Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
α1-adrenoceptors (α1-AR) are
Gq/11-coupled receptors that respond to the neurotransmitters and hormones
norepinephrine and epinephrine to mediate physiological effects such
as prostate smooth muscle contraction and myocardial
hypertrophy by activating phospholipase C and generating
second messengers that release stored intracellular
Ca2+ and stimulate protein kinase
C[1,2]. α1-AR are also involved in the
regulation of growth-promoting responses via the
mitogen-activated protein kinase
family[3].
Three human α1-AR subtypes have been cloned:
α1A-, α1B-, and
1D-AR. Although α1-AR family members are highly
homologous (eg 75% amino acid identity in transmembrane
domains), they share little homology at their amino and
carboxyl termini[3,4], suggesting that
α1-AR subtypes have different expression, function, and subcellular distribution.
α1-AR expression was found in many human tissues such
as the brain, heart, and vascular smooth
muscles[5]. Several reports have shown that
α1B-AR was mainly localized on the cell
surface[6_9], and agonists induced its phosphorylation
and internalization. G-protein-coupled receptors (GPCR),
kinases 2 and 3, seem to be involved in the phosphorylation
of agonist-bound α1B-AR during homologous
desensitization[10,11]. The phosphorylation sites involved have been
located at Ser404, Ser408, and Ser440 in the receptor C
terminus[11]. Several researchers found that
α1A-AR was mainly located on the cell surface and the intracellular compart-
ments[9]. However, α1D-AR was mainly localized in the
intracellular compartments[12], but could be induced to
translocate to the cell surface by co-expressed
α1B-AR[13]. Unfortun-ately, the molecular determinants of desensitization and
internalization for α1A-AR and
α1D-AR were largely unknown. Vazquez-Prado
et al[14] showed that
α1A-AR could undergo agonist-mediated phosphorylation, but not to the same
extent as α1B-AR. Yang and
colleagues[15] stably transfected
fibroblasts with each α1-AR and observed that increases in
inositol phosphates mediated by α1A and
α1B-AR could be desensitized, whereas the increase mediated by
α1D-AR was refractory to agonist-mediated desensitization. In contrast,
Garcia-Sainz et al[16] found that
α1D-AR could be phosphorylated and desensitized. Recent reports showed that cell
trafficking (desensitization and redistribution) was an
important step in the regulation of GPCR, particularly in
response to receptor stimulation by agonists. The ultimate
effect on receptor signaling and the fate of sequestered
receptors varies with receptor type, duration of agonist
exposure, and cellular environment[17]. In this study, we
examined subcellular distribution of the 3
α1-AR subtypes and their internalization and trafficking upon agonist stimulation
in human embryonic kidney (HEK) 293A cells. We also
compared the distribution and trafficking property between total
receptors and functional receptors.
Materials and methods
Flag-tagged human α1A-,
α1B-, and α1D-AR were gifts from
Prof KP MINNEMAN (Emory University, Atlanta, USA).
Dulbecco's modified Eagle's medium (DMEM) and fetal
bovine serum (FBS) were obtained from Hyclone (Rockville,
MD, USA). Geneticin, an anti-flag antibody, and
phenylephrine were obtained from Sigma-Aldrich (St Louis, MO,
USA). [125I]-BE2254 and [3H]-prazosin were obtained from
Amersham Biosciences (Buckinghamshire, UK).
α1-AR green fluorescent protein (GFP) vectors were constructed
by ligating the coding region of human
α1A-,α1B-, and
α1D-AR into the EcoR I_BamH
I site of the basic pEGFP-C2
protein fusion vector (Clontech, USA) as described
previously[7].
Cell culture and transfection HEK293A cells were
cultured in DMEM with sodium pyruvate supplemented with
10% FBS, 100 U/mL streptomycin, and 100 U/mL penicillin in
a humidified atmosphere with 5% CO2. Cells for transient
transfection were grown in culture dishes with a glass
coverslip. When grown to approximately 60% confluence,
cells were transfected with 5 μg cDNA encoding
α1A-, α1B-, α1D-AR/GFP fusion protein using Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA), respectively. Cell lines
stably transfected with α1A-,
α1B-, and α1D-AR/Flag, which
were cloned and established before[18], were grown on 6-well
plates and maintained with geneticin (200 mg/mL). As
previously described, the Bmax of
α1A-AR/Flag, α1B-AR/Flag, and
α1D-AR/Flag were 4.8, 20.8, and 0.24 pmol/mg protein,
respectively, on cell membranes as determined by
[125I]-BE2254 binding
assay[18].
