Homo- and Heterodimerization of Somatostatin Receptor Subtypes

INACTIVATION OF sst3 RECEPTOR FUNCTION BY HETERODIMERIZATION WITH sst2A*

Manuela Pfeiffer, Thomas Koch, Helmut Schröder, Marcus Klutzny, Susanne Kirscht, Hans-Jürgen Kreienkamp, Volker Höllt, and Stefan SchulzDagger

From the Institut für Pharmakologie und Toxikologie, Otto-von-Guericke Universität, 39120 Magdeburg, Germany and Institut für Zellbiochemie und klinische Neurobiologie, Universitätskrankenhaus Hamburg-Eppendorf, Universität Hamburg, 20246 Hamburg, Germany

Received for publication, July 11, 2000, and in revised form, December 1, 2000




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst2A and sst3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and sst3. The sst2A-sst3 heterodimer exhibited high affinity binding to somatostatin-14 and the sst2-selective ligand L-779,976 but not to the sst3-selective ligand L-796,778. Like the sst2A homodimer, the sst2A-sst3 heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgamma S) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst2-selective ligand L-779,976. However, unlike the sst3 homodimer, the sst2A-sst3 heterodimer did not promote GTPgamma S binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst3-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst2A-sst3 heterodimer desensitized at a slower rate than the sst2A and sst3 homodimers. Both sst2A and sst3 homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst2A-sst3 heterodimer separated at the plasma membrane, and only sst2A but not sst3 underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst2A and sst3 results in a new receptor with a pharmacological and functional profile resembling that of the sst2A receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst3 receptor function by heterodimerization with sst2A or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst3-specific binding and signaling in mammalian tissues.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although G protein-coupled receptors (GPCRs)1 generally were believed to act as monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. The existence of homodimers has been shown for several GPCRs including the beta 2-adrenergic receptor (1), delta - and kappa -opioid receptors (2, 3), the metabotrobic glutamate receptor 5 (4), the calcium-sensing receptor (5), the m3 muscarinic receptor (6), and dopamine receptors (7). GPCRs seem to dimerize via different mechanisms. Whereas dimerization of the beta 2-adrenergic receptor (1) and D2 dopamine receptor (8) occurs via their transmembrane regions, dimerization of the delta -opioid receptor involves the carboxyl terminus (2). In contrast, the metabotrobic glutamate receptor 5 (4) and the calcium-sensing receptor (5, 9, 10) appear to be disulfide-linked dimers, and dimerization occurs via their large amino termini. The question of to what extent agonist binding affects dimerization remains controversial. Recent evidence obtained in living cells using bioluminescence resonance energy transfer suggests that the beta 2-adrenergic receptor exists at the cell surface as a constitutive dimer that is stabilized by agonist binding (11). In contrast, agonist stimulation reduced the level of delta -opioid receptor dimers suggesting that monomerization precedes agonist-induced internalization of this receptor (2). Biochemical and functional studies suggest that GPCRs can also assemble as heterodimers with enhanced functional activity (3, 12-20). Formation of heterodimers between two nonfunctional gamma -aminobutyric acid (GABA) receptors, GABABR1 and GABABR2, was necessary for a fully functional GABAB receptor (14-19). delta - and kappa -Opioid receptors form heterodimers with ligand binding and signaling properties resembling that of the kappa 2 receptor (3). Finally, the somatostatin receptor (sst) sst5 and the D2 dopamine receptor heterodimerize to form a new receptor with enhanced activity (20).

The neuropeptide somatostatin (SS-14) is widely expressed throughout the central nervous system and periphery. SS-14 is involved in multiple functions including endocrine and exocrine hormone release, cognition, sleep, and motor activity. In addition, peptide derivatives of somatostatin have successfully been used in the treatment of neuroendocrine malignancies. The actions of SS-14 are mediated via five distinct somatostatin receptor subtypes, termed sst1-sst5, which belong to the superfamily of GPCRs. All somatostatin receptors bind SS-14 with high affinity and inhibit adenylyl cyclase. There is also evidence for different, although not mutually exclusive, pathways of intracellular signaling of somatostatin receptor subtypes, e.g. activation of extracellular signal-regulated kinases (ERK) via sst1, sst3, and sst4; activation of phosphotyrosine phosphatases via sst1, sst2, and sst3; activation of phospholipase A2 via sst4; and modulation of K+ channels via sst2 (21, 22). Individual target cells often express more than one somatostatin receptor, raising the possibility that the functional diversity of these receptors could be expanded by heterodimerization among somatostatin receptor subtypes.

Recently, Rocheville et al. (23) provided evidence, based on photobleaching fluorescence resonance energy transfer, for homodimerization of the sst5 somatostatin receptor. The sst5 receptor also appears to form heterodimers with sst1 but not sst4. In this study, we have examined dimerization of sst2A and sst3 somatostatin receptors. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we show that sst2A and sst3 associate as dimers, both as homodimers and heterodimers. sst2A-sst3 heterodimerization resulted in a new receptor with enhanced sst2A-like and diminished sst3-like activity.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The sst2-selective ligand L-779,976 and the sst3-selective ligand L-796,778 were kindly provided by Dr. Susan Rohrer (24) (Merck). The radioligand 3-[125I]iodotyrosyl-SS-14 (74 TBq/mmol) was from Amersham Pharmacia Biotech, and [35S]GTPgamma S (46.25 TBq/mmol) was from PerkinElmer Life Sciences. Mouse monoclonal anti-T7 tag antibody was obtained from Novagen (Madison, WI), and polyclonal rabbit anti-T7 and anti-c-Myc antibodies were obtained from Gramsch Laboratories (Schwabhausen, Germany). The rabbit anti-sst2A antibody (6291) and the guinea pig anti-sst2A antibody (GP3) were generated to the peptide ETQRTLLNGDLQTSI, which corresponds to residues 355-369 of the carboxyl terminus of the rat/mouse/human sst2A and have been characterized extensively (25, 26). The anti-sst3 antibody (7986) was generated to the peptide TAGDKASTLSHL, which corresponds to residues 417-428 of the carboxyl terminus of the rat/mouse sst3 and has also been characterized previously (27). All polyclonal rabbit antisera were affinity-purified against their immunizing peptides using the Sulfo-Link coupling gel (Pierce) according to the instructions of the manufacturer.

Cell Culture and Transfections-- The wild-type rat sst2A and sst3 receptors were tagged at their amino termini either with the c-Myc epitope tag sequence MEEQKLISEEDLLR or the T7 epitope tag sequence MASMTGGQQMG using polymerase chain reaction. Two more amino acids, KL, were added representing a HindIII cloning site encoded by the nucleotide sequence AAGCTT. The resulting fragments were then subcloned into pcDNA3.1 expression vectors containing either a neomycin or a hygromycin resistance (Invitrogen, Groningen, The Netherlands). The integrity of all constructs was verified by dideoxy sequencing. Human embryonic kidney (HEK) 293 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 10% CO2. Cells were first transfected with plasmids containing the neomycin resistance using the calcium phosphate precipitation method. Stable tranfectants were selected in the presence of 500 µg/ml G418 (Life Technologies, Inc.). To generate lines coexpressing two differentially epitope-tagged receptors, cells were subjected to a second round of transfection using Effectene (Qiagen, Hilden, Germany) and selected in the presence of 500 µg/ml G418 and 300 µg/ml hygromycin B (Invitrogen). Three clones expressing sst2A alone, four clones expressing sst3 alone, and two clones coexpressing sst2A and sst3 were generated. Receptor expression was monitored using saturation ligand binding assays as described below. The Bmax values of all selected clones were in the range of 800-1200 fmol/mg of protein, and KD values were in the range of 0.16-0.24 nM. In addition, quantitative Western blot analysis was carried out to ensure that clones coexpressing ~1:1 ratio of sst2A and sst3 were selected and used throughout this study. Finally, double immunofluorescent staining was performed to validate that sst2A and sst3 were coexpressed within the same cells.

