Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium
Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland
* Author for correspondence (e-mail: e.wyroba{at}nencki.gov.pl)
Accepted 2 February 2004
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Summary |
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Generally receptor sequestration follows its desensitization, which is initiated by receptor phosphorylation by G-protein-coupled receptor kinase. We cloned and sequenced the gene fragment of 407 nucleotides homologous to the ß-adrenergic receptor kinase (ßARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human ßARK2 (GRK3), and may participate in Paramecium ßAR desensitization.
These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate ßAR exists in unicellular Paramecium.
Key words: dynamin, sequestration, desensitization, Paramecium, ß2-adrenergic receptor, GRK, isoproterenol, cloning, confocal imaging, immunological analysis
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Introduction |
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The 69 kDa polypeptide separated by SDS-PAGE in S2 and P2
Paramecium subcellular fractions cross-reacted with antibody directed
against human ß2-adrenergic receptor (ßAR). Quantitative
image analysis of the western blots showed that the ß-selective
adrenergic agonist ()-isoproterenol (Iso), previously shown to enhance
phagocytic activity (Wyroba,
1989), evoked a redistribution of ßAR analogue from the
membranous (P2) to the cytosolic (S2) fractions. The relative increase in
immunoreactive band intensity in the S2 fraction reached 80% and was
paralleled by a 59% decrease in the P2 fraction. Confocal immunofluorescence
studies revealed the ßAR sites on the cell surface and at the ridge of
the cytopharynx, where nascent phagosomes are formed, and the localization of
the ß-immunoanalogue was confirmed by immunoelectron microscopy. These
results indicated that the 69 kDa Paramecium polypeptide
immunorelated to vertebrate ß2AR appeared in this ciliate as a
nutrient receptor. Pretreatment of the cells with 10 µmol
l1 Iso evoked a physiological response, together with a
redistribution of immunoreactivity detected in the subcellular fractions,
suggesting that a desensitization process had occurred.
Signaling by membrane receptors is terminated by endocytosis during the
process of desensitization (Lefkowitz et
al., 1983; Barak et al.,
1994
). One of the initial stages of desensitization is receptor
phosphorylation by GRK kinases (G-protein-coupled receptor kinases), which act
only on agonist-occupied receptor (Premont
et al., 1995
; Zhang et al.,
1997
).
We report here cloning of the gene fragment encoding the putative homologue
of the ßARK=GRK kinase, the enzyme involved in the beta-adrenergic
receptor phosphorylation. The deduced amino acid sequence of this gene
fragment showed 51.6% homology in 126 amino acids that overlapped with the
human ßARK2, including its catalytic and extension domain, and 47.6%
homology to the ßARK1, the first cloned GRK kinase
(Benovic et al., 1989) and the
enzyme that specifically phosphorylates only the agonist-occupied form of the
beta-adrenergic and closely related receptors. We also confirm that this
enzyme is expressed in Paramecium by obtaining the mRNA sequence
(deposited in GenBank, Accession no. AF346411).
In higher eukaryotes, receptor internalization/sequestration was found to
occur via dynamin-dependent and clathrin-mediated endocytosis
(Shetzline et al., 2002;
Braun et al., 2003
). We have
recently cloned the N-terminal and middle domains (1091 nucleotides encoding
363 amino acids) of dynamin in Paramecium. This protein is essential
in different endocytic processes (Damke et
al., 1994
; Schmid et al.,
1998
; Wiejak et al.,
2003
) and we showed the presence of the dynamin immunoanalogue
localized to the transferrin-containing endosomes
(Wiejak and Wyroba, 2002
;
Surmacz et al., 2003
).
Therefore, to elucidate whether receptor sequestration in Paramecium
follows a pathway similar to that observed in mammalian cells, we performed
experiments with antibodies directed against the C termini of human
ß2AR and human dynamin 2. These antibodies were utilized for
dual fluorochrome labeling of isoproterenol-pretreated cells, which were then
examined by laser scanning microscopy and immunogold ultrastructural
localization studies. The anti-dynamin antibody was also used in western blot
analysis of Paramecium subcellular fractions to confirm its
specificity.
Upon isoproterenol treatment the ß-immunoanalogue was redistributed into intracellular vesicles, where its colocalization with dynamin was observed in a series of the confocal optical sections. Detailed immunogold detection by electron microscopy enabled us to identify the small intracellular vesicles, representing the early endosomal compartment, to which the ß-adrenergic receptor and dynamin were localized. This suggests that ß-adrenergic receptor was sequestered in a dynamin-dependent manner. To our knowledge, this represents the first case of dynamin-dependent receptor internalization in unicellular eukaryotes.
On the basis of the above mentioned observations we propose that the molecular machinery for the desensitization/sequestration of the G-protein-coupled receptors exists in this single-cell eukaryote.
