Somatostatin-14 was obtained from Bachem, Torrance, CA. β₂AR agonist formoterol hemifumarate and antagonist ICI-118551 was purchased from Tocris Cookson Inc., Ellisville, Missouri, USA. The non-peptide agonist L-817818 (hSSTR5) was provided by Dr. S.P. Rohrer from Merck & Co 36. Monoclonal and polyclonal antibodies against HA- and cMyc- and β-actin were procured from Sigma-Aldrich, Inc., St. Louis, MO. Fluorescein and rhodamine conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies were purchased from Jackson Immuno Research ON. Polyclonal antibodies for total and phospho-ERK1/2 (phosphorylation site-Thr202/Tyr204) and p38 (phosphorylation site- Thr180/Tyr182) were obtained from Cell Signaling Technology, Danvers, MA. Antibodies for total and phospho-PKA (phosphorylation site-Thr198) and NFAT (phosphorylation site-Ser265) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. cAMP assay kit was purchased from BioVision, Inc. CA, USA. Protein A/G-Agarose beads were procured from Calbiochem, EMD Biosciences, Darmstadt, Germany. Reagents for electrophoresis were purchased from BIO-RAD Laboratories Mississauga ON, Canada. 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride was purchased from Molecular Probes, Inc., Eugene, OR. Reagents for cell culture were purchased from GIBCO, Invitrogen, Burlington, ON, Canada. Other reagents were of AR grade and were procured from various sources.

cMyc-β₂AR in pCDNA3.1⁺/Hygro vector (hygromycin resistance) was purchased from TOP Gene Technologies, Montreal, Canada. Construct of HA-SSTR5 was made using the pCDNA3.1⁺/Neo (neomycin resistance) as previously described 2242537. The stable transfections of HA-hSSTR5 and cMyc- β₂AR in HEK-293 cells were prepared by Lipofectamine transfection reagent as described 12242537. Cotransfection of cMyc-β₂AR in the HEK-293 cells stably expressing HA-hSSTR5 was performed using Lipofectamine transfection reagent and the cells were maintained in Dulbecco's MEM supplemented with 10% fetal bovine serum (FBS), 700 μg/ml neomycin and 400 μg/ml hygromycin as described earlier 12242537.

HEK-293 cells cotransfected with cMyc-β₂AR/HA-hSSTR5 were treated with SST (1 μM) and formoterol (1 μM) alone or in combination for 30 min at 37°C. Membrane protein (250 μg) was solubilized in 1 ml of radioimmune precipitation assay buffer (RIPA Buffer, 150 mM NaCl, 50 mM Tris-HCL, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, pH 8.0) for 1 h at 4°C and Co-IP was performed as previously described 35. Briefly, samples were incubated with 1 μg antibody overnight at 4°C. 25 μl of protein A/G-agarose beads were added to immunoprecipitate antibody for 2 h at 4°C. Beads were washed and solubilized in Laemmli sample buffer (Bio-Rad) and fractionated by electrophoresis on a 10% SDS-polyacrylamide gel. The fractionated proteins were transferred to a 0.2 μm nitrocellulose membrane and blotted with anti-HA or anti-cMyc antibody (dilution 1:500) for the expression of HA-hSSTR5 and cMyc-β₂AR and detected by chemiluminescence using ECL Western blotting detection kit (Amersham) according to the manufacturer's instructions 2437. Images were captured using an Alpha Innotech FluorChem 8800 gel box imager (Alpha Innotech Co., San Leandro, CA).