Immunoprecipitation and Western Blot Analysis-- Cells were plated onto poly-L-lysine-coated 150-mm dishes and grown to 80% confluence. When indicated, cells were exposed to agonist, treated with reducing agents, or incubated with cross-linking agents. Agonist exposure was performed with either SS-14, L-779,976, or L-796,778 at a concentration of 1 µM for 10, 30, or 60 min at 37 °C. Treatment with reducing agents was performed with 1 mM DL-dithiothreitol (DTT) for 30 min at 37 °C. Incubation with cross-linking agents was performed with either 2 mM bis(sulfosuccinimidyl)suberate (BS3) or 5 mM dithiobis-(succinimidylpropionate) (both from Pierce) for 30 or 60 min in phosphate-buffered saline (PBS) at 4 °C. The reaction was quenched by the addition of 50 mM Tris (pH 7.5) for 15 min. Cells were then washed twice with PBS and harvested into ice-cold lysis buffer (10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 3 mM EGTA, 250 mM sucrose, 10 mM iodoacetamide, and the following proteinase inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 10 µg/ml bacitracin). Iodoacetamide was included in each buffer used for protein preparation to prevent nonspecific disulfide linkages. Cells were swollen for 15 min on ice and homogenized. The homogenate was spun at 500 × g for 5 min at 4 °C to remove unbroken cells and nuclei. Membranes were then pelleted at 20,000 × g for 30 min at 4 °C, and pelleted membranes were lysed in detergent buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 4 mg/ml beta -dodecylmaltoside, 10 mM iodoacetamide, and proteinase inhibitors as above) for 1 h on ice. The lysate was centrifuged at 20,000 × g for 30 min at 4 °C. The protein content of the resulting supernatant was determined using the BCA protein assay (Pierce); samples containing equal amounts of protein (300 µg) were then subjected to immunoprecipitation, or glycoproteins were purified using wheat germ lectin.

For enrichment of glycoproteins, one ml of the supernatant was incubated with 100 µl wheat germ agglutinin-agarose beads (Amersham Pharmacia Biotech) for 90 min at 4 °C with continuous agitation. Beads were washed five times with detergent buffer, and adsorbed glycoproteins were either subjected to enzymatic deglycosylation or directly eluted into 200 µl of SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 100 mM DL-dithiothreitol, 0.005% bromphenol blue) at 60 °C for 20 min. Deglycosylation experiments were performed using peptide N-glycosidase F according to the manufacturer's protocol (New England Biolabs, Beverly, MA).

For immunoprecipitation, the lysates were precleared with 50 µl of protein A-agarose beads (Calbiochem) for 2 h. After immunoprecipitation with 10 µg of either mouse monoclonal anti-T7, affinity-purified rabbit anti-c-Myc, affinity-purified rabbit anti-sst2A (6291), or affinity-purified rabbit anti-sst3 (7986) antibodies, immunocomplexes were collected using 100 µl of protein A-agarose beads. Beads were washed five times with detergent buffer, and immunoprecipitates were eluted from the beads with 200 µl of SDS-sample buffer at 60 °C for 20 min. Equal amounts of protein of each sample were then loaded onto regular 6% SDS-polyacrylamide gels, which contain 0.1% SDS. When indicated gels containing 2-fold (0.2%) or 4-fold (0.4%) higher SDS concentrations were run to test the sensitivity of dimers to stronger denaturing conditions. After electroblotting, membranes were incubated with either mouse monoclonal anti-T7, affinity-purified rabbit anti-c-Myc, affinity-purified rabbit anti-sst2A (6291) or affinity-purified rabbit anti-sst3 (7986) antibody at a concentration of 1 µg/ml for 12 h at 4 °C, followed by detection using an enhanced chemiluminescence detection system. Densitometric analysis of Western blots exposed in the linear range of the x-ray film was performed as described (31). The amount of immunoreactive material in each lane was quantified using NIH Image 1.57 software.

Immunocytochemistry-- Cells were grown on poly-L-lysine-treated coverslips overnight and then exposed to agonists. Cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer, pH 6.9, for 40 min at room temperature and washed several times in 10 mM Tris, 10 mM phosphate buffer, 137 mM NaCl, and 0.05% thimerosal, pH 7.4 (TPBS). Specimens were then incubated for 3 min in 50% methanol and for 3 min in 100% methanol and subsequently washed several times in TPBS and preincubated with TPBS and 3% normal goat serum for 1 h at room temperature. For single immunofluorescence, cells were then incubated with either mouse monoclonal anti-T7, affinity-purified rabbit anti-c-Myc, affinity-purified rabbit anti-sst2A (6291), or affinity-purified rabbit anti-sst3 (7986) antibody at a concentration of 1 µg/ml in TPBS and 1% normal goat serum overnight. Bound primary antibody was detected with biotinylated secondary antibodies (1:100; Vector, Burlingame, CA) followed by cyanine 3.18-conjugated streptavidin (Amersham Pharmacia Biotech). For double immunofluorescence, cells were incubated either with a mixture of mouse monoclonal anti-T7 and affinity-purified rabbit anti-c-Myc or guinea pig anti-sst2A (GP3) and affinity-purified rabbit anti-sst3 (7986) antibodies. Bound primary antibodies were detected with biotinylated anti-rabbit antibodies, followed by a mixture of cyanine 2.18-conjugated streptavidin and cyanine 5.18-conjugated anti-mouse or anti-guinea pig antibodies (1:200; Jackson ImmunoResearch, West Grove, PA). Cells were then dehydrated, cleared in xylol, and permanently mounted in DPX (Fluka, Neu-Ulm, Germany). Specimens were examined using a Leica TCS-NT laser-scanning confocal microscope (Heidelberg, Germany) equipped with a krypton/argon laser. Cyanine 2.18 was imaged with 488-nm excitation and 500-560-nm band pass emission filters, cyanine 3.18 with 568-nm excitation and 570-630-nm band pass emission filters, and cyanine 5.18 with 647-nm excitation and 665-nm long pass emission filters. Confocal micrographs were taken by a person blinded to the treatments who was instructed to randomly select one colony of 4-12 cells per coverslip.