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Materials and methods |
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Chemicals
Taq polymerase, deoxynucleotides and agarose were from GibcoBRL
(Gaithersburg, USA), pGEM-T vector, E. coli strain JM 109 and
EcoRI from Promega (Madison, WI, USA), goat anti-dynamin 2 antibody
(Ab) from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA) and all
the other reagents were from Sigma (Steinheim, Germany).
Confocal imaging
Untreated and isoproterenol-treated (10 µmol l1 for 10
min) cells were fixed and processed for confocal imaging as previously
described (Wiejak et al.,
2002). The primary antibodies were: rabbit polyclonal Ab against
the C-terminal 15-amino acid residues of human ß2-adrenergic
receptor (ß2AR: kindly donated by Dr M. Von Zastrow,
University of California, San Francisco, USA;
Von Zastrow and Kobilka, 1992
)
diluted 1:500 in blocking reagent and goat anti-dynamin 2 Ab (1:500). These
were followed by the secondary FITC-conjugated anti-rabbit Ab (1:150) and
TRITC-conjugated anti-goat Ab (1:500), respectively. In the control samples
the primary antibodies were omitted.
The confocal laser scanning system Leica DM IRE2 (oil immersion objectives 63x) was used. Images were collected and processed using Leica confocal software 2.0 and Adobe Photoshop 6.0.
Electron microscopy
Immunoelectron microscopy was performed as described previously
(Wiejak et al., 2002) using
the same set of the primary antibodies (1:250) as in the confocal imaging,
i.e. anti-ß2AR and anti-dynamin. In control experiments the
primary Abs were omitted. The secondary Abs were: anti-rabbit IgG (1:20)
conjugated with colloidal 10 nm gold particles to visualize ßAR and
anti-goat conjugated with colloidal 5 nm gold to visualize dynamin. Ultrathin
sections were observed in a JEM 1200 EX electron microscope.
Immunodetection by confocal and electron microscopy was performed in triplicate.
Western blot analysis
Protein fractionation, SDS-PAGE and western blotting were performed as
described previously (Surmacz et al., 2001). Blots were stained in 0.5%
Ponceau Red in 3% trichloroacetic acid before immunodetection. The recombinant
rat dynamin 2 (kindly donated by Dr D. D. Binns from Department of
Pharmacology, U.T. Southwestern Medical Center, Dallas, USA) was used as a
positive control for immunoblot analysis.
Immunodetection was performed using primary antibody against the C-terminal region of human dynamin 2 (Santa Cruz, CA, USA) at 1:500 (overnight at 4°C) followed by incubation with the horseradish peroxidase-conjugated anti-goat IgG (1:1000) for 1 h and processing for chemiluminescent detection using West Pico (Pierce, USA).
PCR and cloning
Polymerase chain reaction (PCR) was performed on the genomic DNA isolated
from Paramecium (Subramanian et
al., 1994). PCR settings were: denaturation at 94°C (30 s),
annealing at 52°C (30 s) and extension at 72°C (1 min) for 29 cycles
using the PTC-200 DNA Engine from MJ Research. Additional extension at
72°C for 10 min was applied after the last cycle. PCR reactions were
performed in a total volume of 25 µl and the reaction mixture contained
0.75 µg of genomic DNA as a template, 0.4 µmol l1 of
each primer, 100 µmol l1 each of deoxynucleotidyl
triphosphates, 2 mmol l1 of MgCl2, 1x PCR
buffer and 2 units of Taq Polymerase (Amersham, Little Chalfont, UK).
Degenerate primers were synthesized according to Paramecium codon
usage and were originally based on the amino acid sequences of
Paramecium endocytic proteins cloned by us, and public databases of
mammalian species. Forward: 5'-TAATT/CTGT/CTGGAAAATT/CATT/CAA and
backward: 5'-TAATCA/TGCA/TGGAAAATCA/TTC. The control samples not
containing the template did not yield any PCR products. PCR products were
separated by gel electrophoresis (60 V for 2 h) on 1.5% agarose followed by
ethidium bromide staining and a brief rinse in double-distilled water to
visualize DNA under UV light. The band of interest was transferred from the
1.5% agarose gel to DEAE-cellulose, eluted as described in Sambrook et al.
(1989) and subcloned into
pGEM-T Easy vector (according to the manufacturer's instructions; Promega).
Transformation of bacteria (E. coli strain JM 109), selection of
positive clones (white colonies) and isolation of plasmids with insert by
alkaline lysis were performed using standard procedures
(Sambrook et al., 1989
). A
restriction enzyme digest with EcoRI (2 U 11 µg
plasmid at 37°C for 90 min) confirmed the presence of insert of the
correct size.
Sequencing
The isolated plasmid DNA was labeled with Dig-Taq DNA Sequencing Kit
(Boehringer-Manheim, Germany) and sequenced in Sequi-Gen GT Sequencing Cell
(Bio-Rad, Hercules, USA).