HEK-293 cells expressing cMyc- β₂AR/HA-hSSTR5 were grown on poly-D-lysine coated glass coverslips to 60-70% cell confluency. Cells were treated with 1 μM SST and 1 μM β₂ agonist alone or in combination for 15 min at 37°C and fixed with 4% paraformaldehyde for 20 min on ice and were further processed for immunofluorescence immunocytochemistry 243537. Monoclonal anti-HA and polyclonal anti-cMyc primary antibodies were used followed by FITC- and Cy3- conjugated secondary antibodies to create donor-acceptor pair. The plasma membrane region was used to analyze the photobleaching decay on a pixel-by-pixel basis as previously described 2437. The FRET efficiency (E) was calculated in terms of a percent based upon the photo bleaching (Pb) time constants of the donor taken in the absence (D-A) and presence (D+A) of acceptor and relative FRET efficiency was calculated as previously described 35.

To study receptor internalization, HEK-293 cells stably expressing cMyc-β₂AR/HA-hSSTR5 were grown on poly-D-Lysine coated coverslips to 60-70% cell confluency. Cells were treated with SST (1 μM) and formoterol (1 μM) alone or in combination for 15 min at 37°C and were processed for immunocytochemistry as previously described 253537. Receptor expression in both non-permeabilized and permeabilized cells was analyzed by using Leica DMLB microscope attached with the Retiga 2000R camera. DAPI dihydrochloride was used for nuclear staining. Adobe Photoshop was used to construct figure composites and merged images displaying colocalization were generated by using Image J software, NIH.

The changes in cell surface expression of receptors and measurement of FRET was also performed by using a FACS. Approximately 2 × 10⁶ cells were treated with SST, formoterol and CGP alone or in combination for 15 min at 37 C in DMEM. Cells were washed with FACS buffer (PBS pH7.4, 5% FBS, 2 mM EDTA), fixed in 4% paraformaldehyde and were processed for immunostaining. Anti-HA and anti-cMyc primary antibodies were used followed by FITC- and Cy3- fluorescence conjugated secondary antibodies. Non-stained cells were used to setup the background level of fluorescence whereas; control cells stained with either Cy3- or FITC- was used as fluorescence control. A BD LSRII flow cytometer, configured with a 488 nm and 561 nm laser was used for the experiments. The level of Cy3- was monitored using the 561 nm laser and a 610/20 emission filter to directly measure the β₂AR expression levels. The cells were excited with the 488 nm laser first and the emission was detected using a 530/30 filter to detect the SSTR5 expression level whereas a 610/20 filter was used to detect any Cy3 emission due to FRET between SSTR5 and β₂AR. Data analysis was done by using FlowJo 7.6 software.

Briefly, to determine the basal levels of cAMP, transfected cells were incubated for 30 min with receptor specific agonists alone or in combination at 37°C in presence of 0.5 mM 3-isobutyl-1-methylxanthine. Similarly, cells were also incubated for 30 min with receptor specific agonist alone or in combination at 37°C in presence of 20 μM forskolin (FSK) and 0.5 mM 3-isobutyl-1-methylxanthine. Control and treated cells were then scraped in 0.1 N HCl and cAMP was determined by immunoassay using a cAMP Kit from BioVision, Inc. CA, USA according to the manufacturer's guidelines 2437.

To determine the G-proteins coupling with β₂AR and SSTR5 in mono- and/or cotransfected cells, G-Protein antagonizing peptide (GPAP) or Melittin and Pertussis toxin (PTX) were used to inhibit G_s and G_i respectively. Mono-and cotransfected HEK-293 cells expressing cMyc-β₂AR and/or HA-hSSTR5 were grown in 6 well culture plates and used at > 70% cell confluency for cAMP assay 37. In addition to the receptor specific agonist/antagonists, cells were also treated with GPAP (5 μM) and Melittin (1 μM) for 2 h and PTX (100 ng/ml) for 16-18 h in DMEM at 37°C and processed for cAMP estimation.

HEK-293 cells monotransfected with β₂AR or hSSTR5 were treated with receptor specific agonist whereas cells coexpressing β₂AR/hSSTR5 were treated with SST (1 μM), L-817818 (10 nM) and formoterol (1 μM) alone or in combination for 10 min and 30 min at 37°C. Whole cell lysate prepared from cells were fractionated via SDS-PAGE and transferred to a 0.2 μM nitrocellulose membrane. Immunoblotting for ERK1/2 and p38 were performed by using respective phospho-and total specific antibodies and the bands were quantified by densitometry using FluorChem software as described earlier 35. β-actin was used as loading control.