Radioligand Binding Assays-- Cells were harvested into PBS and stored at -80 °C. After thawing, cells were centrifuged at 20,000 × g for 5 min at 4 °C and then homogenized in lysis buffer (50 mM Tris-HCl, 3 mM EGTA, 5 mM EDTA, pH 7.4). Cell membranes were pelleted by centrifugation at 50,000 × g for 15 min at 4 °C, washed with lysis buffer, and resuspended in binding buffer (10 mM HEPES, 5 mM MgCl2, 5 µg/ml bacitracin, pH 7.5). For saturation binding assays, aliquots of the membrane preparations containing 25 µg of protein were incubated with increasing concentrations of [125I-Tyr11]SS-14 ranging from 0.025 to 0.3 nM. Receptor densities (Bmax) and ligand affinities (KD) were calculated as mean ± S.E. from four or five determinations performed in duplicate. Nonspecific binding was defined as that not displaced by 1 µM SS-14. For competition binding assays, aliquots of the membrane preparation containing 25 µg of protein were incubated with 0.05 nM [125I-Tyr11]SS-14 in the presence or absence of either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10-12 to 10-5 M. The experiment for each concentration was performed in triplicate. Assays were performed in 96-well polypropylene plates in a final volume of 200 µl for 45 min at room temperature. The incubation was terminated by vacuum filtration through glass fiber filters presoaked in 0.1% polyethyleneimine using an Inotech cell harvester (Dittikon, Switzerland). Filters were rinsed twice with washing buffer (50 mM Tris-HCl, pH 7.4) and air-dried. Bound radioactivity was determined using a gamma -counter. Protein content was determined by the Lowry method.

GTPgamma S Binding Assays-- Cells were harvested and lysed as described above except that a lysis buffer containing 50 mM Tris and 10 mM EDTA (pH 7.4) was used. The resulting pellet was resuspended in assay buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4), and aliquots containing 25 µg of protein were incubated with 3 µM GDP and 0.05 nM [35S]GTPgamma S in the presence or absence of either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10-12 to 10-6 M. Assays were carried out in a final volume of 1 ml for 30 min at 30 °C under continuous agitation. Nonspecific binding was determined in the presence of 10 µM unlabeled GTPgamma S. The incubation was terminated by vacuum filtration through glass fiber filters as described above. Filters were rinsed twice with washing buffer (20 mM HEPES, pH 7.4). Scintillation mixture was added, and radioactivity was determined using a beta -counter. The maximal effector response (Emax) was determined by stimulation with sst agonists at a concentration of 1 µM.

Measurements of cAMP Accumulation-- Transfected cells were seeded at a density of 1.5 × 105/well onto poly-L-lysine-treated 22-mm 12-well dishes. On the next day, cells were either not preincubated or preincubated with 1 µM SS-14 for 1, 2, 4, or 6 h in Opti-MEM 1 (Life Technologies). The medium was then removed and replaced with 0.5 ml of serum-free RPMI medium containing 25 µM forskolin or 25 µM forskolin plus either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10-12 to 10-6 M. The cells were incubated at 37 °C for 15 min. The reaction was terminated by removal of the culture medium and the subsequent addition of 1 ml of ice-cold HCl/ethanol (1 volume of 1 N HCl, 100 volumes of ethanol). After centrifugation, the supernatant was evaporated, the residue was dissolved in TE buffer (50 mM Tris-EDTA, pH 7.5), and the cAMP content was determined using a commercially available radioimmunoassay kit (Amersham Pharmacia Biotech).

ERK Assays-- Cells were seeded at a density of 1.5 × 105/well onto poly-L-lysine-treated 22-mm 12-well dishes and grown for 2 days in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum. Cells were then exposed to either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10-12 to 10-6 M for 5 min in RPMI medium without fetal calf serum at 37 °C. Incubation was terminated by removal of the culture medium and the subsequent addition of 300 µl of boiling SDS-sample buffer. Samples were heated to 95 °C for an additional 5 min period. Equal amounts of protein of each sample were separated on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. The protein content was determined using the BCA method. After blocking with 5% low fat dried milk dissolved in PBS containing 0.1% Tween 20, membranes were incubated with mouse monoclonal phosphospecific anti-ERK1/2 antibody clone E10 (1:1000; New England Biolabs). Blots were developed using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Densitometric analysis of phospho-ERK1/2 levels on Western blots exposed in the linear range of the x-ray film was performed using NIH Image 1.57 software.

Data Analysis-- Data from ligand binding, GTPgamma S, cAMP, and ERK assays were analyzed by nonlinear regression curve fitting using GraphPad Prism 3.0 software.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homodimerization of the sst2A Receptor-- To examine dimerization of the sst2A receptor, we used HEK 293 cells stably expressing either the T7-tagged sst2A alone (Bmax = 820 ± 19 fmol/mg of membrane protein) or stably coexpressing T7-tagged sst2A and Myc-tagged sst2A receptors were used. Western blot analysis of membrane extracts from these cells with the anti-sst2A antibody (6291) revealed a predominant receptor band migrating at 80 kDa (Fig. 1A). We also observed an additional band with a higher molecular mass migrating at 160 kDa (Fig. 1A). These bands were not only detected with antibodies directed against the carboxyl terminus (6291, GP3) but also with antibodies directed against the amino-terminal added T7 or Myc tag (not shown). A similar ratio of these two immunoreactive bands was observed when the cells had been subjected to cross-linking using the cell-impermeable cross-linker BS3 prior to cell lysis (Fig. 1A). However, enzymatic deglycosylation reduced the size of the 80-kDa protein to 55 kDa and the size of the 160-kDa protein to 110 kDa, suggesting that the band with the higher molecular weight may consist of two sst2A receptor proteins (Fig. 1B). Exposure to SS-14 did not grossly modulate the dimer/monomer ratio (Fig. 1C).



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Fig. 1.   The sst2A somatostatin receptor forms homodimers. A, HEK 293 cells expressing T7 epitope-tagged sst2A receptor were incubated in the absence (-) or presence (+) of 2 mM BS3, and membrane proteins were extracted and immunoblotted using the anti-sst2A antibody (6291) as described under "Experimental Procedures." B, membrane proteins from T7sst2A-expressing cells were extracted and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGF). C, cells were incubated in the absence (-) or presence (+) of 1 µM SS-14 for 30 min. D, cells were incubated in the absence (-) or presence (+) of 1 mM DTT for 30 min, membrane proteins were extracted, and samples were run on regular SDS-polyacrylamide gels (lanes 1 and 2). Alternatively, samples were run on SDS-polyacrylamide gels containing a 4-fold higher concentration of SDS (lane 3). DTT was also present in the SDS-sample buffer. Note that the sst2A dimer is detectable without cross-linking and stable under reducing conditions; however, it is sensitive to higher concentrations of detergents. E, HEK 293 cells expressing either Mycsst2A alone or coexpressing Mycsst2A and T7sst2A were subjected to immunoprecipitation using rabbit anti-c-Myc antibody. Coimmunoprecipitates were immunoblotted using mouse monoclonal anti-T7 antibody. Coimmunoprecipitation of the T7sst2A can be seen only when Mycsst2A and T7sst2A are coexpressed (right lane). The positions of molecular mass markers are indicated on the left (in kDa). The arrows point to the dimeric and monomeric forms of the receptor. Three additional experiments gave similar results.

