Coupling Specificity between Somatostatin Receptor sst2A and G Proteins: Isolation of the Receptor-G Protein Complex with a Receptor Antibody

Yi-Zhong Gu and Agnes Schonbrunn

Department of Integrative Biology, Pharmacology, and Physiology University of Texas Medical School Houston, Texas 77225


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatostatin initiates its actions via a family of seven-transmembrane domain receptors. Of the five somatostatin receptor genes cloned, sst2 exists as two splice variants with the sst2A isoform being predominantly expressed. This receptor is widely distributed in endocrine, exocrine, and neuronal cells, as well as in hormonally responsive tumors, and leads to inhibition of secretion, electrical excitability, and cell proliferation. To investigate the specificity of signal transduction by the sst2A receptor, we developed antibodies against two overlapping peptides located within the C terminus of the receptor protein: peptide 2CSG, containing amino acids 334–348, and peptide 2CER, containing amino acids 339–359. Although antibodies to both peptides bound the inducing antigen with high affinity, only the antibodies against peptide 2CER precipitated the receptor. The best antibody, R2–88, precipitated about 80% of the sst2A receptor-ligand complex solubilized from transfected CHO cells and was specific for the sst2A receptor isotype. Addition of GTP{gamma}S (10 µM) to the immunoprecipitated ligand-sst2A receptor complex markedly accelerated ligand dissociation, indicating that G proteins remained functionally associated with the receptor in the immuno-precipitate. Analysis of the G proteins coprecipitated with the sst2A receptor by immunoblotting with G protein antibodies showed that both G{alpha} and Gß subunits were bound to the hormone-receptor complex. Immunoprecipitation of the receptor was not affected by the presence of bound ligand. However, G protein subunits were coprecipitated only with the hormone-occupied receptor. Thus, the unoccupied receptor has low affinity for G proteins, and hormone binding stabilizes the receptor-G protein complex. Use of subtype-specific G protein antisera further showed that G{alpha}i1, G{alpha}i2, and G{alpha}i3 were complexed with the sst2A receptor whereas G{alpha}o, G{alpha}z, and G{alpha}q were not. Together, these studies demonstrate that the sst2A receptor interacts selectively with G{alpha}i proteins in a hormone-dependent manner. The finding that this receptor couples to all three G{alpha}i subunits may help explain how somatostatin can regulate multiple signaling pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatostatin (SRIF) exists in two biological forms (SRIF-14 and SRIF-28) and is a widely distributed inhibitor of endocrine, exocrine, gastrointestinal, and neural functions (1, 2). The actions of the somatostatin peptides are initiated by a family of seven-transmembrane domain receptors encoded by five distinct genes (3). The sst2 receptor subtype mRNA is abundantly expressed in the pituitary, pancreas, brain, spleen, and gastrointestinal tract as well as in a variety of hormonally responsive tumors. Its wide distribution is consistent with important physiological and pathological roles for this receptor in the control of hormone and exocrine secretion, immune and neural regulation, and tumor cell growth (3, 4, 5). Previous studies showed that the primary transcript from the sst2 receptor gene is alternatively spliced to generate two receptor mRNA isoforms, named sst2A and sst2B, which diverge in their COOH-terminal sequence (6). Moreover, these splice variants appear to differ in their coupling to adenylyl cyclase as well as in their susceptibility to regulation (7, 8) indicating the importance of the COOH terminus in these receptor functions.

Although the somatostatin receptor family is known to regulate multiple signal transduction pathways, signaling by endogenously expressed sst2A receptors have not been clearly identified since this receptor subtype is seldom expressed in the absence of the other sst receptors, and specific agonists are not yet available (3, 9). However, when transfected into receptor-negative host cells, both sst2A and sst2B receptors inhibit adenylyl cyclase (7, 8, 10). Overexpressed sst2A receptors have also been shown to inhibit Ca++ channels (11), stimulate phospholipase C and Ca++ mobilization (12), and stimulate tyrosine phosphatase activity (13). However, the G proteins mediating these effects are unknown.

In this study we have used a novel approach to investigate the specificity of sst2A receptor-G protein coupling. Using a newly developed sst2A receptor-specific antibody, we immunoprecipitated the receptor-G protein complex and identified the G protein subunits associated with the sst2A receptor. This represents the first study to systematically characterize the spectrum of G proteins coupled to this sst receptor isotype.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antibody Preparation and Characterization
Two overlapping peptides, derived from a unique sequence in the carboxy-terminal region of the sst2A receptor, were used as immunogens (Fig. 1Go). The peptide 2CSG corresponds to amino acids 334–348 in the mouse sst2A receptor and differs from both the rat and human receptors by one residue. The peptide 2CER corresponds to amino acids 339–359 in the rat sst2A receptor and is identical in the human, rat, and mouse sst2A proteins (14, 15). The two antigen peptides share a nine-amino acid sequence but contain unique residues at either their amino (2CSG) or their carboxyl (2CER) termini.