To determine the expression of total- and phospho-PKA and NFAT, mono- and/or cotransfected HEK-293 cells were treated with 1 μM SST, 10 nM L-817818, 1 μM formoterol alone or in combination for 10 or 30 min at 37°C in the medium containing 1.8 mM or 2.5 mM Ca²⁺. Cells were further processed and western blot was performed accordingly as described earlier 2437.

Results are presented as mean ± S.E unless otherwise stated. Statistical analysis was carried out using Graph Pad Prism 4.0 and statistical differences were taken at p values < 0.05. The results presented here represent three independent experiments.

To ascertain whether β₂AR and hSSTR5 exists as heterodimers, we first determined heterodimerization using Co-IP in stably cotransfected HEK-293 cells. Membrane preparation from control and treated cells were immunoprecipitated for cMyc-β₂AR and probed with antibody directed against HA to recognize HA-hSSTR5. As shown in Figure 1A, upon treatment with SST and formoterol alone or in combination, a band at ~110 kDa, the expected size of HA-hSSTR5/cMyc-β₂AR heteromeric complex was detected in cMyc immunoprecipitate. To further confirm the specificity of the blot, same membrane was striped and reprobed with the anti-cMyc antibody to determine the expression of cMyc-β₂AR. As shown in Figure 1B, monomers and homodimers of β₂AR as well as heterodimers of hSSTR5/β₂AR were observed at the expected molecular size of ~60 kDa, ~130 kDa and ~110 kDa respectively. The immunoprecipitate prepared from hSSTR5 or β₂AR monotransfected cells probed reciprocally were devoid of any heteromeric complex (Figure 1C and 1D).

Heterodimerization between hSSTR5/β₂AR as seen in Co-IP assay was further confirmed by microscopic Pb-FRET analysis (Figure 2A and 2B). Cells were processed for receptor expression by using monoclonal anti-HA and polyclonal anti-cMyc antibodies followed by FITC- (donor) and Cy3- (acceptor) conjugated secondary antibodies. Cells expressing HA-hSSTR5 and cMyc-β₂AR displayed relative FRET efficiency of 10 ± 0.5%, indicating that hSSTR5/β₂AR exists as preformed heterodimers at basal condition (Figure 2A and 3A). Interestingly, upon treatment with SST or formoterol alone, cells displayed 3.7 ± 1.3% and 4.2 ± 1.1% of relative FRET efficiency respectively in comparison to control. However, co-activation of hSSTR5 and β₂AR exhibited 17.4 ± 1% of relative FRET efficiency, significantly higher than the basal level (Figure 2B and 3A). These data suggest that the simultaneous activation of both the receptors enhanced the formation of heteromeric complex between hSSTR5 and β₂AR. Similar results were observed when directly labeled HA- and cMyc- antibodies were used in FRET analysis to support that FRET signals were not due to the aggregation of antibodies (data not shown).

To further confirm the results obtained from Co-IP and microscopic Pb-FRET analysis, non-invasive, sensitive and quantitative FACS analysis was employed to determine the receptor heterodimerization in cotransfected cells. Cells were processed as described in Materials and methods. To measure the FRET between SSTR5 and β₂AR, Cy3- emission at 610/20 was detected upon excitation of FITC with 488 nm laser in double labeled cells (Figure 3B-D). As shown in the Figure 3B, no significant emission in 610/20 channel was detected when Cy3 labeled cells were excited with 488 nm laser (mean fluorescence intensity = 21.1). In contrast, when both the receptors were labeled with fluorescence conjugated antibodies, excitation with 488 laser in control resulted in the enhanced emission at 610/20 channel displaying mean fluorescence of 26.2, indicating FRET (Figure 3C). Upon treatment with receptors specific agonist alone, cells displayed mean fluorescence comparable to Cy3 labeled cells (~22.0). Interestingly, upon combined agonist treatment, increased FRET was observed with enhanced mean fluorescence of 28.8 indicating fostered heterodimerization (Figure 3D). Taken in consideration, these data indicate that heterodimerization observed between SSTR5 and β₂AR by using Co-IP, Pb-FRET and FACS analysis is receptor specific.