Western blot assay HEK293A cells transiently
expressing α1A-, α1B-, or
α1D-AR were treated with phenylephrine (10
µmol/L) or prazosin and phenylephrine or vehicle. Cells were
lysed with ice in cold lysis buffer containing 20 mmol/L
Tris-HCl (pH 7.4), 150 mmol/L NaCl, 2.5 mmol/L edetic acid, 50
mmol/L NaF, 0.1 mmol/L
Na4P2O7, 1 mmol/L
Na3VO4, 1% TritonX-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid,
1 mmol/L phenylmethylsulfonyl fluoride, and 1 mg/mL
aprotinin for 20 min, sonicated for 20 s, and centrifuged at
12 870×g for 15 min. The supernatant is the whole cell protein.
The lysate [30 µg, extracellular signal-related kinase (ERK)
assay] was separated by electrophoresis using 10%
SDS-PAGE and transferred onto nitrocellulose membrane or
polyvinylidene difluoride membrane. Nonspecific IgG was
blocked with 5% fat-free milk, and the membrane was
incubated with the antibody to phospho-ERK (Cell Signaling
Technology, Inc, Beverly, MA, USA); horseradish
peroxidase-conjugated anti-mouse IgG was used as a second
antibody (Jackson ImmunoResearch Laboratories, West Grove,
PA, USA). The peroxidase reaction products were
visualized by LumiGLO Chemiluminescent Substrate (New
England Biolabs, Beverly, MA, USA). The same membrane
was then stripped and reprobed with anti-ERK to determine
the total protein.
Confocal microscopy and image quantification
HEK293A cells transiently transfected with GFP-tagged construct were
grown on sterile coverslips, fixed for 30 min with 4%
paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4, and rinsed 3
times with PBS. Cells were imaged with a Spectra-Physics
laser scanning confocal microscope with a Plan-Apo 40× oil
immersion objective lens (Leica, Wetzlar, Germany). The
software used to collect the images was the Leica TCS NT,
version 1.6.587. The images were transferred to a computer and
analyzed with Adobe Photoshop version 5.0 (Adobe Systems, Mountain View, CA, USA). The setting on the
laser was constant for all experiments. Fluorescence was
excited using an argon laser at a wavelength of 488 nm, and
the absorbed wavelength was detected at 510_520 nm. The
extent to which the α1-AR/GFP was internalized after
exposure to agonists was quantified by the intracellular
immunofluorescence intensity. The extent of
α1-AR/GFP receptor internalization was defined as the rate of the fluorescence
intensity before and after exposure to agonists.
Internalization assay The HEK293A cells stably
expressing α1-AR were seeded at a density of
2×105/well onto poly-L-lysine-coated 24-well plates. The next day, the cells were
pre-incubated with 1 µg of mouse anti-flag antibody for 1 h
in DMEM free of serum at 37 °C and washed twice using
serum-free DMEM. Cells were then treated with 10 µmol/L
phenylephrine for different time periods in the serum-free
DMEM. Subsequently, the cells were fixed and incubated
with peroxidase-conjugated anti-mouse antibody (1:1000,
Santa Cruz, CA, USA) for 2 h at room temperature. After
washing, the plates were developed with 250 µL of
o-phenylenediamine dihydrochloride (OPD) solution (4 mg OPD
in 10 mL PBS, 15 µL 30%
H2O2). After 10_30 min, 200 µL of
the substrate solution from each well was transferred to
96-well plates and analyzed at 490 nm using a microplate reader
(Bio-Rad, Hercules, CA, USA).