10 µg of plasmid DNA was denatured at 95°C for 3 min. The completed
reactions were resolved on a denaturing gel (6% polyacrylamide, 7 mol
l1 urea, 1x TBE) and run at a constant voltage of 1500
V for various lengths of time. Gel was blotted onto positively charged nylon
membranes (Boehringer-Manheim, Germany) for capillary transfer (45 min).
Chemiluminescent detection was performed with CDP-Star (Boehringer-Manheim,
Germany) at 1:1000 followed by exposure to Hyperfilm ECL (Amersham, Little
Chalfont, UK). Additionally, the isolated plasmid DNA was automatically
sequenced using the standard procedures. Sequences searches were performed by
the BLAST algorithm on the NCBI databases. Sequence alignment was performed
using Clustal W version 1.6 (Thompson et
al., 1994).
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Results |
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Dual fluorochrome immunolocalization was performed in isoproterenol-treated and untreated Paramecium cells and was displayed by superimposing FITC staining (green) representing ß-adrenergic receptor immunoanalogue and TRITC staining representing dynamin distribution (red) (Fig. 2). In isoproterenol-treated cells, colocalization of ßAR and dynamin (yielding a yellow orange image) was observed in consecutive confocal optical sections performed at a vertical resolution of 0.6 µm. Small yellow-orange-fluorescing punctate accumulations were seen (Fig. 2AD, arrows). Such a pattern of localization was not observed in the untreated cells (Fig. 2E). The immunostaining was not detected in the negative control in which primary antibodies were omitted (data not shown).
|
Ultrastructural detection by immunogold electron microscopy revealed that in the isoproterenol-treated cells ßAR and dynamin colocalize in a population of intracellular vesicles approx. 4055 nm in diameter (Fig. 3AD). Almost no ßAR was detected on the membrane, only a single, scarce gold particles may be found (Fig. 3A, arrow). In untreated cells, colocalization of ßAR and dynamin was detected on the surface (Fig. 3E). When ßAR and dynamin were localized separately using anti-ßAR and anti-dynamin antibody, respectively, a significant presence of ßAR on the plasma membrane was observed (Fig. 3F), whereas dynamin was localized on/beneath the membrane (Fig. 3H) and in the coated pits (Fig. 3G).
|
The anti-dynamin antibody (directed against the C-terminal region of human dynamin 2) that was used for confocal and electron microscopic studies was further used for western blot analysis. It revealed one immunoreactive band of approx. 105 kDa (Fig. 4B, lane 1) in the S2 fraction isolated from Paramecium cells and separated by SDS-PAGE (Fig. 4A, lane 1). This result is consistent with the migration pattern of recombinant rat dynamin 2 obtained in the same blot under our experimental conditions (Fig. 4A,B, lane 2).
|
The molecular basis for the initiation of desensitization/sequestration process in Paramecium seems to be the putative ßAR kinase. Its gene fragment was identified serendipitously by us during PCR cloning of genes involved in endocytic processes.
The PCR product of 400 bp (Fig.
5A) was excised from the gel, eluted and reamplified, yielding a
band of the same molecular size (Fig.
5B). These DNA species were purified (as described in Materials
and methods), subcloned into pGEM-T vector and used for transformation of
bacteria. Plasmids isolated from the positive clones were digested with
EcoRI and revealed the presence of the insert of the correct size of
420 bp (Fig. 5C). The
identified gene fragment of 407 nucleotides contained one short intron of 25
bp (data not shown). The computer-assisted alignment of the deduced amino acid
sequence revealed a high homology to the human ßARK2 the enzyme
involved in the phosphorylation of the agonist-occupied beta-adrenergic
receptor. This sequence displayed a 23.8% identity, 51.6% homology and 62.7%
similarity to this enzyme including its catalytic and extension domains in a
126-amino-acid overlap (Fig.
6). Alignment with ßARK1, the first identified
beta-adrenergic receptor kinase (Benovic et
al., 1989
), revealed 24.6% identity, 47.6% homology and 61.9%
similarity.
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Discussion |
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The desensitization process is defined as attenuation of the receptor
responsiveness upon agonist stimulation and it is a consequence of combination
of different mechanisms (January et al.,
1997; Ferguson,
2001
). It is initiated by receptor phosphorylation including by
G-protein-coupled receptor kinase (GRK) that acts only on agonist-occupied
receptor (Premont et al.,
1995
; Claing et al.,
2002
; Sorkin and Von Zastrow,
2002
). We reported here the partial cloning of putative ßAR
kinase in Paramecium cell. Its deduced amino acid sequence shows
51.6% homology to the human ßARK2 in 126 amino acid overlap including its
catalytic and extension domains, and 47.6% homology to the first cloned
ßARK1 from the bovine brain (Benovic
et al., 1989
). We also proved that this putative enzyme is
expressed in Paramecium by obtaining the corresponding mRNA sequence
(GenBank: accession no. AF346411; data not shown).