Previous studies have shown that hSSTR5 and β₂AR displayed internalization upon agonist treatment 2838. Here, we determined the expression pattern of hSSTR5 and β₂AR at the cell surface and intracellularly in cotransfected cells following treatment with the receptor specific agonists by using indirect immunofluorescence microscopy and FACS analysis. In control cells, hSSTR5 and β₂AR exhibited strong colocalization at the cell surface than intracellularly (Figure 4A). Three different receptor populations were detected intracellularly i.e., either expressing hSSTR5 or β₂AR and colocalization. hSSTR5 membrane expression was decreased upon treatment with SST and resulted in the loss of receptor colocalization at the cell surface. Increased expression of hSSTR5 was observed intracellularly without any significant effect on β₂AR membrane expression (Figure 4A). In contrast, upon treatment with formoterol, β₂AR internalized and resulted with the loss of receptor expression at cell surface. This resulted in the loss of colocalization with SSTR5 and increased intracellular expression of β₂AR (Figure 4A). Importantly, simultaneous activation of both receptors displayed no significant changes in the membrane or intracellular expression as well as in colocalization when compared to the control.

Microscopic immunofluorescence internalization was further confirmed by using FACS analysis (Figure 4B, a-h). In control cells, both the receptors were well expressed on the membrane (Figure 4B, a and 4b). As shown in panel c, a significant decrease in the SSTR5 expression (FITC intensity) upon treatment with SST was observed, without any change in β₂AR expression (d). Upon treatment with formoterol, β₂AR expression (Cy3 intensity) was reduced (f), whereas, SSTR5 expression remained unaffected (e). In combined treatment with both the agonist, no significant change in the emission at 530/30 and 610/20 filters was observed in comparison to control (Figure 4B, g and 4h). Taken together, results from immunofluorescence and FACS analysis indicate that agonist induced internalization of β₂AR and SSTR5 is receptor specific and is independent of each other.

To better understand the molecular mechanism of this interaction, we first determined the changes in receptor coupling to AC in mono and/or cotransfected cells. In basal condition, cells transfected with β₂AR showed relatively higher cAMP formation (2.99 ± 0.2 picomole/mg protein) in comparison to the cells transfected with hSSTR5 (1.3 ± 0.3 picomole/mg protein) (Figure 5A). In the presence of FSK, cells expressing β₂AR or hSSTR5 displayed 7.9 ± 0.3 and 5.07 ± 0.41 picomole/mg protein of cAMP respectively. Monotransfected cells expressing hSSTR5 displayed inhibition of FSK stimulated cAMP levels by 55 ± 3% and 54 ± 3% upon treatment with SST and hSSTR5 specific agonist (L-817818) respectively (Figure 5A). In contrast, level of cAMP increased by ~3 folds (8.23 ± 0.5 picomole/mg protein) in β₂AR monotransfected cells upon formoterol treatment. In cotransfected cells, the basal intracellular cAMP level (1.65 ± 0.2 picomole/mg protein) was elevated by ~4 folds in the presence of formoterol (8.4 ± 0.4 picomole/mg protein) (Figure 5B). Upon treatment with SST or L-817818, FSK stimulated cAMP was inhibited by 8.6 ± 0.3% and 9 ± 1.5% respectively. Conversely, following treatment with formoterol, FSK stimulated cAMP was increased by 6 ± 1%. Upon combined treatment with SST or L-817818 and formoterol cells displayed 7 ± 0.5% and 8.6 ± 1% increased cAMP in the presence of FSK. Most importantly, blockade of β₂AR with receptor specific antagonist ICI-118551 (5 nM) in presence of SST resulted in enhanced inhibition of FSK stimulated cAMP levels by 23 ± 1.6% (Figure 5C). These data suggest that blockade of β₂AR is essential for the inhibitory role of SST in cotransfected cells.