Whole cell binding assay To further determine the
subcellular distribution of the 3 α1-AR subtypes prior to, or after
incubation with phenylephrine for 1 h, radioligand binding
was performed on whole cells stably transfected with the
flag-tagged 3 α1-AR constructs as described by Eason MG
et al[19]. The cells were re-suspended in ice-cold DMEM
medium, then incubated with increasing concentrations (0.01_2 nmol/L) of [3H]-prazosin alone, or in the presence of
the lipophilic competitor, phentolamine (10 µmol/L), or in the
presence of the hydrophilic competitor, adrenaline (100 mmol/L), for 4 h at 4 °C. With the intact cells, specific binding,
as defined with phentolamine, represented the total cellular
amount of α1-AR, because it is able to penetrate the plasma
membrane and scatter about the whole cell, whereas specific
binding, as defined with adrenaline, represents only cell
surface receptors owing to its hydrophilic properties, that is,
adrenaline is unable to penetrate the plasma membrane,
especially at 4 °C. The intracellular pool of receptors is
defined as the total number of receptors minus those on the
cell surface. Saturation and displacement curves were
analyzed by non-linear regression analysis with Prism (GraphPad
Software, San Diego, CA, USA).
Statistical analysis Data from confocal quantity were
analyzed using the unpaired t-test with statistical
significance defined as P<0.05. Data from Western and
internalization assays were analyzed by GraphPad Prism using the 2-tailed paired t-test. Significant differences were defined as
P<0.05.
Results
ERK 1/2 were activated by phenylephrine in the HEK293A cells transiently transfected with
α1-AR/GFP fusion protein To demonstrate that the
α1-AR/GFP fusion proteins expressed in HEK293A cells were functional, the
HEK293A cells transfected with α1A-,
α1B-, and α1D-AR/GFP, respectively, were treated with phenylephrine, and the
phosphorylation of ERK 1/2 was measured by Western blot.
The levels of the phospho-ERK1/2 in HEK293A cells transfected with α1A-, α1B-, or
α1D-AR/GFP significantly increased after treatment with phenylephrine (PE, 10
µmol/L), an agonist of α1-AR, for 30 min. Pretreatment with prazosin
(1 µmol/L), an antagonist of α1-AR, however, greatly
attenuated the effects of phenylephrine on the activation of
ERK1/2 in the transfected HEK293A cells. These results suggested
that the α1-AR/GFP fusion proteins expressed in the
HEK293A cells were biologically active (Figure 1).
Agonist-induced localization changes of
α1-AR subtypes in HEK293A cells
By real-time confocal microscopy, we found that the incubation of HEK293A cells transiently
expressing α1A- or α1B-AR/GFP with 10 µmol/L
phenylephrine caused changes of receptor localization (Figures 2, 3).
An accumulation of α1-AR in vesicle-like structures was
found in the cytoplasm, and the membrane receptors
became uneven. Because coated vesicles are the prelude of
GPCR internalization by agonist stimulation, these results
suggested that agonist stimulation could induce
α1A- and α1B-AR internalization. The receptor localization in cells
expressing α1D-AR, however, was unchanged after
phenylephrine stimulation (Figure 4).
We then measured the signal intensity of intracellular
fluorescence to estimate the rate of receptor internalization.
The fluorescent intensity of α1A- and
α1B-AR was significantly increased in the cytoplasm after phenylephrine
stimu-lation, and α1B-AR occurred in a very rapid manner (20 min
after phenylephrine stimulation, Figure 5A), while
α1A-AR occurred in a slower manner (25 min, Figure 5B). HEK293A
cells expressing α1D-AR, however, showed no translocation
of the receptors on the cell surface after phenylephrine
stimulation (Figure 5C). These results indicated that the location
changes of the receptors after phenylephrine stimulation were
due to receptor internalization.
Pretreatment with prazosin prior to phenylephrine
stimulation inhibited α1A- and
α1B-AR translocation and decreased the fluorescent intensity of cytoplasmic receptors in the
transfected HEK293A cells (Figure 5), suggesting that
phenylephrine acted directly on α1A- and
α1B-AR to induce receptor translocation, a phenomenon called homologous
desensitization or internalization.
α1-AR internalization induced by phenylephrine in
HEK293A cells We next examined the effects of
phenylephrine stimulation on cell surface receptor internalization using
HEK293A cells stably expressing α1A-,
α1B-, or α1D-AR by intact cell enzyme-linked immunosorbent assay (ELISA). Five
minutes after phenylephrine stimulation, the amounts of
α1B-, α1A-, and
α1D-AR on the cell surface decreased by about 29.2%, 9.7%, and 11.3%, respectively. One hour after the
stimulation, they further decreased by about 64.8%, 36.4%,
and 32.1%. These results indicated that phenylephrine could
induce the internalization of α1A,
α1B, and α1D-AR, and the internalization of
α1B-AR occurred faster than that of
α1A- and α1D-AR (Figure 6).