The sst2A dimer was stable under reducing conditions. Similar levels of sst2A dimers were detected after incubation of the cells with 1 mM DTT for 30 min prior to cell lysis (Fig. 1D). The addition of DTT to the SDS-sample buffer also did not reduce the level of sst2A dimers detected on Western blots. sst2A dimers were also stable in the presence of 2% SDS (e.g. heating to 60 °C for 20 min in SDS-sample buffer and loading onto regular SDS-polyacrylamide gels). However, when these samples were run on SDS-polyacrylamide gels containing 2- or 4-fold higher SDS concentrations, the sst2A dimer was destabilized in a concentration-dependent manner, and only the monomeric form of the receptor was detectable under these conditions (Fig. 1D). This suggests that the sst2A dimer is formed by noncovalent interactions of two receptor proteins.

To directly examine the presence of sst2A receptor dimers, we used coimmunoprecipitation and Western blotting of differentially epitope-tagged sst2A receptors. HEK 293 cells coexpressing Mycsst2A and T7sst2A were lysed, and the receptors from these cells were immunoprecipitated using anti-c-Myc antibody. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-T7 antibody. As depicted in Fig. 1E, anti-T7 antibody detected a single band migrating at 160 kDa, which represents the Mycsst2A-T7sst2A dimer. Receptor monomer was not detectable, suggesting that the coprecipitated sst2A dimers were stable during sample preparation and SDS-PAGE. The fact that T7sst2A and Mycsst2A were coimmunoprecipitated as part of a dimer complex from membrane extracts of untreated HEK 293 cells indicates that the sst2A receptor exists as a constitutive homodimer in these cells. No bands were detectable in immunoprecipitates prepared under identical conditions from cells expressing only Mycsst2A (Fig. 1E).

Homodimerization of the sst3 Receptor-- To examine dimerization of the sst3 receptor, we used HEK 293 cells stably expressing the T7-tagged sst3 (Bmax 1182 ± 31 fmol/mg of membrane protein). Western blot analysis of membrane extracts from these cells with the anti-sst3 antibody (7896) revealed a predominant receptor band migrating at 80 kDa (Fig. 2A). We also observed an additional band with a higher molecular mass migrating at 160 kDa (Fig. 2A). These bands were also detected with antibodies directed against the amino-terminal added T7 tag (not shown). Treatment of cells with the cross-linker BS3 prior to cell lysis resulted in an increase of the 160-kDa band, suggesting that cross-linking can stabilize the sst3 dimer (Fig. 2A). However, no further increase in the intensity of the 160-kDa band was found when cells were exposed to SS-14 prior to cross-linking. (Fig. 2A). Enzymatic deglycosylation reduced the size of the 80-kDa protein to 70 kDa and the size of the 160-kDa protein to 140 kDa, suggesting that the band with the higher molecular weight may consist of two sst3 receptor proteins (Fig. 2B).



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Fig. 2.   Characterization of sst3 somatostatin receptor homodimers by immunoblotting. A, HEK 293 cells expressing T7 epitope-tagged sst3 receptor were incubated in the absence (-) or presence (+) of 2 mM BS3 and/or SS-14 (+). Membrane proteins were extracted and immunoblotted using the anti-sst3 antibody (7986) as described under "Experimental Procedures." B, membrane proteins from T7sst3-expressing cells were extracted and subjected to enzymatic deglycosylation using peptide N-glycosidase F (PNGF). C, cells were incubated in the absence (-) or presence (+) of 1 mM DTT for 30 min. Membrane proteins were extracted, and samples were run on regular SDS-polyacrylamide gels (lanes 1 and 2). Alternatively, samples were run on SDS-polyacrylamide gels containing a 4-fold higher concentration of SDS (lane 3). DTT was also present in the SDS-sample buffer. Note that the sst3 dimer is detectable without cross-linking and stable under reducing conditions; however, it is sensitive to higher concentrations of detergents. D, HEK 293 cells expressing either the wild-type sst3 receptor (T7sst3) or a C-terminal truncated sst3 receptor (T7sst3Delta 75) were immunoblotted using mouse monoclonal anti-T7 antibody. The positions of molecular mass markers are indicated on the left (in kDa). The arrows point to the dimeric and monomeric forms of the receptor. Four additional experiments gave similar results.

Similar to that observed for the sst2A dimer, the sst3 dimer was stable under reducing conditions. The levels of sst3 dimers were unchanged after incubation of cells with 1 mM DTT for 30 min prior to cell lysis (Fig. 2C). The addition of DTT to the SDS-sample buffer also did not reduce the levels of sst3 dimers detected on Western blots. sst3 dimers were also stable in the presence of 2% SDS (e.g. heating to 60 °C for 20 min in SDS-sample buffer and loading onto regular SDS-polyacrylamide gels). However, when these samples were run on SDS-polyacrylamide gels containing 2- or 4-fold higher SDS concentrations, the sst3 dimer was destabilized in a concentration-dependent manner, and only the monomeric form of the receptor was detectable under these conditions (Fig. 2C). This suggests that the sst3 dimer is formed by noncovalent interactions of two receptor proteins.

To examine the role of the carboxyl terminus for sst3 dimerization, we performed Western blotting of HEK 293 cells expressing an sst3 receptor with a deletion of the entire cytoplasmic tail (T7sst3Delta C75). In membrane extracts from these cells, we detected a predominant receptor band migrating at 70 kDa and a second band migrating at 140 kDa, suggesting that the carboxyl terminus was not absolutely required for sst3 receptor homodimerization (Fig. 2D).

Heterodimerization of the sst2A and sst3 Receptors-- First, we determined the levels of sst2A and sst3 receptor proteins in coexpressing HEK 293 cells using quantitative Western blot analysis. Since sst2A and sst3 were tagged with different epitopes, it was not possible to compare their expression levels directly using different antibodies. Therefore, equal amounts of membrane protein extracted from HEK 293 cells stably expressing the T7-tagged sst2A receptor alone (Bmax 820 ± 19 fmol/mg) or the T7-tagged sst3 receptor alone (Bmax 1182 ± 31 fmol/mg) or coexpressing T7-tagged sst2A and c-Myc-tagged sst3 receptors (Bmax 1088 ± 66 fmol/mg) were subjected to SDS-PAGE and subsequently probed with both anti-sst2A (6291) and anti-sst3 (7986) antibodies (Fig. 3A). Densitometric scanning of the resulting Western blots revealed that virtually identical levels of sst2A receptor protein were present in HEK 293 cells coexpressing sst2A and sst3 as compared with cells expressing sst2A alone (Fig. 3A, left panel). Conversely, virtually identical levels of sst3 receptor protein were present in HEK 293 cells coexpressing sst3 and sst2A as compared with cells expressing sst3 alone (Fig. 3A, right panel). Given the fact that T7sst2A and T7sst3 cells expressed similar numbers of somatostatin binding sites, we conclude that cotransfected cells expressed T7sst2A and Mycsst3 receptor proteins in a ratio of ~1:1. However, it should be noted that the higher levels of total somatostatin receptor proteins (sst2A plus sst3) present in coexpressing cells were not associated with an equivalent increase in the total number of somatostatin binding sites detected in saturation binding assays (Bmax for T7sst2A-Mycsst3 1088 ± 66 fmol/mg as compared with Bmax for T7sst2A 820 ± 19 fmol/mg and Bmax for T7sst3 1182 ± 31 fmol/mg).



