Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium

Using cell fractionation, SDS-PAGE, quantitative western blot, confocal immunolocalization and immunogold labeling techniques we identified the immunoanalogue of vertebrate β₂-adrenergic receptor in the unicellular eukaryote Paramecium(Wiejak et al., 2002). This provided a molecular basis for the previously reported physiological response of this cell to the beta-adrenergic ligands(Wyroba, 1989).

The 69 kDa polypeptide separated by SDS-PAGE in S2 and P2 Paramecium subcellular fractions cross-reacted with antibody directed against human β₂-adrenergic receptor (βAR). Quantitative image analysis of the western blots showed that the β-selective adrenergic agonist (–)-isoproterenol (Iso), previously shown to enhance phagocytic activity (Wyroba,1989), evoked a redistribution of βAR 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 βAR sites on the cell surface and at the ridge of the cytopharynx, where nascent phagosomes are formed, and the localization of the β-immunoanalogue was confirmed by immunoelectron microscopy. These results indicated that the 69 kDa Paramecium polypeptide immunorelated to vertebrate β₂AR appeared in this ciliate as a nutrient receptor. Pretreatment of the cells with 10 μmol l^–1 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 βARK=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 βARK2, including its catalytic and extension domain, and 47.6%homology to the βARK1, 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 Parameciumfollows a pathway similar to that observed in mammalian cells, we performed experiments with antibodies directed against the C termini of humanβ ₂AR 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 β-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 β-adrenergic receptor and dynamin were localized. This suggests thatβ-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.

Paramecium aurelia strain 299s (5-day-old axenic cultures) were cultivated, collected and starved aseptically for 18 h as described previously(Wiejak et al., 2002).

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).

Untreated and isoproterenol-treated (10 μmol l^–1 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 β₂-adrenergic receptor (β₂AR: 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 63×) was used. Images were collected and processed using Leica confocal software 2.0 and Adobe Photoshop 6.0.

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-β₂AR 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 βAR 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.

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).

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 l^–1 of each primer, 100 μmol l^–1 each of deoxynucleotidyl triphosphates, 2 mmol l^–1 of MgCl₂, 1× 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 1^–1 μg plasmid at 37°C for 90 min) confirmed the presence of insert of the correct size.

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 l^–1 urea, 1× 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).

The β₂-adrenergic receptor immunoanalogue in Paramecium cells was demonstrated using immunodetection in confocal laser scanning and electron microscopy to be localized mainly on the cell surface, as reported previously (Wiejak et al., 2002). In the presence of the β-selective adrenergic agonist isoproterenol (10 μmol l^–1), the β-adrenergic sites were translocated from the cell surface(Fig. 1A) to the cytoplasmic compartment (Fig. 1B). Such a pattern of localization suggested that the beta-immunoanalogue had been internalized, so anti-dynamin Ab was applied to investigate whether the dynamin-dependent process had occurred.

Dual fluorochrome immunolocalization was performed in isoproterenol-treated and untreated Paramecium cells and was displayed by superimposing FITC staining (green) representing β-adrenergic receptor immunoanalogue and TRITC staining representing dynamin distribution (red)(Fig. 2). In isoproterenol-treated cells, colocalization of βAR 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. 2A–D, 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 βAR and dynamin colocalize in a population of intracellular vesicles approx. 40–55 nm in diameter(Fig. 3A–D). Almost noβAR was detected on the membrane, only a single, scarce gold particles may be found (Fig. 3A, arrow). In untreated cells, colocalization of βAR and dynamin was detected on the surface (Fig. 3E). WhenβAR and dynamin were localized separately using anti-βAR and anti-dynamin antibody, respectively, a significant presence of βAR 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 βAR 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 βARK2 – 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 βARK1, the first identified beta-adrenergic receptor kinase (Benovic et al., 1989), revealed 24.6% identity, 47.6% homology and 61.9%similarity.

Dual fluorochrome immunostaining in confocal laser microscopy and immunogold detection in electron microscopy suggest the existence of the dynamin-dependent agonist-mediated sequestration of the beta-adrenergic receptor immunoanalogue in Paramecium. Upon isoproterenol treatmentβAR redistributed from the cell surface to the cytoplasmic compartment,undergoing internalization. Its partial colocalization with dynamin was detected in the small punctate accumulations in the series of the consecutive optical confocal sections from cells labeled simultaneously with antibodies against the human βAR and dynamin. This receptor distribution was further examined at higher resolution by immunogold technique in transmission electron microscopy: tiny endocytic vesicles were revealed in which βAR colocalized with dynamin. The diameter of the vesicles (40–55 nm) to which βAR homologue is translocated corresponds to the early endosomal compartment observed in transferrin-internalizing Paramecium cells by immunoelectron microscopy (J. Wiejak et al., unpublished observations). We have also identified dynamin in Paramecium by western blot analysis using the same antibody as for confocal and electron microscopic studies. Its apparent molecular mass in migration in SDS gel analysis and localization confirm that the protein is dynamin or an immunologically related protein.

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 βAR kinase in Paramecium cell. Its deduced amino acid sequence shows 51.6% homology to the human βARK2 in 126 amino acid overlap including its catalytic and extension domains, and 47.6% homology to the first clonedβARK1 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 β₂-adrenergic receptor. Such an antibody is suitable for examining the effect of agonists since receptor phosphorylation by βAR 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 βAR redistribution in Paramecium upon isoproterenol treatment were consistent with those reported by Barak et al.(1997) and Waldo et al.(1983). The translocation ofβ ₂AR from the plasma membrane to an intracellular compartment occurred very rapidly, exhibiting a t_1/2 of ∼2 min in 1321N1 human astrocytoma cells (Waldo et al., 1983). Barak et al.(1997) reported that, upon desensitization, a functionally intact β₂AR–green 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 β₂AR 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 β₂-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 β₂AR. They reported that expression of dynamin K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, prevented endocytosis of theβ ₂AR. In HEK293 cells this dynamin mutant profoundly inhibited agonist-induced internalization and downregulation of the β₂AR(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 humanβ ₂-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. Kallal et al.(1998) reported thatβ ₂AR-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 someβAR 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 dynamin–dependent 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), andβAR 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.

This study was supported by the statutory funds to the Nencki Institute of Experimental Biology. The technical assistance of Mr K. Krawczyk in photographic documentation and Mr L. Kilianek, MSc in confocal imaging is acknowledged.