GPCR coupling to AC is regulated by different G proteins including G_s, G_i, G_q and G₁₂ that play determinant role on intracellular cAMP levels. β-ARs typically interact with G_s whereas SSTRs have been shown to couple G_i. Excellent observations from Lefkowitz's laboratory and others have described that β₁AR/β₂AR also couple to G_i and activate signaling pathways in distinct manner than G_s1739. To ascertain the specificity of G proteins involved in β₂AR and SSTR5 mediated regulation of cAMP, we used G_s inhibitors GPAP and Melittin and G_i inhibitor PTX 404142. Mono and/or cotransfected cells were treated with GPAP, Melittin and PTX as described in methods section. As shown in Figure 6A, cells expressing β₂AR displayed increased levels of cAMP by ~125% in presence of FSK or formoterol, the effect which was completely abrogated upon treatment with GPAP and Melittin. β₂AR stimulated cAMP was not affected in presence of PTX in monotransfected cells (Figure 6A). SST mediated effect on cAMP inhibition was completely abrogated upon PTX treatment in SSTR5 monotransfected cells, whereas, GPAP and Melittin had no significant effect on cAMP levels (Figure 6A). These data in concurrence with previous studies indicate that β₂AR and SSTR5 are coupled to G_s and G_i respectively 1743. In cotransfected cells, increased level of cAMP upon treatment with formoterol was abolished in presence of G_s inhibitors GPAP and Melittin but not with PTX whereas, SST mediated inhibition of cAMP in presence of β₂AR antagonist ICI was blocked with PTX (Figure 6B). Interestingly, SST displayed inhibition of cAMP by 25% and 27% in the presence of GPAP and Melittin respectively. These observations indicate that β₂AR is predominantly coupled to G_s in monotransfected as well as in cotransfected cells whereas, SSTR5 mediated regulation of cAMP is mediated by G_i protein.

Previous studies have suggested the critical role of PKA in switching of G protein coupling of β₂AR 44. PKA upon activation phosphorylates β₂AR at specific C-terminal domains which in turn promotes switching of receptor coupling from G_s to G_i, thus activating different signaling cascade 44. To activate cAMP/PKA pathway mono-and/or cotransfected cells were grown in presence of basal Ca²⁺ (1.8 mM) as well as in high concentration of Ca²⁺ (2.5 mM) and cells were processed to determine the status of PKA phosphorylation. As shown in Figure 7A and 7B, β₂AR or hSSTR5 monotransfected cells displayed receptor and agonist specific changes on PKA phosphorylation in presence of basal Ca²⁺. In β₂AR monotransfected cells, formoterol decreased PKA phosphorylation upon 10 and 30 min treatment in comparison to control. In contrast, cells expressing hSSTR5 exhibited significantly decreased PKA phosphorylation in presence of SST at 10 min, whereas upon 30 min treatment phospho-PKA was comparable to control. In cotransfected cells, PKA phosphorylation was decreased upon 10 min treatment with formoterol alone or in combination with SST or SSTR5 agonist in comparison to control. However, following treatment for 30 min, the level of phospho-PKA was decreased significantly in comparison to control (Figure 7B). These results suggest that in cotransfected cells, β₂AR mediated inhibition of phospho-PKA was predominant.

In comparison, at high Ca²⁺ concentration in culture medium, the level of phospho-PKA increased in cells expressing β₂AR and decreased in hSSTR5 transfected cells when compared to basal Ca²⁺ concentration. SST or formoterol caused decrease in PKA phosphorylation in monotransfected cells (Figure 7C). However, cotransfected cells displayed complete inhibition of phospho-PKA upon treatment with SST, L-817818 (SSTR5 agonist) and formoterol alone or in combination. The effect observed in presence of high Ca²⁺ indicates the blockade of cAMP/PKA mediated pathway with predominant role of β₂AR.