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Fig. 3.   Characterization of sst2A-sst3 heterodimers by coimmunoprecipitation. A, membrane proteins from HEK 293 cells expressing either T7sst2A (Bmax = 820 ± 19 fmol/mg) or T7sst3 (Bmax = 1182 ± 31 fmol/mg) alone or coexpressing T7sst2A and Mycsst3 (Bmax = 1088 ± 66 fmol/mg) were extracted and immunoblotted using either the anti-sst2A antibody (6291) or the anti-sst3 antibody (7986) as described under "Experimental Procedures." B, HEK 293 cells coexpressing T7sst2A and Mycsst3 were subjected to immunoprecipitation using either anti-sst2A antibody (6291) or anti-sst3 antibody (7986), and immunoprecipitates were immunoblotted using anti-sst2A antibody (6291). C, HEK 293 cells expressing either T7sst2A or T7sst3 alone or coexpressing T7sst2A and Mycsst3 or a mixture of cells expressing either T7sst2A or Mycsst3 alone was subjected to immunoprecipitation using rabbit anti-c-Myc antibody, and coimmunoprecipitates were immunoblotted using mouse monoclonal anti-T7 antibody. Coimmunoprecipitation of the T7sst2A can be seen only when T7sst2A and Mycsst3 are coexpressed in the same cell (lane 3) but not when cells expressing T7sst2A or Mycsst3 individually were mixed prior to immunoprecipitation (lane 4). D, HEK 293 cells coexpressing T7sst2A and Mycsst3 were incubated in the absence (-) or presence (+) of 1 µM SS-14 for 30 min and then subjected to immunoprecipitation using anti-sst3 antibody (7986). Coimmunoprecipitates were immunoblotted using anti-sst2A antibody (6291). Note that 1) cotransfected cells expressed T7sst2A and Mycsst3 receptor proteins in a ratio of ~1:1, 2) sst2A-sst3 heterodimers can be coprecipitated using a variety of antibody combinations directed against different epitopes, and 3) agonist treatment destabilized the sst2A-sst3 heterodimer, resulting in the appearance of a monomeric receptor form. The positions of molecular mass markers are indicated on the left (in kDa). The arrows point to the dimeric and monomeric forms of the receptor. Three additional experiments gave similar results.

We next examined the ability of sst2A and sst3 to assemble as heterodimers in coexpressing cells. As shown in Fig. 3B, the C-terminal anti-sst2A antibody (6291) detected a band migrating at 160 kDa in material immunoprecipitated using the C-terminal anti-sst3 antibody (7986), suggesting that this band represents a T7sst2A-Mycsst3 heterodimer. sst2A-sst3 Heterodimers can be immunoprecipitated under a variety of conditions (e.g. using the anti-c-Myc antibody or the anti-T7 antibody and immunoblotted vice versa) (Fig. 3C). All of these preparations revealed a single 160-kDa band, suggesting that stable sst2A-sst3 heterodimers were precipitated under these conditions and that sst2A-sst3 heterodimers were not subject to monomerization during sample preparation. However, when T7-tagged sst2A receptors were immunoprecipitated from T7sst2A-Mycsst3 cells using anti-sst2A antibody (6291) and then immunoblotted with anti-sst2A antibody (6291), both dimeric and monomeric forms of the receptor could be detected (Fig. 3B). Essentially identical results were obtained when Myc-tagged sst3 receptors were immunoprecipitated from T7sst2A-Mycsst3 cells using anti-sst3 antibody (7986) and then immunoblotted with anti-sst3 antibody (7986) (not shown). These results are in agreement with those seen on Western blots from membrane extracts of T7sst2A-Mycsst3 cells, suggesting that the receptor proteins existed in both monomeric and dimeric form in these cells (Fig. 3A).

As shown in Fig. 3C, anti-T7 antibody detected a band migrating at 160 kDa in material immunoprecipitated using anti-c-Myc antibodies specific for the sst3 receptor, suggesting that this band represents a T7sst2A-Mycsst3 heterodimer. In contrast, no bands were detectable in immunoprecipitates prepared under identical conditions from cells expressing only T7sst2A or T7sst3 or from a mixture of T7sst2A- and Mycsst3-expressing cells (Fig. 3C). These data strongly suggest that sst2A-sst3 heterodimers preexisted in cells prior to cell lysis and were not artificially formed during sample preparation.

Agonist exposure had little effect on the level of sst2A-sst3 heterodimer (Fig. 3D). However, we observed that after exposure of coexpressing cells, not only heterodimers but also traces of the monomeric receptor forms became detectable (Fig. 3D). The simplest explanation for this finding is that during agonist exposure a proportion of the sst2A-sst3 heterodimer was destabilized, presumably due to a change in the conformation and/or phosphorylation of the receptor that still allowed coimmunoprecipitation of sst2A-sst3 heterodimers but facilitated separation of the receptors during sample preparation and gel electrophoresis.

Endocytotic Trafficking of the sst2A-sst3 Heterodimer-- We next examined the effect of sst2A-sst3 heterodimerization on agonist-induced endocytosis using HEK 293 cells expressing either T7sst2A or T7sst3 or coexpressing both T7sst2A and Mycsst3. Cells were exposed to either 100 nM SS-14, 10 nM L-779,976 (sst2-selective agonist), or 100 nM L-796,778 (sst3-selective agonist) for 30 min. Cells were subsequently fixed, permeabilized, and fluorescently labeled with sst2A and/or sst3-specific antibodies. The subcellular distribution of receptor proteins was then analyzed by confocal microscopy. As depicted in Fig. 4, upper panel, sst2A-like immunoreactivity was clearly confined to the plasma membrane in untreated cells. After 30 min of exposure to SS-14, a dramatic loss of sst2A-like immunoreactivity from the plasma membrane with a concomitant accumulation in vesicle-like structures within the cytoplasm was observed. In addition, treatment with the sst2-selective ligand L-779,976 but not with the sst3-selective ligand L-796,778 induced robust internalization of the sst2A receptor. Similarly, sst3-like immunoreactivity was present at the plasma membrane in the absence of agonist and was redistributed into vesicle-like structures in the presence of SS-14 (Fig. 4, lower panel). Treatment with the sst3-selective ligand L-796,778 but not with the sst2-selective ligand L-779,976 induced intracellular trafficking of the sst3 receptor.



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Fig. 4.   Comparison of agonist-induced endocytosis of sst2A and sst3 homodimers. HEK 293 cells expressing either T7sst2A (upper panel) or T7sst3 (lower panel) were either not exposed (Control) or exposed to 100 nM SS-14 or 10 nM L-779,976 (sst2-selective agonist) or 100 nM L-796,778 (sst3-selective agonist) for 30 min. Cells were subsequently fixed and fluorescently labeled with either anti-sst2A antibody (6291) or anti-sst3 antibody (7986), and the subcellular distribution of receptor proteins was examined by confocal microscopy. Note that in untreated cells, both sst2A and sst3 were almost exclusively confined to the plasma membrane. SS-14 induced robust internalization of the sst2A as well as the sst3 receptor. While the sst2-selective agonist L-779,976 promoted endocytosis of sst2A but not sst3, the sst3-selective agonist L-796,778 promoted endocytosis of sst3 but not sst2A. Shown are representative results from one of three independent experiments performed in duplicate. Scale bar, 20 µm.