Agonist-induced distribution changes in binding of
functional α1-AR subtypes in HEK293A
cells To test whether membrane and cytoplasm functional receptors changed
under phenylephrine stimulation, whole cell
[3H]-prazosin binding assay was used. Functional
α1B-AR was mainly expressed on the surface of transfected HEK293A cells, which
was about 92.4% of the total receptor.
α1D-AR was mainly expressed in the cytoplasm, about 69.5% of total receptor,
and α1A-AR was expressed both on the cell surface and in
the cytoplasm, which was consistent with the results of the
confocal analysis. One hour after phenylephrine stimulation,
functional α1D-AR did not change compared with the control.
α1A- and α1B-AR on the cell surface, however, decreased
significantly: α1A-AR from 53.7% to 35.5% of the total
receptor, and α1B-AR from 92.4% to 30.2% of the total
receptor, which suggested that phenylephrine stimulation
could induce the internalization of functional
α1A-AR and α1B-AR proteins (Table 1).
Discussion
In this report, we investigated the trafficking profile of
the 3 α1-AR. We created a fusion construct consisting of N-terminal GFP-tagged α1-AR and transfected this construct
into HEK293A cells, which allowed us not only to identify
the 3 subtype receptors by confocal microscopy, but also to
compare the surface and total receptor by intact cell ELISA
and [3H]-prazosin binding assay before and after
phenylephrine stimulation.
As described in the Methods and in our previous work,
the Bmax of α1B-AR on the membrane was found to be the
highest, followed by α1A-AR and
α1D-AR, respectively[18].
These results are consistent with the previous report in which
the functional binding of wild type α1-AR was
examined[20].
Previous studies with the α1B-AR/GFP construct
demonstrated that it was fully functional and internalized in the
same manner as a non-GFP tagged α1B-AR
construct[21]. Both the α1A- and
α1B-AR were associated with the activation of
ERK[22]. In this study, all the
α1A-, α1B-, and
α1D-AR, when coupled to GFP, promoted the increase in ERK1/2
phosphorylation, suggesting that the α1-AR were functional
and retained their ability to activate cellular signaling when
conjugated to the GFP.
Cellular localization and trafficking is very important for
α1-AR to accomplish their physiological functions. Results
obtained over the past years have shown that there is a
significant difference in the subcellular distribution among
the α1-AR subtypes. Fonseca et
al[6] observed that α1B-AR
was expressed predominantly on the cell membrane and
α1A-AR was expressed both on the membrane and cytoplasm in
the stably-transfected fibroblasts by immunocytochemistry.
This was confirmed by Awaji et
al[21] who used
α1B-AR/GFP fusion proteins to verify cell membrane localization of
α1B-AR in COS-7 cells. McGrath et
al[23] used BODIPY-FL-labeled prazosin to image
α1-AR subtypes in cultured prostate smooth muscle cells and fibroblasts stably transfected
with each subtype and found that the 3
α1-AR subtypes were expressed in the cytoplasm of fibroblasts. Very little
α1D-AR expression was detected on the cell surface. In this
study, we transfected HEK293A cells with cDNA encoding
α1-AR/GFP. Living cells were then visualized by real-time
laser scanning confocal microscopy. We found that
α1B-AR/GFP fluorescence was predominant on the cell surface,
whereas the α1A-AR expression was detected both on the
cell surface and in the cytoplasm. α1D-AR was mainly
localized in intracellular compartments, suggesting that the
localization of the 3 α1-AR subtypes was different in the
transfected HEK293A cells.
α1-AR are subject to dynamic regulation by a variety of
mechanisms, including phosphorylation, protein-protein
interactions, protein trafficking, and transcription. One of
the most intensive studies of these mechanisms is
internalization and desensitization, a general phenomenon in which
the intensity of a biological response wanes over time,
despite continued stimulus. Desensitization can be further
characterized as either homologous, where receptor response
wanes upon continuous exposure to its agonist, or
hetero-logous, where agonist-mediated stimulation of a receptor
can attenuate the response by other receptors mediating
similar cellular events[24]. With regard to the
α1-AR subtypes, the desensitization, downregulation, and internalization
characteristics of the α1B-AR have been extensively examined.