The MAPK represents a critical signal transduction cascade involved in multitude of cellular processes and upon distinctive activation plays an important role in responding to the growth and stress stimuli 45. To ascertain whether receptor heterodimerization and the changes in cAMP/PKA regulate downstream signaling cascades, we next examined the status of key MAPKs i.e., ERK1/2 and p38 in mono-and cotransfected HEK-293 cells. As shown in Figure 8A, the activation of β₂AR and hSSTR5 enhanced phosphorylation of ERK1/2 in monotransfected cells. This effect was relatively more pronounced in SSTR5 transfected cells following treatment with SST for 10 and 30 min (Figure 8A and 8B). In comparison to monotransfectant, the cells coexpressing both the receptors, ERK1/2 phosphorylation was receptor and time dependent with the prominent effect of SST or SSTR5 agonist in combination with formoterol upon 10 min treatment (Figure 8A and 8B). In contrast, upon 30 min treatment with SST, L-817818 and formoterol alone or in combination, cells displayed decreased expression of phospho-ERK1/2 in comparison to 10 min treatment (Figure 8B). These results strongly support the G_i mediated activation of phospho-ERK1/2 at early time point (10 min) whereas upon treatment for 30 min phospho-ERK1/2 is predominantly regulated by β₂AR in heteromeric complex. Densitometric analysis was performed to quantify changes in phospho-ERK1/2 expression is presented in Figure 8C and 8D.

Several previous studies have described that changes in the p38 expression are associated with pro and/or anti-apoptotic effects in receptor and tissue specific manner 45. β₂AR coupling to G_i has been appreciated for its role in cell survival pathway due to anti-apoptotic action other than G_s mediated signaling. In contrast, hSSTR5 is shown to exert antiproliferative effects. Whether or not β₂AR encounter or potentiate this effect of hSSTR5 in cotransfected cells is not known. Here, we examined the status of phospho-p38 in mono-and cotransfected cells expressing β₂AR and hSSTR5. Monotransfected cells displayed weak expression of phospho-p38 upon receptor specific activation at 10 min in comparison to control (Figure 8E). Following 30 min incubation in presence of receptor specific agonists phosphorylated p38 was not detected despite the expression of total p38 as indicated (Figure 8F). The phospho-p38 was not detected in cotransfected cells with or without receptor specific activation alone or in combination following 10 or 30 min treatments, whereas expression level of total p38 remained comparable in all the conditions as indicated in Figure 8E and 8F.

Calcium mediated activation of Calcineurin induces dephosphorylation of NFAT transcription factor in the cytoplasm and increased dephosphorylation persuade its nuclear translocation which is associated with activation of several gene expression 46. Accordingly, in this experiment we compared the expression of phospho-NFAT in mono-and/or cotransfected HEK-293 cells. As shown in Figure 9A, no significant changes in the expression of pNFAT were observed when β₂AR or hSSTR5 monotransfected cells were treated with receptor specific agonists in presence of basal Ca²⁺. Comparable expression of phospho-NFAT was observed in β₂AR and hSSTR5 cotransfected cells in control as well as treated conditions as indicated. However, the status of NFAT phosphorylation was elevated in HEK-293 cells expressing hSSTR5 or β₂AR in presence of high Ca²⁺ with no significant effect of receptor specific activation (Figure 9B). In cotransfected cells, Formoterol treatment enhanced the phosphorylation of NFAT significantly whereas SST or L-817818 alone or in combination with Formoterol the expression of phospho-NFAT was comparable to control. To quantify the changes in phospho-NFAT expression, densitometric analysis was performed (Figure 9C and 9D). These results provide direct evidence that β₂AR and hSSTR5 ablated calcineurin mediated dephosphorylation and nuclear translocation of NFAT.