In cells coexpressing T7sst2A and Mycsst3, both sst2A-like immunoreactivity and sst3-like immunoreactivity were seen at the cell surface, revealing extensive colocalization of sst2A and sst3 receptor proteins (Fig. 5, Control). Interestingly, after 30 min of SS-14 exposure, the sst2A receptor underwent robust internalization, whereas the sst3 receptor remained almost exclusively confined to plasma membrane (Fig. 5, SS-14). However, internalization of the sst2A receptor was not complete, which may explain the fact that sst2A-sst3 heterodimers could also be coimmunoprecipitated after treatment with SS-14 (Fig. 3C). The sst3 receptor did not undergo endocytosis even after prolonged SS-14 exposure (up to 4 h, not shown). Similarly, after treatment with the sst2-selective ligand L-779,976, only the sst2A and not the sst3 receptor was internalized (Fig. 5, L-779,976). In contrast, treatment with sst3-selective ligand L-796,778 did not induce substantial internalization of either sst2A or sst3 (Fig. 5, L-796,778).



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Fig. 5.   Differential agonist-induced endocytosis of sst2A-sst3 heterodimers. HEK 293 cells coexpressing T7sst2A and Mycsst3 were either not exposed (Control) or exposed to 100 nM SS-14 or 10 nM L-779,976 (sst2-selective agonist) or 100 nM L-796,778 (sst3-selective agonist) for 30 min. Cells were subsequently fixed and fluorescently labeled with a mixture of guinea pig anti-sst2A antibody (GP3) and rabbit anti-sst3 antibody (7986). The subcellular distribution of receptor proteins was examined by confocal microscopy. Upper panel, sst2A-like immunoreactivity is shown in red. Middle panel, sst3-like immunoreactivity is shown in green. Lower panel, overlay of sst2A-like immunoreactivity (red) and sst3-like immunoreactivity (green). Note that in untreated cells both sst2A and sst3 were almost exclusively confined to the plasma membrane, revealing extensive colocalization. Exposure to SS-14 destabilized the sst2A-sst3 heterodimer and induced robust internalization of sst2A only but not of sst3. Similarly, treatment with the sst2-selective agonist L-779,976 promoted endocytosis of sst2A but not sst3. In contrast, the sst3-selective agonist L-796,778 did not promote substantial endocytosis of either receptor. Shown are representative results from one of three independent experiments performed in duplicate. Scale bar, 20 µm.

Ligand Binding Properties of the sst2A-sst3 Heterodimer-- We compared ligand-binding properties of sst2A-sst3 heterodimers with those of sst2A and sst3 homodimers and examined the ability of subtype-selective agonists to compete with [125I-Tyr11]SS-14 binding in membranes prepared from cells expressing either T7sst2A or T7sst3 or coexpressing both T7sst2A and Mycsst3. We found that T7sst2A cells had high affinities for SS-14 and the sst2-selective agonist L-779,976. T7sst3 cells had high affinities for SS-14 and the sst3-selective agonist L-796,778 as well as moderate affinities for the sst2-selective agonist L-779,976. However, T7sst2A-Mycsst3 cells had high affinities only for SS-14 and the sst2-selective agonist L-779,976 but not for the sst3-selective agonist L-796,778 (Fig. 6, A-C). Specifically, the cells coexpressing sst2A and sst3 exhibited a 100-fold lower affinity for the sst3-selective agonist L-796,778 than cells expressing sst3 alone (Table I). These findings imply that sst2A-sst3 heterodimerization results in a new binding site with a pharmacological profile resembling that of the sst2A receptor. Although sst2A and sst3 receptor proteins were expressed in a 1:1 ratio, the sst2A-sst3 heterodimer showed no significant affinity for the sst3-selective ligand L-796,778.



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Fig. 6.   Ligand binding properties, stimulation of GTPgamma S binding, and inhibition of adenylyl cyclase by sst2A-sst3 heterodimers. A-C, competition of [125I-Tyr11]SS-14 binding by SS-14 (black-square), sst2-selective agonist L-779,976 (black-down-triangle ), and the sst3-selective agonist L-796,778 () in membranes from HEK 293 cells expressing either T7sst2A (A) or T7sst3 (B) alone or coexpressing T7sst2A and Mycsst3 (C). D-E, stimulation of [35S]GTPgamma S binding by SS-14 (black-square), the sst2-selective agonist L-779,976 (black-down-triangle ), and the sst3-selective agonist L-796,778 () in membranes from HEK 293 cells expressing either T7sst2A (D) or T7sst3 (E) alone or coexpressing T7sst2A and Mycsst3 (F). G-I, inhibition of forskolin-stimulated cAMP accumulation by SS-14 (black-square), the sst2-selective agonist L-779,976 (black-down-triangle ), and the sst3-selective agonist L-796,778 () in HEK 293 cells expressing either T7sst2A (G) or T7sst3 (H) alone or coexpressing T7sst2A and Mycsst3 (I). Results are mean of triplicate (A-F) or duplicate (G-I) measurements. S.E. were smaller than 10%. Three replicate experiments gave similar results.


                              
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Table I
Ligand binding and functional properties of sst2A-sst3 heterodimers
Radioligand binding studies and cAMP and ERK assays were carried out as described under "Experimental Procedures." The half-maximal inhibitory concentrations (IC50) for competition binding and cAMP assays and the half-maximal effector concentration (EC50) for ERK activation were analyzed by nonlinear regression curve fitting using the computer program GraphPad Prism 3.0. Data are presented as the mean of three or four independent experiments. S.E. values were smaller than 15%.

Stimulation of [35S]GTPgamma S Binding by sst2A-sst3 Heterodimers-- We next compared effector coupling efficiencies of sst2A-sst3 heterodimers with those of sst2A and sst3 homodimers and examined the ability of different agonists to stimulate [35S]GTPgamma S binding in membranes prepared from cells expressing either T7sst2A or T7sst3 or coexpressing both T7sst2A and Mycsst3. SS-14 induced a concentration-dependent stimulation of [35S]GTPgamma S binding in cells expressing either T7sst2A or T7sst3 as well as in cells coexpressing T7sst2A and Mycsst3 (Fig. 6, D-F). Notably, 1 µM SS-14 produced a more robust effector response in cells coexpressing T7sst2A and Mycsst3 (Emax = 297%) than in cells expressing only T7sst2A (Emax = 193%). The sst2-selective agonist L-779,976 stimulated strong [35S]GTPgamma S binding in cells expressing T7sst2A and in cells coexpressing T7sst2A and Mycsst3. In addition, L-779,976 produced a similar stimulatory response as L-796,778 (sst3-selective agonist) in cells expressing T7sst3, indicating limited sst2 selectivity over sst3 of L-779,976 in concentrations exceeding 100 nM (Fig. 6E). In contrast to that seen in T7sst3 cells, the sst3-selective agonist L-796,778 produced no substantial [35S]GTPgamma S binding in cells coexpressing T7sst2A and Mycsst3 (Fig. 6, E and F).