For example, agonist-mediated phosphorylation and
internalization of the α1B-AR have been demonstrated, and the
domains of the receptors involved in internalization have
been identified[6,10,11]. We know much less about the
molecular determinants of desensitization, downregulation, and internalization for α1A- and
α1D-AR. Here, we transfected HEK293A cells with cDNA encoding
a-AR/GFP. Living cells were then visualized by real time laser scanning confocal
microscopy under phenylephrine stimulation. Stimulation
of α1-AR with phenylephrine induced the changes of
localization of α1A-AR and α1B
-AR, but not the localization of α1D-AR.
Confocal image quantification analysis found that
stimulation of α1B-AR with phenylephrine promoted a rapid
internalization of α1B-AR, which began at about 20 min, and a
slower internalization of α1A-AR, which began at about 25
min. However, α1D-AR internalization did not occur after
phenylephrine stimulation. We only assayed the changes
of fluorescence intensity of the plasma, but
α1D-AR was mainly localized in intracellular compartments. Therefore,
this method can not determine whether
α1D-AR was internalized under phenylephrine stimulation.
To confirm the results of confocal microscopy, the
internalization of membrane receptors was detected by intact cell
ELISA. This does not change the permeability of cell
membrane, so the antibodies to α1-AR subtypes only bind
to the membrane surface of stably-transfected HEK293A
cells. The internalization percentage of
α1A-AR was about 9.7% at 5 min and about 36.4% at 1 h after phenylephrine
stimulation. The internalization percentage of
α1B-AR was about 29.2% at 5 min and about 64.8% at 1 h after
phenylephrine stimulation. Intact cell ELISA assay also demonstrated
α1D-AR internalization after phenylephrine stimulation. The
internalization percentage of α1D-AR was about 11.3% at 5
min and about 32.1% at 1 h after phenylephrine stimulation.
Although Garcia-Sainz et al[22] showed that
α1D-AR could be phosphorylated and internalized, many other studies reported
that α1D-AR could not be internalized under agonist
stimulation. Our results by ELISA are the same as
Garcia-Sainz et al. As mentioned earlier, cell-intact ELISA only
detects the receptors located on the outside surface of the
membrane, but confocal imaging analysis mainly assesses
the changes of fluorescence intensity of the plasma. When
internalization occurs, α1D-AR may just move from the
outside to the inside of the membrane. Although some
α1D-AR may translocate from the membrane to the plasma, the
internalized α1D-AR are negligible compared with the total
α1D-AR because most α1D-AR are expressed in the plasma. This
may explain why α1D-AR internalization can be detected by
ELISA rather than confocal imaging analysis. However,
further studies are needed to elucidate this discrepancy.
The confocal imaging and ELISA analyses demonstrated
the localization changes and internalization of the 3
α1-AR subtypes. Receptor proteins include receptors with binding
activity and receptors without binding activity. Past studies
generally did not differentiate these 2 categories. In this
study, we detected the Bmax of the total cell surface and
intracellular functional α1-AR by whole cell
[3H]-prazosin binding assay. We found that the total
Bmax were the same in the 3 subtypes, and not different before and after phenylephrine
stimulation. α1A functional receptors were not only on the
cell surface, but also in intracellular compartments.
α1B functional receptors were predominantly on the cell surface;
α1D functional receptors were mainly in intracellular
compart-ments. In general, AR are G-protein-coupled membrane
receptors. From the previous point of view, only the
receptors on the membrane have the binding function, whereas
the receptors in the cytoplasm are immature and do not have
binding ability. However, the present study demonstrated
that there were functional receptors in the cytoplasm.
Stimulation of α1A- and α1B-AR with phenylephrine for 1 h
promoted internalization of α1A- and
α1B-AR, but not α1D-AR. This apparent discrepancy in
α1D-AR internalization, as determined by different methods, may be partly explained by
the different observed parameters. Both confocal imaging
and internalization assay determine receptor protein, whereas
whole cell binding assay determines functional receptors.