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Figure 1. Alignment of Peptide Antigens with the Carboxy-Terminal Region of sst Receptors

Amino acids in the carboxy-terminal region of rat sst receptor subtypes were aligned starting from the seventh putative transmembrane domain. Single-letter code is used to denote the amino acids. Gaps were introduced to maximize alignement. Boxed areas indicate conserved amino acid residues between the two peptide antigens and the sst receptor subtypes. Nucleotide sequences for the rat sst receptors were obtained from Genbank using the following accession numbers: rat sst1: M97656, rat sst2A: M93273, rat sst3: X63574, rat sst4: M96544 and rat stt5: L04535 and X74828. sst2B is a splice variant of sst2A (57)

 
Peptides were coupled to keyhole limpet hemocyanin (KLH) through an amino-terminal cysteine, and each peptide was used to immunize two rabbits. Immune sera were assayed by enzyme-linked immunosorbent assay, and the dilution of antiserum giving half-maximal binding to adsorbed peptide was subsequently used to determine the relative affinity of each antibody for the inducing antigen. Antisera R2–87 and R2–88 bound the immunizing peptide 2CER with ED50 values of 5.1 ± 2.5 nM (not shown) and 4.4 ± 2.9 nM (Fig. 2Go), respectively. Antisera R2–204 and R2–206 bound the immunizing peptide 2CSG with ED50 values of 2.7 ± 1.1 nM (not shown) and 1.5 ± 0.3 nM (Fig. 2Go), respectively. Interestingly, peptide 2CSG did not compete for binding to antiserum R2–88 (Fig. 2Go), suggesting that this antiserum recognized the unique C-terminal sequence of the immunizing peptide 2CER rather than the region it shared with the peptide 2CSG.



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Figure 2. Affinity and Peptide Specificity of Antisera

Fifty nanograms of peptide 2CER (• and {blacksquare}) or peptide 2CSG ({circ}) was adsorbed onto each well of a 96-well plate and incubated with antiserum and the concentrations of competing peptide shown on the abscissa. ELISA assays were performed as described in Materials and Methods. Antiserum R2–88 (• and {blacksquare}) was used at a dilution of 1:25,000 and competed with either the homologous peptide 2CER (•) or with the heterologous peptide 2CSG ({blacksquare}). Antiserum R2–206 was used at a dilution of 1:8,000 and was competed with the homologous peptide 2CSG ({circ}). Each point shows the mean ± SEM of triplicate samples.

 
Specific Immunoprecipitation of sst2A Receptors
CHO-K1 cells stably transfected with the rat sst2A receptor gene (CHO-R2A) (10) were used to determine which antisera recognized the receptor protein. After preincubation of CHO-R2A membranes with the radiolabeled somatostatin analog [125I-Tyr11]SRIF, the ligand-receptor complex was solubilized with dodecyl-ß-D-maltoside (DßM) (16). The soluble [125I-Tyr11]-SRIF-receptor complex was quantitated by precipitation with polyethylene glycol, and the amount of the complex bound by each antibody was then determined (Fig. 3Go, upper panel). Previous studies had shown that the [125I-Tyr11]SRIF-receptor complex is extremely stable at 4 C (16), and we confirmed that less than 10% of the bound ligand dissociated from the sst2A receptor during an overnight incubation with antiserum at 4 C. The results in Fig. 3Go (upper panel) demonstrate that the sst2A receptor was immunoprecipitated by both antisera against peptide 2CER (R2–87 and R2–88) but was not significantly precipitated by either of the antisera against peptide 2CSG (R2–204 and R2–206). Because antiserum R2–88 was most effective, it was characterized in greater detail.



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Figure 3. Immunoprecipitation of the sst2A Receptor

Upper panel, CHO-R2A membranes (37 µg/ml) were incubated with [125I-Tyr11]SRIF (304, 900 cpm/ml, 0.084 nM) in the absence or presence of 100 nM SRIF for 2 h at 30 C. After binding, membranes were separated from free ligand by centrifugation and solubilized for 1 h at 4 C. The amount of intact ligand-receptor complex in the soluble fraction was determined by precipitating an aliquot with 40% PEG as described in Materials and Methods. Immunoabsorption was performed by incubating soluble receptor with antisera for 3 h at 4 C followed by precipitation with Protein A-Sepharose. Antiserum R2–88 was used at a final dilution of 1:1000 whereas other antisera were diluted 1:100. The graph shows the amount of the [125I-Tyr11]SRIF-receptor complex immunoprecipitated out of 14,400 cpm added to the assay. Lower panel, CHO-R2A membranes (63 µg/ml) were bound with [125I-Tyr11]SRIF (792, 800 cpm/ml, 0.22 nM) and then solubilized for 1 h at 4 C. Soluble receptor was either precipitated with PEG to quantitate the ligand-receptor complex (Total) or incubated with antiserum R2–88 at different dilutions for 3 h at 4 C. After precipitation with Protein A-Sepharose, specific binding was measured in the immunoprecipitate. For both panels, the data represent the mean ± SEM.

 
Immunoprecipitation of the sst2A receptor-ligand complex by R2–88 was concentration dependent with a maximal 75–80% immunoprecipitation achieved at antiserum dilutions of 1:1000 or less (Fig. 3Go, lower panel). Whereas immune serum precipitated 80% of the soluble [125I-Tyr11]SRIF-sst2A receptor complex, preimmune serum precipitated only 2% of the receptor (Fig. 4Go, upper panel). Moreover, addition of 10 µM antigen peptide to the incubation with antiserum completely inhibited receptor immunoprecipitation (Fig. 4Go, upper panel).



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Figure 4. Specificity of the sst2A Receptor Antiserum R2–88

Upper panel, Membranes from CHO-R2A cells (86 µg/ml) were incubated with [125I-Tyr11]SRIF (568, 620 cpm/ml, 0.157 nM) in the absence or presence of 100 nM SRIF for 2 h at 30 C. After receptor solubilization, the amount of ligand-receptor complex in the soluble fraction (Total) was determined by PEG precipitation. For immunoabsorption, soluble receptors were incubated with antiserum R2–88 (1:100) at 4 C for 3 h. After precipitation of the immune complex with Protein A-Sepharose, specific binding in the pellets was measured. Lower panel, Membranes (80–130 µg/ml) from CHO cells expressing sst1, sst2A, sst2B, or sst4 were incubated with [125I-Tyr11]SRIF for 2 h at 30°C and then solubilized. The amount of soluble receptor-ligand complex was determined for each receptor subtype by PEG precipitation (Total) and ranged from 7,060 to 10,010 cpm. After incubation with R2–88 (dilution = 1:100–1:400) for 3 h at 4 C, specific binding in the immunoprecipitate was measured and expressed as a percent of the total soluble receptor-ligand complex added. For both panels the data represent mean ± SEM for specific binding in triplicate samples.