Inhibition of Adenylyl Cyclase by sst2A-sst3 Heterodimers-- The activation of somatostatin receptors by agonists results in decreased levels of intracellular cAMP. We therefore examined the ability of sst agonists to inhibit forskolin-stimulated cAMP accumulation in cells expressing either T7sst2A or T7sst3 or coexpressing both T7sst2A and Mycsst3. SS-14 induced an inhibition of adenylyl cyclase with half-maximal inhibitory concentrations (IC50) in the low nanomolar range in T7sst2A cells and T7sst3 cells as well as in cells coexpressing both T7sst2A and Mycsst3 (Fig. 6, G-I, Table I). The sst2-selective agonist L-779,976 produced similar robust responses in T7sst2A cells as in cells coexpressing T7sst2A and Mycsst3. In contrast, sst3-selective agonist L-796,778 inhibited forskolin-stimulated cAMP accumulation in T7sst3 cells only, but essentially no inhibition was found in cells coexpressing T7sst2A and Mycsst3 (Fig. 6, H and I, Table I). We also investigated the possibility of a partial agonism by L-796,778. If the sst3-selective ligand L-796,778 would be a partial agonist at the sst3 dimer or antagonist at the sst2A-sst3 heterodimer, it would be expected to block the SS-14-mediated inhibition of cAMP accumulation. However, SS-14-mediated responses were not attenuated in the presence of L-796,778 either on T7sst3 cells or on cells coexpressing both T7sst2A and Mycsst3, suggesting that L-796,778 is a pure sst3 agonist (data not shown).

ERK Activation by sst2A-sst3 Heterodimers-- The activation of somatostatin receptors by agonists results in a rapid and transient stimulation of ERK1/2 phosphorylation. We next examined the ability of several agonists to increase the levels of phosphorylated ERK1/2 in cells expressing either T7sst2A or T7sst3 or coexpressing both T7sst2A and Mycsst3. As shown in Fig. 7, 5-min exposure to 100 nM SS-14 produced a robust increase in ERK1/2 phosphorylation in T7sst2A cells and T7sst3 as well as in cells coexpressing both T7sst2A and Mycsst3. The sst2-selective agonist L-779,976 (10 nM) promoted a similar response in T7sst2A cells as in cells coexpressing T7sst2A and Mycsst3. In contrast, the sst3-selective agonist L-796,778 (100 nM) stimulated ERK1/2 activity in T7sst3 cells only but not in cells coexpressing T7sst2A and Mycsst3 (Fig. 7, Table I). These findings imply that sst2A-sst3 heterodimerization results in a new receptor with a functional profile (e.g. [35S]GTPgamma S binding, inhibition of adenylyl cyclase, and ERK activation) resembling that of the sst2A receptor. Although sst2A and sst3 receptor proteins were expressed in a 1:1 ratio, the sst2A-sst3 heterodimer showed no significant functional response to the sst3-specific ligand L-796,778.



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Fig. 7.   Activation of ERK1/2 by sst2A-sst3 heterodimers. HEK 293 cells expressing either T7sst2A (upper panel) or T7sst3 alone (middle panel) or cells coexpressing Mycsst2A and T7sst3 (lower panel) were either not treated (-) or treated with 100 nM SS-14 (+), 10 nM L-779,976 (+) (sst2-selective agonist), or 100 nM L-796,778 (+) (sst3-selective agonist) for 5 min. Cells were lysed, equal amounts of protein were resolved by SDS-PAGE, and levels of phosphorylated ERK1/2 were determined by immunoblotting as described under "Experimental Procedures." The arrows indicate the positions of phospho-ERK1/2. Three additional experiments gave similar results.

Desensitization of the sst2A-sst3 Heterodimer-- Finally, we compared agonist-induced desensitization of sst2A dimer and sst2A-sst3 heterodimer. Cells expressing T7sst2A or coexpressing both T7sst2A and Mycsst3 were preincubated with 1 µM SS-14 for 0, 1, 2, 4, or 6 h. The medium was removed, and the ability of SS-14 to inhibit forskolin-stimulated cAMP accumulation was examined. The sst2A dimer underwent a rapid time-dependent loss of coupling to adenylate cyclase with a maximum desensitization at 6 h (Fig. 8A). In contrast, the sst2A-sst3 heterodimer appeared to be more resistant to agonist-induced desensitization with a strong receptor response retained as long as 6 h (Fig. 8B). To test the possibility that the delayed desensitization of the sst2A-sst3 heterodimer may be related to agonist-induced monomerization and subsequent gain of function of the sst3 receptor, cells coexpressing T7sst2A and Mycsst3 were challenged with the sst3-selective ligand L-796,778 after extended exposure with SS-14. However, the sst3-selective ligand was not able to inhibit forskolin-induced cAMP accumulation in T7sst2A-Mycsst3 cells whether or not these cells had been preincubated with SS-14 (data not shown).



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Fig. 8.   Comparison of agonist-induced desensitization of sst2A homodimers and sst2A-sst3 heterodimers. HEK 293 cells expressing T7sst2A (A) or cells coexpressing Mycsst2A and T7sst3 (B) were either not preincubated (black-square, solid line) or preincubated with 1 µM SS-14 for 6 h (, dotted line). Cells were washed, and inhibition of forskolin-stimulated cAMP accumulation by SS-14 was determined. Results are means of duplicate measurements. S.E. values were smaller than 10%. Two replicate experiments gave similar results.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Western blot and coimmunoprecipitation experiments carried out in the present study clearly demonstrate that the sst2A receptor as well as the sst3 receptor exist as constitutive homodimers at the plasma membrane when expressed alone and as heterodimers when coexpressed in HEK 293 cells. Both sst2A and sst3 dimers were resistant to reducing agents but sensitive to higher concentrations of detergents, suggesting that dimerization involves noncovalent hydrophobic interactions of the receptor proteins (Figs. 1D and 2C). Formation of homodimers between truncated sst3 receptors suggests that the cytoplasmic tail was not necessary for dimerization (Fig. 2D). Several dimerization interfaces have been proposed for other GPCRs such as the extracellular amino-terminal domain for the glutamate and calcium-sensing receptors, the intracellular third loop, and the VIth transmembrane region for the dopaminergic and beta 2-adrenergic receptor and the C-terminal tail for the delta -opioid and GABAB receptor (1, 2, 4, 5, 7, 14-19).

To study the sst2A-sst3 heterodimer directly, we performed coimmunoprecipitation experiments using HEK 293 cells coexpressing T7sst2A and Mycsst3. These studies clearly showed that the sst2A-sst3 heterodimers can be coimmunoprecipitated using a variety of antibody combinations (Fig. 3). To exclude the possibility that sst2A-sst3 heterodimers were artifactually formed during the preparation of cell lysates and sample processing, coimmunoprecipitation studies were also carried out using a mixture of cells expressing T7sst2A and Mycsst3 individually. Under these conditions, heterodimers could not be immunoprecipitated, strongly suggesting that sst2A-sst3 heterodimers were formed in vivo prior to cell lysis (Fig. 3A). Interestingly, when the material coimmunoprecipitated using an anti-sst2A antibody was probed with an anti-sst3 antibody, we detected only a single high molecular weight band corresponding to the sst2A-sst3 heterodimer. This suggests that heterodimers were stable during cell lysis and reducing SDS-PAGE. When material coimmunoprecipitated using an anti-sst2A antibody was probed with an anti-sst2A antibody, we detected an additional lower molecular weight band that corresponds to the sst2A monomer, indicating that, similar to the results seen on Western blots, both monomeric and heterodimeric forms of the receptor proteins were present at the plasma membrane. However, the possibility that sst2A and sst3 may exist as high molecular weight hetero-oligomeric arrays in coexpressing cells cannot be excluded.