Since only a few α1D-AR are located on the cell surface, in
order to maintain the reactivity as strongly as possible, most
of them are probably functional receptors. On the contrary,
since both α1A- and α1B-AR on the cell surface are quite
abundant, the amount of their functional receptors would be
far more than that of α1D-AR, even though their functional
receptors may only account for a small fraction of the whole
receptors. It is supposed that when internalization occurs,
there would be as many mature functional
α1D-AR as possible sorted on the cell surface to complement the
internalized ones in order to maintain the maximal reactivity. However,
even if quite a lot of functional α1A- or
α1B-AR were internalized upon phenylephrine stimulation, the residual functional
receptors on the cell surface would be enough to maintain
the same reactivity as before, so it would not be necessary
to sort the mature functional receptors on the cell membrane.
Therefore, internalization could only be observed on
α1A- and α1B-AR, but not
α1D-AR by confocal imaging. However, the exact mechanism remains to be explored.
These results also explain why the total amount of the 3
α1-AR subtypes was equal by whole cell
[3H]-prazosin binding assay, while their densities on the membrane were quite
different when detected by traditional
[125I]-BE2254 binding assay. Because
[125I]-BE2254 binding assay only reflected
the receptors on the membrane, a part of the whole cell
receptors, [3H]-prazosin binding assay reflected the total
amount of receptors, including both receptors on the cell
membrane and those in the cytoplasm. The discrepancy
between the 2 methods offers evidence that the 3
α1-AR subtypes have different distribution, localization, and
trafficking characteristics, which is consistent with that
examined by other methods in this study.
In summary, in transfected HEK293A cells,
α1A-AR evenly distribute in the cytoplasm and membranes, but
phenylephrine stimulation causes their internalization.
α1B-AR are mainly located on the membrane, however, phenylephrine
can result in rapid internalization, and the number of
internalized α1B-AR is more than
α1A-AR. α1D-AR predominantly
exist in the cytoplasm, and their internalization upon
phenylephrine stimulation could be detected only by intact-cell
ELISA, but not by other methods. Therefore, a definitive
answer regarding the internalization characteristics of
α1D-AR requires additional study. Furthermore, we compared
the distribution between functional receptors and total
receptor proteins. Because there were functional receptors in
the cytoplasm, we suggested that the receptors in the plasma
act as a receptor's storage pool.
References
1 Milligan G. Constitutive activity and inverse agonists of G
protein-coupled receptors: a current perspective. Mol Pharmacol
2003; 64: 1271_6.
2 Graham RM, Perez DM, Hwa J, Piascik MT.
α1-Adrenergic receptor subtypes. Molecular structure, function and signaling.
Circ Res 1996; 78: 737_49.
3 Zhong H, Minneman KP. Differential activation of
mitogen-activated protein kinase pathways in PC12 cells by closely
related alpha1-adrenergic receptor subtypes. J Neurochem 1999;
72: 2388_96.
4 Schwinn DA, Johnston GI, Page SO, Mosley MJ, Wilson KH,
Worman NP, et al. Cloning and pharmacological
characterization of human α1-adrenergic receptors: sequence corrections
and direct comparison with other species homologues. J
Pharmacol Exp Ther 1995; 272: 134_42.
5 Rokosh DG, Stewart AF, Chang KC, Bailey BA, Karliner JS,
Camacho SA, et al. α1-Adrenergic receptor subtype mRNAs are
differentially regulated by α1-adrenergic and other hypertrophic
stimuli in cardiac myocytes in culture and in
vivo. J Biol Chem 1996; 271: 5839_43.
6 Fonseca MI, Button DC, Brown RD. Agonist regulation of
α1B-adrenergic receptor subcellular distribution and function. J Biol
Chem 1995; 270: 8902_9.
7 Hirasawa A, Sugawara T, Awaji T, Tsumaya K, Ito H, Tsujimoto
G. Subtype-specific differences in subcellular localization of
α1-adrenoceptors (ARs): chloroethylclonidine preferentially
alkylates the accessible surface α1-ARs irrespective of subtype. Mol
Pharmacol 1997; 52: 764_70.