 
The receptor specificity of antiserum R2–88 was characterized using CHO-K1 cells stably transfected with individual sst receptor subtypes (Fig. 4Go, lower panel). Sst2B receptors lack any sequence homology with the peptide used for immunization, and, as expected, this receptor was not recognized by antiserum R2–88 (Fig. 4Go, lower panel). Moreover, neither sst1 nor sst4 receptors were immunoprecipitated even though the immunizing peptide 2CER contained a three- amino acid sequence also present in these receptor subtypes (Fig. 4Go, lower panel). In separate experiments, we further found that this antiserum did not immunoprecipitate the sst3 or sst5 receptors (data not shown). Therefore, antiserum R2–88 specifically recognizes the sst2A receptor and precipitates it with high efficiency. This antiserum was used in all subsequent experiments.

Biochemical Characterization of Endogenously Expressed sst2A Receptors
GH4C1 rat mammosomatotropic pituitary cells, AtT-20 mouse corticotropic pituitary cells, and AR4–2J rat pancreatic acinar cells have been widely used for studies of sst receptors and SRIF signal transduction (17, 18, 19, 20). However, these cell lines express mRNA for multiple sst receptor subtypes, and the level of individual sst receptor proteins is unknown (19, 21, 22). To covalently radiolabel sst receptors in these cells, membranes from each cell line were incubated with the photolabile analog [125I-Tyr11, ANB-Lys4]SRIF (5'-azido-2'-nitrobenzoyl Lys4, Tyr11) SRIF and then irradiated with UV light (23). Analysis of the affinity-labeled membranes by SDS-PAGE and autoradiography showed that [125I-Tyr11, ANB-Lys4]SRIF covalently labeled a broad, 60- to 80-kDa band in CHO-R1 cells and a broad 80- to 100-kDa band in CHO-R2A cells (Fig. 5Go, upper panel), confirming previous observations (21). Addition of 100 nM SRIF inhibited photoaffinity labeling of these bands as expected for saturable receptors (Fig. 5Go, upper panel). In the three cell lines known to express sst receptors endogenously, the most heavily labeled proteins also migrated as broad bands between 80–100 kDa, as previously reported (23). Although the molecular mass predicted for sst receptor proteins from their DNA sequence is 41 kDa, these receptors contain potential glycosylation sites. The migration of photoaffinity-labeled sst receptors as broad, high molecular mass bands is consistent with glycosylation. Two other bands were weakly labeled in AtT-20 membranes: one at approximately 55 kDa and one at approximately 32 kDa. Photoaffinity labeling of these bands was also inhibited by excess SRIF consistent with their being either sst receptors or receptor degradation products.



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Figure 5. Specific Immunoprecipitation of Photoaffinity-Labeled sst2A Receptors

Top panel, Membranes (23–66 µg/ml) from CHO-R1, CHO-R2, AR4–2J, AtT-20, or GH4C1 cells were incubated in the dark for 2 h at 30 C with [125I-Tyr11, ANB-Lys4]SRIF (253, 410 cpm/ml, 0.07 nM) in the absence or presence of 100 nM SRIF. After centrifugation to remove unbound ligand, membranes were resuspended in HEPES binding buffer and irradiated with UV light for 10 min. Membranes were then solubilized in sample buffer and analyzed by SDS-PAGE and autoradiography. Bottom panel, Membranes (30–120 µg/ml) from each cell line were incubated with [125I-Tyr11,ANB-Lys4]SRIF (326,040 cpm/ml, 0.089 nM) and extracted with DßM/CHS, and the resulting soluble receptors were then irradiated as described in Materials and Methods. Photoaffinity-labeled receptors were subsequently incubated for 3 h at 4°C with antiserum R2–88 (1:1000 final dilution) in the presence or absence of the peptide antigen 2CER (5 µM). After precipitation with Protein A-Sepharose, the pellets were dissolved in sample buffer and analyzed by SDS-PAGE and autoradiography.

 
We next used the R2–88 antiserum to isolate the photoaffinity-labeled sst2A receptor subtype from each cell line. Membranes were incubated with [125I-Tyr11, ANB-Lys4]SRIF, solubilized, irradiated with UV light, and incubated with receptor antiserum in the presence or absence of excess antigen peptide (Fig. 5Go, lower panel). No labeled bands were observed in immunoprecipitates from CHO-R1 cells. However, a broad~ 85-kDa band was precipitated from CHO-R2A cells, and immunoprecipitation of this band was completely inhibited by 5 µM antigen peptide, confirming that R2–88 specifically recognized the sst2A receptor.

We next determined whether any of the photoaffinity-labeled receptors in AR4–2J, AtT-20, or GH4C1 cells represented the sst2A receptor. A broadly migrating 85-kDa band was observed in the immunoprecipitates from all three cell lines (Fig. 5Go, lower panel). Neither of the smaller photoaffinity-labeled membrane proteins from AtT-20 cells were immunoprecipitated, indicating that these proteins lacked the sst2A C-terminal epitope. These results demonstrate that the sst2A receptor is endogenously expressed in AR4–2J, AtT-20, and GH4C1 cells and migrates at 80–100 kDa on SDS polyacrylamide gels. The subtle variation in the apparent molecular mass of this receptor indicates that it may be glycosylated differently in the three cell types.