Agonist treatment had little effect on the levels of sst2A and sst3 homo- and heterodimers detected on Western blots (Figs. 1C, 2A, and 3D). Interestingly, agonist exposure of the delta -opioid receptor was found to induce monomerization, which precedes receptor internalization (2). In contrast, recent studies using fluorescence resonance energy transfer suggest that dimers formed by the beta 2-adrenergic receptor and the sst5 somatostatin receptor are stabilized by agonist binding (1, 11, 20). On the other hand, agonist stimulation of the m3 muscarinic receptor and the calcium-sensing receptor neither promotes nor destabilizes receptor dimer formation (5, 6, 9, 10). It remains to be clarified whether the observed differences in agonist regulation of receptor dimerization reflect intrinsic differences in the structural properties of the studied receptor proteins or are due to variations in experimental conditions.

Although sst2A and sst3 receptor proteins were expressed approximately in a 1:1 ratio in T7sst2A-Mycsst3 cells as determined by quantitative Western blot analysis and saturation binding assays, membrane preparations from these cells had greatly reduced affinity for the sst3-selective ligand L-796,778. In contrast, T7sst2A-Mycsst3 cells exhibited high ligand binding affinities for SS-14 and the sst2-selective ligand L-779,976. Like the sst2A homodimer, the sst2A-sst3 heterodimer stimulated GTPgamma S binding, inhibition of adenylyl cyclase and activation of ERK1/2 after exposure to the sst2-selective ligand L-779,976. However, unlike the sst3 homodimer, the sst2A-sst3 heterodimer did not promote GTPgamma S binding, adenylyl cyclase inhibition, or ERK activation in the presence of the sst3-selective ligand L-796,778 (Figs. 6 and 7, Table I). These findings suggest that heterodimerization of sst2A and sst3 results in a new receptor with a pharmacological profile resembling that of the sst2A receptor. However, the sst2A-sst3 heterodimer and the sst2A homodimer differ in that the sst2A-sst3 heterodimer appears to be more resistant to agonist-induced desensitization of coupling to adenylyl cyclase than the sst2A homodimer (Fig. 8). Several recent studies have reported on heterodimerization among GPCRs (e.g. the GABAB heterodimers, kappa -delta heterodimers, and D2R-sst5 heterodimers) (3, 12-20). In each case, heterodimerization results in a new receptor with enhanced functional activity. Here, we provide the first evidence for inactivation of a fully functional sst3 receptor by heterodimerization with the sst2A receptor.

The fact that the sst3 receptor is rendered nonfunctional after heterodimerization with sst2A may provide a plausible explanation for the unexpectedly low number of somatostatin binding sites on HEK 293 cells coexpressing sst2A and sst3. Comparative Western blot analysis revealed that virtually identical levels of sst2A and sst3 receptor protein were present in coexpressing cells as compared with cells expressing these receptors alone. Saturation binding assays revealed that T7sst2A (Bmax = 820 ± 19 fmol/mg) and T7sst3 cells (Bmax = 1182 ± 31 fmol/mg) exhibited similar densities of somatostatin binding sites. This suggests that sst2A and sst3 receptor proteins were expressed approximately in a 1:1 ratio in T7sst2A-Mycsst3 cells. This also suggests that higher levels of total somatostatin receptor proteins (sst2A plus sst3) were present in coexpressing cells. However, this doubling of somatostatin receptor protein was not associated with an equivalent increase in the total number of detectable somatostatin binding sites in these cells (Bmax = 1088 ± 66 fmol/mg).

The functional inactivation of sst3 by heterodimerization with sst2A may also explain the observed destabilization of the sst2A-sst3 heterodimer and selective internalization of sst2A after SS-14 treatment. Agonist exposure may induce a conformational change of the sst2A-sst3 heterodimer that favors G protein-coupled receptor kinase-mediated phosphorylation of sst2A while sst3 remains in a nonphosphorylated state. Phosphorylation of intracellular domains of the sst2A receptor would then be expected to facilitate binding of beta -arrestin and preferential targeting of this receptor to the endocytotic machinery. Nevertheless, internalization of the sst2A receptor was not complete (Fig. 5), and sst2A-sst3 heterodimers could also be coimmunoprecipitated from membrane preparations of SS-14-treated cells (Fig. 3D). However, the agonist-induced conformational change appeared to destabilize the sst2A-sst3 heterodimer to an extent that still allowed coimmunoprecipitation of sst2A-sst3 heterodimers but facilitated separation of a proportion of heterodimers during sample preparation and gel electrophoresis and, hence, permitting detection of monomeric receptor forms on Western blots (Fig. 3D).

sst2A and sst3 are widely distributed throughout the central nervous system and periphery (25-27). sst2A-sst3 heterodimerization may also occur in vivo, since colocalization of these receptors has been observed in several tissues including pancreatic islands and the anterior lobe of the pituitary. Interestingly, particular high levels of sst3 expression have been detected in the cerebellum; however, radioligand binding studies largely failed to detect sst3 receptor binding sites in this region (27-29). Thus, functional inactivation of the sst3 receptor by heterodimerization with sst2A or other GPCRs may provide a possible explanation for some of the difficulties in detecting sst3-specific binding and signaling in mammalian tissues.

The physical interaction between somatostatin receptors may have direct implications for the treatment of neuroendocrine malignancies that frequently overexpress several subtypes of somatostatin receptors (30, 31). Based on the present findings, a tumor coexpressing sst2A and sst3 would be expected to respond to treatment with sst2-selective but not with sst3-selective agonists. In contrast, a tumor with isolated expression of sst3 would be expected to respond to treatment with sst3-selective but not with sst2-selective agonists. Evaluation of the somatostatin receptor status in a given tumor may therefore provide valuable predictive information for the treatment of human tumors with somatostatin receptor subtype-specific ligands (24, 31).

In conclusion, we provide biochemical and functional evidence for homo- and heterodimerization of the sst2A and sst3 somatostatin receptors. We show that heterodimerization results in a new receptor with a pharmacological and functional profile resembling that of the sst2A receptor, however with a greater resistance to agonist-induced desensitization. The sst2A-sst3 receptor is the first heterodimer that results in inactivation of a fully functional receptor.


    ACKNOWLEDGEMENTS

We thank Dana Mayer, Evelyn Kahl, and Inge Schwarz for excellent technical assistance.


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grants SCHU 924/4-3 (to S. S.), SFB 426/A2 (to V. H.), and SFB 545/B7 (to H.-J. K.) and European Commission Grant QRTL-1999-00908 (to S. S.).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.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Leipziger Str. 44. Tel.: 49-391-671-5881; Fax: 49-391-671-5869; E-mail: Stefan.Schulz@medizin.uni-magdeburg.de.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006084200


    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; BS3, bis(sulfosuccinimidyl)suberate; DTT, DL-dithiothreitol; ERK, extracellular signal-regulated kinases; GABA, gamma -aminobutyric acid; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SS-14, somatostatin; sst, somatostatin receptor; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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