Prado and colleagues (14). Both studies observe that exposure of the 1aAR to NE causes rapid
receptor phosphorylation (1 to 2 min), generating a 5-10-fold increase in phosphorylation compared with basal. Vazquez-Prado and colleagues (14) also suggested that phosphorylation of 1aAR
occurred primarily in the carboxyl-terminal tail; however, the evidence
presented was indirect. While phosphorylation of the chimeric
1baAR (consisting of the unphosphorylatable
transmembrane region of 1bAR (12) and carboxyl
terminus of 1aAR) does imply phosphorylation of the
terminus, it does not demonstrate this phosphorylation is dominant or
functionally significant. Indeed, no functional assays were performed
using the chimeric protein. On the other hand, data presented in our
current study are clear and direct; we demonstrate that the truncated
1aAR (HA-T348) is phosphorylated in response to NE
stimulation in the same rapid manner as full-length receptor and
furthermore, has identical desensitization properties. Although, phosphorylation of the carboxyl terminus may occur, it is not involved
in agonist-mediated desensitization as deletion of the tail did not
change any characteristic of desensitization.
In conclusion, our findings demonstrate that 1aAR
signaling is subject to acute desensitization in response to NE as well as PMA exposure. In agreement with current paradigms, acute
desensitization appears to be mediated by receptor phosphorylation
since both occur simultaneously within 2 min of NE exposure. The GRK
family of kinases is probably responsible for agonist-mediated
phosphorylation as PKC was not involved and GRK2 was capable of
desensitizing the 1aAR. Study of a truncated
1aAR provides definitive evidence that
1aAR phosphorylation is not localized to the carboxyl
terminus alone, but that other receptor regions are also phosphorylated (presumably one or more intracellular loops). In addition,
characterization of the functional consequences of carboxyl-terminal
truncation of the human 1aAR also shows that this
receptor region is not necessary for full agonist-induced receptor
desensitization and internalization. Not only does carboxyl-terminal
independent regulation of 1aARs represent a significant
mechanistic difference between 1a and
1bAR family members; it also suggests a common
regulatory mechanism between 1aAR splice variants.
Although differences may be discovered between 1aAR
isoform regulation upon closer examination of individual
1aAR splice variants, our data suggest a common behavior
is possible and perhaps likely. Our findings also underscore the
importance of examining receptor-specific regulation, particularly in
cases where distinct differential agonist-mediated regulatory processes
are known to occur such as within the 1AR family. The
challenge now is to identify specific amino acid residues that are
involved in human 1aAR desensitization and begin to
unravel the relationship of acute desensitization to other regulatory
pathways such as receptor trafficking and gene transcription.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Sylvia Hill and Mark K. Dole for
excellent technical assistance and Drs. Eric Roush and Madan Kwatra for
scientific discussions. We also thank Dr. Anthony Ford (Roche
Bioscience, Palo Alto, CA) for the kind gift of 1aAR
carboxyl-terminal splice variant cDNA constructs and Dr. Robert
Lefkowitz for GRK cDNAs.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grants AG00745 and HL49103 (to D. A. S.). Human tissues were obtained via the Duke Rapid Autopsy Program (supported by National Institutes of Health Grant AG05128 and GlaxoWellcome, Inc.) and the Duke General Clinical Research Center (NIH-M01 RR30).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: GlaxoSmithKline, Inc., RTP, NC 27709.
Present address: School of Veterinary Medicine, University of
Pennsylvania, Philadelphia, PA 19104.
Senior fellow in the Center for the Study of Aging and Human
Development, Duke University Medical Center. To whom correspondence should be addressed: Professor of Anesthesiology, Pharmacology/Cancer Biology, and Surgery, Box 3094, Dept. of Anesthesiology, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-4781; Fax:
919-681-4776; E-mail: schwi001@mc.duke.edu.
Published, JBC Papers in Press, January 7, 2002, DOI 10.1074/jbc.M111762200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
1aAR, 1a-adrenergic receptors;
GPCR, G
protein-coupled receptor;
GRK, G protein-coupled receptor kinase;
NE, norepinephrine;
PMA, phorbol 12-myristate 13-acetate;
DMEM, Dulbecco's
modified Eagle's medium;
HA, hemagglutinin;
PBS, phosphate-buffered
saline;
IP, inositol phosphate.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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