Effect of Ligand Binding on Receptor Immunoprecipitation
In the previous experiments, receptor immunoprecipitation was measured by the coprecipitation of either noncovalently or covalently bound radiolabeled ligand. To determine whether the presence of ligand affected the ability of the antibody to bind the receptor, we developed a Western blot procedure for receptor detection (Fig. 6Go). Proteins from CHO-R2A membranes were subjected to SDS-PAGE and immunoblotted with antiserum R2–88. The antiserum stained a broad band centered around 85 kDa, and this staining was abolished by the addition of 1 µM antigen peptide (Fig. 6Go, left panel). The comigration of this immunoreactive band with the photoaffinity-labeled receptor from CHO-R2A membranes indicates that it corresponds to the sst2A receptor. Since noncovalently bound ligand is dissociated from SRIF receptors during SDS-PAGE (23) the R2–88 antibody can recognize the denatured receptor on immunoblots in the absence of bound ligand.



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Figure 6. Effect of Ligand Binding on Receptor Immunoprecipitation

Left panel, CHO-R2A membrane proteins (10 µg/lane) were separated by electrophoresis in a 10% polyacrylamide SDS gel. After transfer of proteins to a PVDF membrane, the membrane was incubated with antiserum R2–88 (final dilution 1:20,000) in the absence (-) or presence (+) of 1 µM peptide 2CER. Immunoreactive proteins were visualized as described in Materials and Methods. Right panel, CHO-R2A membranes were incubated in the absence (-) or presence (+) of 100 nM SRIF at 30 C for 2 h. After solubilization, proteins (from 8.5 µg of membranes) were incubated with the covalently coupled antiserum R2–88-Protein A-Sepharose complex in the absence or presence of peptide 2CER (1 µM). Immunoprecipitated proteins were solubilized in sample buffer, electrophoresed on a 10% SDS-acrylamide gel, and transfered to a PVDF membrane. The sst2A receptor was detected by immunoblotting with R2–88 antiserum (1:10,000 final dilution).

 
To determine the effect of hormone binding on receptor immunoprecipitation, CHO-R2A membranes were incubated in the absence or presence of 100 nM SRIF, solubilized, and immunoprecipitated with antibody R2–88 covalently coupled to Protein A-Sepharose (Fig. 6Go, right panel). Subsequent analysis of the immunoprecipitated proteins by SDS-PAGE and immunoblotting with receptor antiserum showed that the 85-kDa receptor protein was present in the immunoprecipitates from both SRIF-treated and control membranes (Fig. 6Go, right panel). Therefore, antiserum R2–88 recognized the sst2A receptor either with or without bound ligand.

Coprecipitation of G Proteins with the sst2A Receptor
The sst2A receptor has been shown to couple to inhibition of adenlyl cyclase via pertussis toxin-sensitive G proteins in the CHO-R2 cell strain used in our studies (10). Consistent with these observations, we found that pretreating the cells for 16 h with 100 ng/ml pertussis toxin produced an 84% decrease in [125I-Tyr11]SRIF binding to the sst2A receptor in membranes (data not shown). To determine whether G proteins were immunoprecipitated with the sst2A receptor, we took advantage of the known ability of GTP analogs to stimulate ligand dissociation from the G protein- coupled form of SRIF receptors (16). After immunoprecipitation with receptor antibody, the Sepharose-bound [125I-Tyr11]SRIF-sst2A receptor complex was resuspended in buffer with or without 10 µM GTP{gamma}S and incubated at 25 C. At various times the radioactive ligand that remained associated with the antibody-bound receptor was quantitated after recentrifugation (Fig. 7Go). The amount of receptor-[125I-Tyr11]SRIF complex was unaltered during a 10-min incubation in the absence of guanine nucleotide (Fig. 7Go). However, GTP{gamma}S markedly stimulated ligand dissociation such that more than 50% of the receptor-bound peptide had dissociated after 10 min (Fig. 7Go). Thus, G proteins are functionally associated with the receptor-ligand complex in the immunoprecipitate.



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Figure 7. Effect of GTP{gamma}S on Ligand Dissociation from Immunoprecipitated Receptor

CHO-R2A membranes (100 µg/ml) were incubated [125I-Tyr11]SRIF (308, 950 cpm/ml, 0.085 nM) for 2 h at 30 C. After solubilization, receptors were incubated with antiserum R2–88 (1:200 final dilution) and precipitated with Protein A-Sepharose. The washed immunoprecipitate was then resuspended and incubated at 25 C in HEPES buffer containing 0.25 mg/ml DßM and 10 nM SRIF, with or without 10 µM GTP{gamma}S. At the times shown, aliquots were removed from the reaction and centrifuged at 4 C. The [125I-Tyr11]SRIF specifically bound in the immunoprecipitate was measured and expressed as a fraction of the initial binding (1440 cpm). Each point shows the mean ± SEM of triplicate samples.

 
Specificity and Ligand Dependence of sst2A Receptor-G Protein Interactions
Before examining the coupling of the sst2A receptor to specific G proteins, we determined which G protein {alpha}-subunits were present in CHO membranes using immunoblot analysis. Specific immunoreactivity was detected at 40–43 kDa with antibodies to G{alpha}i2, G{alpha}i3, and G{alpha}q/11 (data not shown). However, antibodies to G{alpha}i1, G{alpha}o, and G{alpha}z failed to detect any specific bands in 50 µg of CHO membrane protein suggesting that, if expressed at all, these G proteins are present at low levels. These results are consistent with published observations in CHO cells (10, 24, 25, 26).

To identify the specific G proteins coupled to the sst2A receptor, membranes from CHO-R2A cells were incubated with or without ligand, solubilized, and immunoprecipitated with receptor antiserum (Fig. 8Go). The G protein subunits copurifying with the receptor were detected by immunoblotting with antibodies specific for different {alpha}- or ß-subunits. A rat brain cholate extract, known to be enriched in G proteins, was used as a positive control for the blotting antisera.