We performed immunocytochemical localization of the beta-immunoanalogue in
Isopreterol-treated Paramecium cells using rabbit polyclonal antibody
(Ab), raised by Von Zastrow and Kobilka
(1992), against the C-terminal
region of human ß2-adrenergic receptor. Such an antibody is
suitable for examining the effect of agonists since receptor phosphorylation
by ßAR kinase occurs at the C-terminal Ser/Thr residues of the
agonist-occupied receptor (Barak et al.,
1994
; Premont et al.,
1995
; Krupnick and Benovic,
1998
).
Agonist treatment leads to the redistribution of the receptors away from
the cell surface by a process of endocytosis, also known as internalization or
sequestration (Chuang and Costa,
1979a,b
;
January et al., 1997
;
Pierce et al., 2002
).
Our findings of ßAR redistribution in Paramecium upon
isoproterenol treatment were consistent with those reported by Barak et al.
(1997) and Waldo et al.
(1983
). The translocation of
ß2AR from the plasma membrane to an intracellular compartment
occurred very rapidly, exhibiting a t1/2 of
2 min in
1321N1 human astrocytoma cells (Waldo et
al., 1983
). Barak et al.
(1997
) reported that, upon
desensitization, a functionally intact ß2ARgreen
fluorescent protein conjugate was localized on endosomal membranes within
minutes of agonist treatment.
There is evidence that the clathrin- and dynamin-dependent machinery of
internalization is involved in the response to the ß2AR
agonist isoproterenol (Zhang et al.,
1996,
1997
;
Gagnon et al., 1998
;
Laporte et al., 1999
;
Walker et al., 1999
;
Seachrist et al., 2000
;
Ferguson, 2001
;
Paing et al., 2002
;
Pierce et al., 2002
). Walker
et al. (1999
) reported that
some G-protein-coupled receptors, such as the ß2-adrenergic
receptor, internalized in clathrin-coated vesicles and this process was
mediated by G-protein-coupled receptor kinases (GRKs), beta-arrestin and
dynamin. Zhang et al. (1996
)
demonstrated that dynamin, a GTPase that regulates the formation and
internalization of clathrin-coated vesicles, is essential for the
agonist-promoted sequestration of the ß2AR. They reported that
expression of dynamin K44A, a dominant-negative mutant of dynamin that
inhibits clathrin-mediated endocytosis, prevented endocytosis of the
ß2AR. In HEK293 cells this dynamin mutant profoundly inhibited
agonist-induced internalization and downregulation of the ß2AR
(Zhang et al., 1996
;
Gagnon et al., 1998
) whereas
in COS-1 and HeLa cells it attenuated these processes
(Gagnon et al., 1998
). Von
Zastrow and Kobilka (1992
)
observed isoproterenol-regulated internalization and recycling of human
ß2-adrenergic receptors between the plasma membrane and
endosomes containing transferrin receptors. Kallal et al.
(1998
) reported that
ß2AR-GFP in HeLa cells following a short agonist exposure
distributed into early endosomes and colocalized with rhodamine-labeled
transferrin. These results seem to be consistent with our data that some
ßAR was sequestered into vesicles corresponding to the early endosomes.
Dynamin was localized to such endosomes in Paramecium cells during
transferrin internalization (Surmacz et
al., 2003
).
As far as we know, there is no proof of the existence of
dynamindependent sequestration in the unicellular eukaryotes, though
protozoa have been found to be sensitive to a variety of neurotransmitters and
neuropeptides (Le Roith et al.,
1980; O'Neill et al.,
1988
; Wyroba,
1989
; Renaud et al.,
1995
; Yang et al.,
1997
; Christensen et al.,
1998
; Vallesi et al.,
1998
; Csaba and Kovacs,
2000
; Iwamoto et al.,
2000
; Delmonte Corrado et al.,
2001
). Some cases of receptor desensitization have been reported
in protozoa but not the presence of G-protein-coupled kinase
(Ayala and Kierszenbaum, 1990
;
Van Haastert et al., 1992
;
Xiao et al., 1999
).
Paramecium emerged early in evolution, about 1.5 billion years
ago, prior to the divergence of plants, animals and yeast
(Sogin and Elwood, 1986), and
ßAR appeared in this evolutionary ancient cell as a nutrient receptor.
Taking into account that the beta-adrenergic system began to diverge about 0.6
billion years ago (Fryxell,
1995
), it seems that the desensitization/sequestration mechanism
developed early in evolution almost parallel to the appearance of this
receptor in unicellular eukaryotes.
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Acknowledgments |
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