8 Hrometz SL, Edelmann SE, McCune DF, Olges JR, Hadley RW,
Perez DM, et al. Expression of multiple alpha1-adrenergic
receptors on vascular smooth muscle: correlation with the
regulation of contraction. J Pharmacol Exp Ther 1999; 290:
452_63.
9 Sugawara T, Hirasawa A, Hashimoto K, Tsujimoto G.
Differences in the subcellular localization of alpha1-adrenoceptor
subtypes can affect the subtype selectivity of drugs in a study with
the fluorescent ligand BODIPY FL-prazosin. Life Sci 2002; 70:
2113_ 24.
10 Diviani D, Lattion AL, Larbi N, Kunapuli P, Pronin A, Benovic
JL, et al. Effect of different G protein-coupled receptor kinases
on phosphorylation and desensitization of the
alpha1B-adrenergic receptor. J Biol Chem 1996; 271: 5049_58.
11 Diviani D, Lattion AL, Cotecchia S. Characterization of the
phosphorylation sites involved in G protein-coupled receptor
kinase- and protein kinase C-mediated desensitization of the
alpha1B-adrenergic receptor. J Biol Chem 1997; 272: 28712_9.
12 Chalothorn D, McCune DF, Edelmann SE, Garcia-Cazarin ML,
Tsujimoto G, Piascik MT. Differences in the cellular
localization and agonist-mediated internalization properties of the
α1-adrenoceptor subtypes. Mol Pharmacol 2002; 61: 1008_16.
13 Hague C, Uberti MA, Chen Z, Hall RA, Minneman KP. Cell
surface expression of α1D-adrenergic receptors is controlled by
heterodimerization with α1B-adrenergic receptors. J Biol Chem
2004; 279: 15 541_9.
14 Vazquez-Prado J, Medina LC, Romero-Avila MT,
Gonzalez-Espinosa C, Garcia-Sainz JA. Norepinephrine and phorbol
ester-induced phosphorylation of α(1a)-adrenergic receptor. J Biol
Chem 2000; 275: 6553_9.
15 Yang M, Ruan J, Voller M, Schalken J, Michel MC. Differential
regulation of human α1-adrenoceptor subtypes.
Naunyn-Schmiedeberg's Arch Pharmacol 1999; 359: 439_46.
16 Garcia-Sainz JA, Vazquez-Cuevas FG, Romero-Avila MT.
Phosphorylation and desensitization of α1D-adrenergic receptors.
Biochem J 2001; 353: 603_10.
17 Ferguson SS. Evolving concepts in G protein-coupled receptor
endocytosis: the role in receptor desensitization and signaling.
Pharmacol Rev 2001; 53: 1_24.
18 Wang S, Song Y, Xu M, Hao T, Han Q, Zhang Y. Redistribution
of three α1-adrenergic receptor subtypes in the stably
transfected HEK 293A cells upon agonist stimulation. Acta Physiol
Sin 2004; 57: 480_5.
19 Eason MG, Liggett SB. Subtype-selective desensitization of
alpha 2-adrenergic receptors. Different mechanisms control short
and long term agonist-promoted desensitization of alpha 2C10,
alpha 2C4, and alpha 2C2. J Biol Chem 1992; 267: 25 473_9.
20 Minneman KP, Esbenshade TA. Alpha 1-adrenergic receptor
subtypes. Annu Rev Pharmacol Toxicol 1994; 34: 117_33.
21 Awaji T, Hirasawa A, Kataoka M, Shinoura H, Nakayama Y,
Sugawara T, et al. Real-time optical monitoring of
ligand-mediated internalization of α1b-adrenoceptor with green fluorescent
protein. Mol Endocrinol 1998; 12: 1099_111.
22 Garcia-Sainz JA, Vazquez-Prado J, Villalobos-Molina R.
α1-adrenoceptors: subtypes, signaling and roles in health and disease.
Arch Med Res 1999; 31: 449_58.
23 McGrath JC, Mackenzie JF, Daly CJ. Pharmacological
implications of cellular localization of alpha1-adrenoceptors in native
smooth muscle cells. J Auton Pharmacol 1999; 19: 303_10.
24 Bunemann M, Lee KB, Pals-Rylaarsdam R, Roseberry AG, Hosey
MM. Desensitization of G-protein-coupled receptors in the
cardiovascular system. Annu Rev Physiol 1999; 61: 169_92.
|