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Figure 8. Identification of G Proteins Coprecipitated with the sst2A Receptor

CHO-R2A membranes were incubated with or without 100 nM SRIF. After solubilization, receptors were immunoprecipitated in the absence or presence of 7 µM peptide 2CER with antiserum R2–88 covalently coupled to Protein A-Sepharose. Precipitated proteins were solubilized in sample buffer and separated on a 12% acrylamide-SDS gel. After tranfer to PVDF membranes, G protein subunits were identified by immunoblotting with specific G protein antibodies (anti-G{alpha}i1 at 1:1,000; J-883 against G{alpha}i2 at 1:1,000; EC against G{alpha}i1/{alpha}o at 1:2,000; U-46 against G{alpha}o at 1:2,000; P-961 against G{alpha}z at 1:1,000, anti-G{alpha}q/11 at 1:2, 500, SW1 against Gß at 1:20,000).

 
Figure 8Go (top panel) shows that antibody EC, which recognizes both {alpha}i3 and {alpha}o (27), detected a 41-kDa band in immunoprecipitates from SRIF preincubated membranes. This protein was absent when 1 µM antigen peptide was added during the incubation with antiserum, demonstrating that it was dependent on receptor immunoprecipitation. Moreover, this protein was only precipitated when the receptor was preincubated with ligand (Fig. 8Go, top panel). Thus, although hormone binding did not affect receptor immunoprecipitation (Fig. 6Go), it did determine whether the G{alpha} subunit was coprecipitated (Fig. 8Go). The protein recognized by antibody EC is G{alpha}i3 rather than G{alpha}o 1) because its 41-kDa molecular mass was closer to that of G{alpha}i3 (41 kDa) than that of G{alpha}o (39 kDa) (27), and 2) because it was not recognized by the antibody U-46, which is specific for G{alpha}o (Fig. 8Go, bottom panel). As with the G{alpha} subunit, Gß was precipitated with the sst2A receptor in a ligand- dependent manner (Fig. 8Go, top panel). Therefore, both G protein {alpha}- and ß-subunits form a stable complex with the sst2A receptor only when it is occupied by hormone.

To further characterize the specificity of sst2A receptor-G protein interactions, we determined whether other G proteins copurified with the receptor. The results in Fig. 8Go (bottom panel) show that G{alpha}i1 and G{alpha}i2 also complexed with the receptor whereas G{alpha}q, G{alpha}o, and G{alpha}z did not. Hence the sst2A receptor exhibits strong G protein selectivity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The specificity of signal transduction by heptahelical receptors is determined by the G proteins with which they interact, since it is the receptor-activated G protein {alpha}- and ß{gamma}-subunits that regulate effector enzymes and ion channels (28, 29, 30). SRIF receptors have been shown to signal via both pertussis toxin-sensitive and -insensitive G proteins, and sst receptor-G protein coupling specificity has received considerable attention in the attempt to delineate the molecular mechanisms involved (4, 31). However, most previous studies were conducted with tissues or cell lines that are now known to express multiple sst receptor subtypes (16, 32, 33, 34, 35, 36). Thus, although the spectrum of G proteins with which the sst receptor family can couple has been examined, the G protein coupling specificity of individual sst receptor subtypes remains poorly understood. To investigate sst2A receptor-G protein coupling, we developed antibodies specific for this receptor isoform and used these antibodies to isolate and characterize the sst2A receptor-G protein complex.

Antisera were generated against two overlapping peptide antigens corresponding to unique sst2A sequences. Although all the antisera bound the corresponding immunizing peptide with comparable affinities, the two antisera against peptide 2CER were both able to immunoprecipitate the receptor, whereas neither of the antisera against peptide 2CSG could do so. The failure of the 2CSG antisera to recognize the receptor was not due to interference by receptor-associated G proteins because these antisera also did not precipitate photoaffinity-labeled receptors preincubated with GTP{gamma}S (data not shown). In the case of the best antiserum, R2–88, the epitope region was deduced to be COOH-terminal to amino acid 348 because the peptide 2CER (amino acids 339–359) bound to the antibody with nanomolar affinity, whereas the overlapping peptide 2CSG (amino acids 334–348) did not bind. Interestingly, sequence analysis by the method of Hopp and Woods (37) predicted that the region spanned by amino acids 334–348 would be more hydrophilic/antigenic than that spanned by amino acids 349–359. Thus, although choice of the peptide used for immunization proved critical for receptor recognition by the antisera, hydrophilicity analysis did not identify the most useful sequence in this instance.

Several observations proved that antiserum R2–88 recognized the sst2A receptor specifically. The antibody immunoprecipitated more than 70% of the [125I-Tyr11]SRIF-sst2A receptor complex solubilized from CHO-R2 cells. It also immunoprecipitated the 85-kDa photoaffinity-labeled sst2A receptor and recognized the denatured receptor on immunoblots. Both the immunoprecipitation and the immunoblotting of the sst2A receptor were blocked by micromolar concentrations of antigen peptide, and preimmune serum was inactive. Finally, the antiserum did not bind any of the other sst receptor subtypes.

We next identified the endogenous sst2A receptor protein in differentiated cell lines that had previously been shown to express sst2 mRNA, along with mRNA for other sst receptors (19, 21, 22). Photoaffinity-labeled receptors, migrating as broad {approx} 85 kDa bands, were immunoprecipitated from AR4–2J pancreatic acinar cells, AtT-20 corticotrophic pituitary cells, and GH4C1 mammosomatotrophic pituitary cells. Thus all three of these cell lines express the sst2A receptor protein. The molecular mass of the sst2 receptor is predicted to be 41 kDa from its amino acid sequence; however, previous studies with GH4C1 cells showed that this receptor is glycosylated (38). Hence, added carbohydrates probably account for the increased mass of the photoaffinity-labeled sst2A receptor as well as its broad migration pattern on polyacrylamide gels. The subtle differences in the apparent size and breadth of the immunoprecipitated sst2A receptor band in the cell lines that we examined suggest that receptor glycosylation varies among cell types. More than 70% of the ligand-receptor complex could be immunoprecipitated from AR4–2J cells (data not shown). This is similar to the immunoprecipitation efficiency observed with receptors from CHO-R2A cells, which express only the sst2A subtype, and indicates that sst2A is the major sst receptor in this acinar cell line. Only 30–40% of the ligand-receptor complex was precipitated from AtT-20 and GH4C1 cells (data not shown), consistent with the known presence of other sst receptors isotypes in these pituitary cells (19, 21).

Two other groups have described the biochemical properties of sst2 receptors by immunoblotting with antipeptide antibodies. Patel et al. (19) generated an antiserum (EE704) to an extracellular amino-terminal segment common to the sst2A/B receptors and detected a broad, 72-kDa protein in membranes from sst2-transfected CHO and AtT-20 cells (19). This size is close to that of the photoaffinity-labeled receptor immunoprecipitated from CHO-R2 and AtT20 cells by our sst2A-specific antibody. In contrast, Theveniau et al. (39) stained distinctly different molecular mass proteins with an antibody (2e3) generated to amino acids in the third extracellular domain common to the sst2A/B receptors. The 2e3 antibody detected a sharp 93-kDa band in sst2-expressing CHO membranes and a sharp band of 148 kDa in AR4–2J cell membranes. These bands differ from the broad, photoaffinity-labeled 85 kDa band that we identified as the sst2A receptor in CHO and AR4–2J cells by immunoprecipitation. Since the 93- and 148-kDa proteins were not shown to bind SRIF, their identity as sst receptors, rather than related proteins, remains to be established. The antibody described here is the first to specifically recognize the sst2A receptor subtype in immunoprecipitation and immunoblotting, as well as immunohistochemical, assays (40) and provides a valuable new reagent for studies of the sst2A receptor protein.

To determine whether immunoprecipitation could be used to elucidate the G protein-coupling specificity of the sst2A receptor, we first determined the effect of ligand and G protein association on antibody binding to the receptor. Our results showed that the antibody efficiently immunoprecipitated both the hormone-receptor complex and the unoccupied receptor. To determine whether G proteins were physically associated with the receptor in the immune complex, we examined the effect of GTP{gamma}S on the rate of ligand dissociation from the immunoprecipitated receptor. To favor the formation of the agonist-receptor-G protein ternary complex in the native membrane environment, membrane receptors were first incubated with radiolabeled hormone in the absence of guanine nucleotides (28) and then solubilized under conditions that maintain a functional interaction between receptor and G proteins (16). Since GTP{gamma}S markedly stimulated hormone dissociation from the immunoprecipitated receptor, G proteins must remain coupled, and the carboxy-terminal receptor epitope recognized by the antibody cannot be essential for functional interaction between the sst2A receptor and G proteins. We previously found that this was also true for the sst1 receptor subtype (21). Further analysis of the immunoprecipitated complex showed that both {alpha}- and ß- G protein subunits were coprecipitated with the sst2A receptor. Since the ß-subunit is usually found tightly complexed with the {gamma}-subunit, it is likely that the latter is also present. The physical association between these G protein subunits and the receptor was absolutely dependent on ligand occupancy, showing that hormone binding stabilizes the receptor-G protein complex and that little of the receptor is precoupled to G proteins in the absence of agonist activation.

The G protein {alpha}-subunits physically associated with the sst2A receptor were subsequently identified by immunoblotting of the immunoprecipitated complex. Immunoblotting of CHO membranes indicated that G{alpha}i2 and G{alpha}q/11 were abundantly expressed whereas G{alpha}i3, although readily detectable, was less intensely stained (data not shown). G{alpha}i1, G{alpha}o, and G{alpha}z were not detected. Our results are consistent with the quantification of G{alpha}i subunits reported by Gettys et al. (26) who showed that G{alpha}i1 was not detectable (<0.1 pmol/mg) in CHO-K1 membranes and that G{alpha}i2 (5 pmol/mg) was present at much higher levels than G{alpha}i3 (0.6 pmol/mg). Other groups have also reported that G{alpha}o and G{alpha}z are not detectable in CHO-K1 cells (25).

Whereas the three G{alpha}i subunits copurified with the sst2A receptor, G{alpha}q/11, G{alpha}o, and G{alpha}z were not found in the receptor complex. The observation that G{alpha}o and G{alpha}z were not associated with the receptor probably results from the absence of these G proteins from CHO cells, although we cannot rule out the possibility that they are expressed at levels below the limits of immunodetection. However, because G{alpha}q/11 is abundant, its absence from the receptor complex demonstrates that the sst2A receptor exhibits strong G protein specificity. Although the receptor was physically associated with all three G{alpha}i subunits, it had the highest affinity for G{alpha}i1, since this is the least abundant G{alpha}i in CHO-K1 membranes and showed the greatest enrichment in the receptor complex.

In contrast to our observations, Law et al. (41) concluded that the sst2A receptor couples to G{alpha}i3 but not to G{alpha}i1 or G{alpha}i2 when expressed in CHO-DG44 cells, a cell line that contains all three G{alpha}i subunits. This difference may reflect functional differences in coupling in the two cell lines. In the same transfected CHO-K1 cell line used in our study, the sst2A receptor was shown to inhibit adenylyl cyclase via pertussis toxin-sensitive G proteins (10). In contrast, the transfected sst2A receptor did not inhibit adenylyl cyclase in CHO-DG44 cells (42). However, critical differences between the protocols used in the two studies could also explain the disparate results obtained. In our experiments, hormone binding was carried out in membranes to stabilize specific receptor-G protein complexes within the normal lipid environment of the receptor. The ternary complex was then solubilized under conditions in which it is known to remain stable (16). By comparison, Law et al. (41) solubilized the unoccupied receptor and then examined the ability of G protein antibodies to inhibit hormone binding in the detergent extract. The presence of detergent in the binding reaction may well alter the specificity of receptor-G protein coupling from that which occurs in the plasma membrane.

Transfected sst2 receptors stimulate tyrosine phosphatases and phospholipase C as well as inhibit calcium channels and adenylyl cyclase (11, 12, 13). Whereas the inhibition of cAMP accumulation is completely reversed by pertussis toxin treatment, SRIF-induced activation of tyrosine phosphatase and phospholipase C are either insensitive or only partially inhibited by the toxin (12, 13). Hence, the sst2 receptor has been proposed to act via pertussis toxin-insensitive as well as -sensitive G proteins. We did not detect any interaction between the sst2A receptor and the pertussis toxin- insensitive G proteins G{alpha}q/11 and G{alpha}z, even though the former is abundant in the CHO-K1 cells. Thus, the pertussis toxin-insensitive pathways activated by the sst2A receptor remain to be identified.

It is now widely recognized that seven-transmembrane domain receptors are capable of coupling with multiple but selected G proteins either of the same or different subgroups, thus permitting activation of several distinct signal transduction pathways. This coupling is believed to play an important role in determining cell-specific receptor functions. Our studies demonstrate that in a normal membrane environment, hormone binding causes the sst2A receptor to couple specifically to the three Gi proteins, with highest affinity for G{alpha}i1. The availability of an antibody that selectively immunoprecipitates this receptor subtype now makes it possible to explore the coupling of endogenously expressed sst2A receptors irrespective of the presence of other sst isoforms and allow identification of its signal transduction pathways in different cellular environments.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antisera Preparation and Assay
Antigen peptides were synthesized with an N-terminal cysteine and conjugated to KLH through the cysteine sulfhydryl with m-maleimidobenzoyl-N-hydroxysuccinimide (43). New Zealand white male rabbits (Ray Nichols, Lumberton, TX) were immunized with each KLH-coupled peptide antigen as previously described (21). The titers and binding affinities of each antiserum for the corresponding peptide antigen were measured by ELISA (21).

Covalently coupled Protein A-Sepharose was prepared by incubating Protein A (20 mg) with CNBr-activated Sepharose 4B (1.75 g) according to manufacturer’s instructions (Pharmacia-LKB Biotechnology, Rockville, MD). IgG and Protein A-Sepharose 4B were coupled with dimethyl pimelimidate as described by Harlow and Lane (44). Any noncovalently bound IgG was eluted from the Protein A-Sepharose by incubating the beads in 10 volumes of 0.1 M glycine, pH 2.8, at room temperature for 5 min. After centrifugation, the pellet was extensively washed with PBS, pH 7.5, and then stored at 4 C in PBS with 0.05% NaN3.

Cell Culture and Membrane Preparation
CHO-K1 cell lines transfected to stably express individual sst receptor subtypes were obtained from the following sources: rat sst1 (CHO-R1) and rat sst2A (CHO-R2A) receptor-expressing cells were provided by Dr. Philip Stork (10), mouse sst2B receptor-expressing cells (CHO-R2B) were provided by Dr. Voker Hollt (6), human sst3 receptor-expressing cells were provided by Dr. Yogesh Patel (45), and rat sst4- expressing cells were provided by Drs. Jack Bruno and Michael Berelowitz (46). CHO-K1 cells stably expressing the human sst5 receptor were generated from a plasmid provided by Dr. Susumu Seino (47). A 1.7-kb DNA fragment containing the entire human sst5 coding sequence was excised from the pCMV6c plasmid by digestion with EcoRI/SalI and ligated into the EcoRI/XhoI sites of plasmid pcDNA3. Cells were selected in 250 µg/ml G418 and cloned by serial dilution.

GH4C1, AR4–2J, AtT-20, RINm5F, and CHO cell lines were grown and subcultured as previously described (10, 23, 48). Pertussis toxin treatment was performed by incubating cells with fresh medium containing 100 ng/ml pertussis toxin for 16–24 h before membrane preparation.

Cell membranes were prepared and stored as previously published (23). Briefly, cells from either monolayer or suspension cultures were collected, washed with PBS (10 mM Na2HPO4, 150 mM NaCl, pH 7.4), and homogenized at 4 C in Tris buffer (10 mM Tris-Cl, 2 mM MgCl2, 2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, pH 7.6). After centrifugation at 500 x g for 10 min, the supernatant was collected and centrifuged again at 10,000 x g for 30 min. The membrane pellet was stored at -70 C.

Ligand Binding and Receptor Photoaffinity Labeling
[Tyr11]SRIF and the photoactive SRIF analog [Tyr11, ANB-Lys4]SRIF were iodinated with Chloramine-T, and the radiolabeled peptides were purified by reverse-phase HPLC to a specific activity of 2,200 Ci/mmole (23). Binding reactions were performed as previously described (23). Briefly, membranes were incubated at 30 C for 2 h in HEPES binding buffer (50 mM HEPES, 7 mM MgCl2, 2 mM EDTA, and 2 U/ml of bacitracin) containing radiolabeled peptide (0.05–0.15 nM) with or without 100 nM unlabeled SRIF. After dilution with cold binding buffer, samples were centrifuged at 30,000 x g for 15 min, and the radioactivity associated with the pellets was measured. Specific binding was calculated as the difference between the amount of radioactivity bound in the absence and in the presence of 100 nM unlabeled SRIF. Curve-fitting and data analysis was carried out as described previously (23).

Receptor photoaffinity labeling was performed with either membranes or solubilized receptors (21, 23). Membranes were incubated in the dark with [125I-Tyr11, ANB-Lys4]SRIF, centrifuged, and then resuspended in cold HEPES binding buffer to a final concentration of 0.1 mg/ml. Irradiation was at 254 nm on ice for 10 min (Mineralight model R-52, Ultraviolet Products Inc., San Gabriel, CA). After the addition of 1 M Tris·Cl, pH 7.6, membranes were pelleted, solubilized in sample buffer (62.5 mM Tris·Cl, pH 6.8, 2% SDS, 20% glycerol, and 50 mM dithiothreitol), and subjected to SDS-PAGE according to the method of Laemmli (49). The dried gels were exposed to Amersham Hyperfilm at -70 C. For immunoprecipitation experiments, [125I-Tyr11, ANB-Lys4]SRIF binding was carried out in the dark as described above, but irradiation was performed after solubilization of the ligand-receptor complex with DßM/cholesterol hemisuccinate (CHS). The photoaffinity-labeled receptor was then subjected to immunoprecipitation and analysis by SDS-PAGE and autoradiography.

Immunoprecipitation
Unless stated otherwise, cell membranes, prebound with a radiolabeled SRIF analog, were solubilized for 1 h at 4 C in HEPES binding buffer containing 1 mg/ml DßM, 0.2 mg/ml CHS, 10 µg/ml soybean trypsin inhibitor, 50 µg/ml bacitracin, and 10 µg/ml leupeptin as described previously (16). After centrifugation at 100,000 x g, the solubilized ligand-receptor complex was either quantitated by polyethylene glycol 8000 (PEG) precipitation (16) or subjected to immunoprecipitation.

For immunoprecipitation, antiserum was added to the solubilized receptor to the final concentration indicated and incubated at 4 C for 3–20 h. Protein A-Sepharose-4B was then added, and the incubation was continued for another hour at 4 C. After centrifugation at 10,000 x g for 2 min, the pellet was washed with cold HEPES binding buffer containing 0.25 mg/ml DßM, and the precipitated radioactivity was measured.

To determine the rate of ligand dissociation from the immunoprecipitated receptor, cell membranes were incubated with [125I-Tyr11]SRIF in the absence and presence of 100 nM SRIF for 2 h at 30 C. After solubilization and immunoprecipitation, the precipitate was washed, resuspended in HEPES binding buffer containing 10 nM SRIF, and 0.25 mg/ml DßM with or without 10 µM GTP{gamma}S, and then incubated at 25 C. At indicated times, duplicate aliquots of the suspension were removed and centrifuged, and the specifically bound ligand remaining associated with the immunoprecipitated receptor was quantitated.

Immunoblotting
After receptor immunoprecipitation, the pellet was resuspended in sample buffer and subjected to SDS-PAGE. Rat brain cholate extract (50 µg) was used as a positive control for all G protein blotting experiments. The resolved proteins were transferred to polyvinylidene difluoride (PVDF) membrane at 100 mA for 30 min and 500 mA for 1.5 h. The membrane was then blocked with 5% nonfat dry milk at room temperature for 2 h and subsequently incubated at 4 C overnight either with receptor antiserum or with G protein subtype-specific antisera. PVDF membranes were washed twice with PBS containing 0.05% Tween 20, incubated with goat anti-rabbit antibody conjugated with horse radish peroxidase for 1 h, and washed again. Immunoreactive proteins were detected by chemiluminescence using enhanced chemiluminescence (ECL) substrate according to manufacturer’s instructions (Amersham, Arlington Heights, IL).

Antisera against different G protein subunits were obtained from the following sources: G{alpha}i1 (internal 159–168) (51) and G{alpha}i3 (C-terminal 345–354) (52) purchased from Calbiochem (San Diego, CA); antiserum J-883 against G{alpha}i2 and antiserum U-46 against G{alpha}o from Dr. Suzanne Mumby (53); the G{alpha}z antiserum P-961 from Dr. Patrick Casey (54); antiserum EC against G{alpha}i3/G{alpha}o and antiserum SW1 against Gß from Dr. Allen Spiegel (27, 55); antiserum against G{alpha}q/11 from Dr. Thomas F. J. Martin (56).


    ACKNOWLEDGMENTS
 
We thank Ms. Fengyu Jiang for excellent technical assistance, Ms. Yining Wang for generating the CHO-R5 cells, Dr. Philip Stork for providing CHO-R1and CHO-R2A cells, Drs. Jack Bruno and Michael Berelowitz for providing CHO-R4 cells, Dr. Volker Hollt for providing CHO-R2B cells, Dr. Yogesh Patel for providing CHO-R3 cells, and Dr. Susumu Seino for providing human sst5 receptor plasmid. We also thank Drs. Suzanne Mumby, Patrick Casey, Paul Sternweis, Allen Spiegel, and Thomas F. J. Martin for providing G protein subtype-specific antibodies and Dr. William T. Moore of the Analytical Chemistry Center at the University of Texas Medical School and Drs. Kaijun Ren and Timothy P. Kogan of Texas Biotechnology Corporation for peptide synthesis.


    FOOTNOTES
 
Address requests for reprints to: Agnes Schonbrunn, Department of Pharmacology, University of Texas Health Sciences Center, Houston, P.O. Box 20708, Houston, Texas 77225.

This investigation was supported by Research Grant DK-32234 from the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases.

Received for publication January 16, 1997. Revision received February 7, 1997. Accepted for publication February 10, 1997.


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